Materials and methods for respiratory disease control in canines.

(54) Title: MATERIALS AND METHODS FOR THE CONTROL OF RESPIRATORY DISEASES IN CANINES. (54) Title: MATERIALS AND METHODS FOR RESPIRATORY DISEASE CONTROL IN CANINES.

(57) Summary

The present invention relates to isolated influenza viruses that are capable of infecting canids, and causing respiratory disease in the canid; The present invention also pertains to compositions and methods for inducing an immune response against an influenza virus of the present invention; The present invention also pertains to compositions and methods for identifying a virus of the invention, and for diagnosing infection of an animal with a virus of the invention.

(57) Abstract

The subject invention pertains to isolated influenza virus that is capable of infecting canids and causing respiratory disease in the canid. The subject invention also pertains to compositions and methods for inducing an immune response against an influenza virus of the present invention. The subject invention also pertains to compositions and methods for identifying a virus of the invention and diagnosing infection of an animal with a virus of the invention.

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Mexican Property

Industrial

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of

PATENT TITLE NO. 339202

Headlines):

Home:

Denomination:

UNIVERSITY OF FLORIDA RESEARCH FOUNDATION, INC .; THE GOVERNMENT OF THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES, CENTERS FOR DISEASE CONTROL AND PREVENTION; CORNELL RESEARCH FOUNDATION, INC.

223 Grinter Hall, Gainesville, Florida, 32611, USA; 4770 Buford Hwy (K79), Atlanta, Georgia, 30341, USA; 20 Thornwood Drlve, Suite 105, Ithaca, New York, 14850, USA

MATERIALS AND METHODS FOR DISEASE CONTROL

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8fcAWBDRD PAUL J. GIBBS; EDWARD J.WSCVfc i BEN O NIS; JACQUELINE KATZ; ALEXANDER I. KLIMOV. WILLtAM L. CA LEMAN; cox

REQUEST

International filing date:

April 2006 ional of Patent Numbers 306534 PRIORITY

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of

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Date:

April 2005

Number:

60/673,443

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V, 6th fraction Ht, and Headquarters Layde lAustrial Property.

Reference patent is granted on the basis of

Proprietary Law of the application ii of twenty years imprt rate to maintain vile lables, intes those based on the provisions of the articles ^ s ^ S 'sections III and 7 ° bis 2 of the Bey de la

ΙβΒΛΜΜΗΜ ^ subsection a), sub subsection iii) 4th and 12th fractions I and III of the Regulations of the Mexican Institute of Industrial Property (DOF 12/14/1999, amended on 07/01/2002, 07/15/2004, 07/28/2004 and 09/07/2007); Articles 1, 3, 4, 5, section V, subsection a), sub subsection iii), 16 sections I and III and 30 of the Organic Statute of the Mexican Institute of Industrial Property '(DO; F. 12/27/1999, amended on 10/10/2002, 07/29/2004, 08/04/2004 and 09/13/2007); 1, 3 and 5 Subsection a) and antepenultimate paragraph of the Agreement that delegates powers to the Deputy Directors General, Coordinator, Divisional Directors, Heads of Regional Offices, Divisional Deputy Directors, Departmental Coordinators and other subordinates of the Mexican Institute of Property Industrial. (DOF 12/15/1999, amended on 02/04/2000, 07/29/2004, 08/04/2004 and 09/13/2007).

Issue Date: May 13, 2016

DIVISIONAL DEPUTY DIRECTOR OF EXAMINATION OF PATENT FUND, AREAS k, ELECTRICAL AND REGISTRIES-OF INDUSTRIAL DESIGNS AND

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Arenai No. 550. Floor 1,

Coi. Puebío Santa María Tepepan,

Xochimilco, CP&gt; 6020,

Mexico City

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MATERIALS AND METHODS FOR DISEASE CONTROL

RESPIRATORY ENCANINES

RECIPROCAL REFERENCE TO RELATED APPLICATION

This application claims the benefit of US provisional application serial number 60 / 673,443, filed on April 21, 2005, which is incorporated herein by reference in its entirety, including any figures, tables, nucleic acid sequences, sequences amino acids and drawings.

BACKGROUND OF THE INVENTION

Canine cough, or infectious tracheobronchitis (ITB), is an acute contagious respiratory infection in dogs characterized primarily by cough (Ford et al., 1998). Canine ITB is considered one of the most frequent infectious respiratory diseases of dogs worldwide, and outbreaks can reach epidemic proportions when dogs are housed in high-density population environments, such as kennels. Most outbreaks are due to direct dog- to-dog contact or dispersal of respiratory secretions in a gas (Ford et al., 1998). Clinical signs are caused by infection with one or a combination of bacterial and viral agents that colonize the epithelium of the

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INDUSTRIAL upper and lower respiratory tract. ParainfhiAnza ranina virus (CPiV) and Bordetella bronchiseptica bacteria are the most common organisms isolated from affected dogs, but several other viruses such as canine distemper virus (CDV) and canine adenovirus 1 and 2 (CAV-1 , CAV-2), together with bacteria such as Streptococcus sp., Pasteurella multicoda and Escherichia coli, can influence the course and clinical outcome (Ford et al., 1998). While outbreaks occur more efficiently and quickly in high-density populations with high morbidity, complicated respiratory infections and death are not uncommon. Although life-threatening secondary bacterial pneumonia may develop, most cases of ITB are self-limiting, and resolve without treatment (Ford et al., 1998).

In July 1992, a suspected respiratory infection was "canine cough", it became epidemic in several greyhound tracks in New

England, Florida, West Virginia, Wisconsin, Kansas, Colorado, Oklahoma and Arizona. According to veterinarians, most affected dogs had a mild cough that resolved, but more than a dozen greyhounds developed acute hemorrhagic pneumonia followed by rapid death (Greyhound Daily News, 1999).

In late 1998 and early 1999, several outbreaks of "canine cough" occurred in racing greyhound kennels across the country, resulting in the forced closure of the tracks, and quarantine of all racing greyhounds in the United States by several weeks (Greyhound Daily

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News, 1999). On one track in Florida (Palm Beach Kennel Club), cough was recorded in almost 40% of the dog population in a single day (personal communication from Dr. William Duggar). Similar to the 1992 outbreak, cough resolved in most greyhounds, but 10 dogs in Florida died from a hemorrhagic pneumonia syndrome not characteristic of "canine cough" (Putnam, 1999).

From March to April 2003, another outbreak of "canine cough" occurred on greyhound tracks in the eastern United States. The outbreak is believed to have originated from kennels on four tracks in Florida, causing the dogs to be suspended and quarantined for nearly three weeks. Approximately 25% of the dogs on the track in West Palm Beach were affected, while almost 50% of the 1,400 dogs at Derby Lane in St. Petersburg developed a cough. Again, most of the dogs recovered, but several dogs died from the respiratory infection. The estimated economic impact of the respiratory outbreak on the Derby Lane track alone was $ 2 million.

There are no published reports documenting the etiology or clinical pathology of the "canine cough" epidemic in racing greyhound kennels in 1992, 1998-1999 or 2003. The assumption has been that the infections were due to CPiV and / or B Bronchiseptica, the two most common causes of canine cough. Unconfirmed communications such as websites have attributed the fatal hemorrhagic pneumonia reported in some of the coughing dogs to infection with Streptococcus equi subspecies.

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Transmission of viruses from one host species to another is a crucial feature of the ecology and epidemiology of the influenza virus (Webster, 1998). Two basic mechanisms of influenza virus transmission between species are possible (Webster et al., 1992; Lipatov et al., 2004).

One is the direct transfer of an essentially unaltered virus from one species to another. Examples of this mechanism include recent human infections with the H5N1 avian influenza virus subtype (Subbarao et al., 1998; Peiris et al., 2004; Guan et al., 2004), and possibly the 1918 pandemic, known like Spanish influenza (Reid et al., 2004). The second mechanism is a consequence of the segmented nature of the influenza genome. Co-infection of a host with viruses of different species can result in a rearrangement of the segmented viral genes and the generation of a recombinant with the ability to infect other species. For example, novel viruses generated by gene rearrangement between avian and human influenza viruses resulted in human influenza pandemics in 1957 and 1968 (Webster et al., 1992; Lipatov et al., 2004; Kawaoka et al. , 1989).

Most direct transmissions of unaltered influenza viruses from the natural host species to a different species are terminal events because sustained transmission between individuals of the new species does not occur. Multiple interactions are necessary between

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INDUSTRIAL virus and host for replication and horizontal transmission, and provide a formidable barrier to the perpetuation of influenza viruses in the new host (Webby et al., 2004). Therefore, the establishment of new influenza virus host-specific lineages is not uncommon, and has only occurred in domestic poultry, pigs, horses, and humans (Webster et al., 1992; Lipatov et al., 2004 ).

Due to the serious nature of influenza virus infection, there is a need for methods to diagnose, prevent, and treat infection caused by influenza virus.

BRIEF DESCRIPTION OF THE INVENTION

The present invention pertains to isolated influenza viruses that are capable of infecting canids and causing respiratory disease in the canid. The present invention also pertains to compositions and methods for inducing an immune response against an influenza virus of the present invention. The present invention also pertains to compositions and methods for identifying a virus of the invention, and for diagnosing infection of an animal with a virus of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The application or patent file contains at least one

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drawing executed in color. Copies of this patent or publication of the patent application with color drawings will be provided by the Patent Office upon request and payment of the necessary fees.

Figures 1A-1B show phylogenetic relationships between the hemagglutinin genes. Figure 1A shows a representative HA gene tree of canine, human, bird, pig and equine isolates, including A / australian parakeet / Hokkaldo / 1/77 (H4) as the external group. Figure 1B shows a tree of canine influenza virus HA genes with contemporary and longer equine HA genes, using

A / duck / Ukraine / 63 (H3) as an external group. Phylogenetic trees were inferred from nucleotide sequences by the neighbor binding method, and initiation analysis values&gt; 90% are shown. The bar denotes the number of nucleotide changes per unit length of the horizontal branches of the tree.

Figures 2A-2B show immunohlstochemical detection of Influenza H3 antigen in the lungs. Lung tissue sections were scanned with a mouse monoclonal antibody to H3 hemagglutinin, and binding was detected by reaction with immunoperoxidase (brown precipitate). Figure 2A shows the bronchial epithelium of a greyhound with spontaneous disease. Viral H3 antigen was detected in the cytoplasm of bronchial epithelial cells and in macrophages in lumens and in alveolar spaces of the airways. Figure 2B shows a dog's bronchial epithelium 5 days after inoculation with A / canino / Florida / 43/04 (H3N8). I know

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detected viral H3 antigen in the cytoplasm of bronchial epithelial cells. Graphic scale, 66 pm.

Figures 3A-3B show the characteristic histological changes in the greyhound bronchi that died of hemorrhagic pneumonia associated with influenza virus infection. The tissues are stained with hematoxylin and eosin. Upper panel: normal bronchus with ciliated epithelial cells, mucous cells, and basal cells. Lower panel: bronchus of a greyhound with spontaneous influenza. There is necrosis and aerosion of the bronchial hair epithelial cells. Graphic scale, 100 pm.

Figures 4A-4B show phylogenetic relationships between the hemagglutinin H3 genes. Figure 4A shows a phylogenetic tree of canine influenza virus HA genes with contemporaneous and longer equine HA genes. Figure 4B shows a phylogenetic tree of the contemporary and older equine HA canine influenza virus HA protein. Phylogenetic trees were inferred from genetic or amino acid sequences by the neighbor binding method and initiation analysis values&gt; 80% are shown. The bar denotes the number of amino acid changes per unit length of the horizontal branches of the tree.

Figure 5 shows the influenza virus H3 protein in bronchial epithelial cells and bronchial glands in lungs of dogs that died of pneumonia associated with influenza virus infection. Superior panels: erosion of bronchial epithelial cells

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ciliated in bronchi. The tissues were stained with hematoxyUoa-y «eoa¡üaL Lower panels; H3 protein from influenza virus in the cytoplasm of bronchial epithelial cells (left) and bronchial glands (right). Tissues were stained with a monoclonal antibody to influenza H3 detected by reaction with immunoperoxidase (brown precipitate), and counterstained with hematoxylin.

Figures 6A-6D show amplification charts of matrix protein and H3 genes (Figure 6A and Figure 6B) obtained from amplification of serially diluted in vitro transcribed RNA standards 10-fold. Standard curves of matrix protein and H3 genes (Figure 6C and Figure 6D) constructed by plotting the logarithm of starting RNA concentrations against the threshold cycle (Ct) obtained from each dilution.

Figure 7 shows that the sensitivity of Directigen Flu A was tested with serially diluted virus stocks 10 times, including

A / Wyoming / 3/2003 and A / canino / FL / 242/2003. The purple triangle indicates a positive result.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is a nucleotide sequence of a canine influenza virus (Florida / 43/04) encoding a PB2 protein that can be used in accordance with the present invention.

SEQ ID NO: 2 is the amino acid sequence encoded by

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INDUSTRIAL

SEQ ID NO: 1.

SEQ ID NO: 3 is a nucleotide sequence of a canine influenza virus (Florida / 43/04) encoding a PB1 protein that can be used in accordance with the present invention.

SEQ ID NO: 4 is the amino acid sequence encoded by

SEQ ID NO: 3.

SEQ ID NO. 5 is a nucleotide sequence of a canine influenza virus (Florida / 43/04) encoding a PA protein that can be used in accordance with the present invention.

SEQ ID NO: 6 is the amino acid sequence encoded by

SEQ ID NO: 5.

SEQ ID NO: 7 is a nucleotide sequence of a canine influenza virus (Florida / 43/04) encoding an NS protein that can be used in accordance with the present invention.

SEQ ID NO: 8 is the amino acid sequence encoded by

SEQ ID NO: 7.

SEQ ID NO: 9 is a nucleotide sequence of a canine influenza virus (Florida / 43/04) encoding an NP protein that can be used in accordance with the present invention.

SEQ ID NO: 10 is the amino acid sequence encoded by

SEQ ID NO: 9.

SEQ ID NO: 11 is a nucleotide sequence of a canine influenza virus (Florida / 43/04) that encodes an NA protein that

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SEQ ID NO: 11.

SEQ ID NO: 13 is a nucleotide sequence of a canine influenza virus (Florida / 43/04) encoding an MA protein that can be used in accordance with the present invention.

SEQ ID NO: 14 is the amino acid sequence encoded by

SEQ ID NO: 13.

SEQ ID NO: 15 is a nucleotide sequence of a canine influenza virus (Florida / 43/04) encoding an HA protein that can be used in accordance with the present invention.

SEQ ID NO: 16 is the amino acid sequence encoded by

SEQ ID NO: 15.

SEQ ID NO: 17 is a nucleotide sequence of a canine influenza virus (FL / 242/03) that encodes a PB2 protein that can be used in accordance with the present invention.

SEQ ID NO: 18 is the amino acid sequence encoded by

SEQ ID NO: 17.

SEQ ID NO: 19 is a nucleotide sequence of a canine influenza virus (FL / 242/03) that encodes a PB1 protein that can be used in accordance with the present invention.

SEQ ID NO: 20 is the amino acid sequence encoded by

SEQ ID NO: 19.

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SEQ ID NO: 21 is a nucleotide sequence of a canine influenza virus (FL / 242/03) that encodes a PA protein that can be used in accordance with the present invention.

SEQ ID NO: 22 is the amino acid sequence encoded by

SEQ ID NO: 21.

SEQ ID NO: 23 is a nucleotide sequence of a canine influenza virus (FL / 242/03) encoding an NS protein that can be used in accordance with the present invention.

SEQ ID NO: 24 is the amino acid sequence encoded by

SEQ ID NO: 23.

SEQ ID NO: 25 is a nucleotide sequence of a canine influenza virus (FL / 242/03) that encodes an NP protein that can be used in accordance with the present invention.

SEQ ID NO: 26 is the amino acid sequence encoded by

SEQ ID NO: 25.

SEQ ID NO: 27 is a nucleotide sequence of a canine influenza virus (FL / 242/03) that encodes an NA protein that can be used in accordance with the present invention.

SEQ ID NO: 28 is the amino acid sequence encoded by

SEQ ID NO: 27.

SEQ ID NO: 29 is a nucleotide sequence of a canine influenza virus (FL / 242/03) encoding an MA protein that can be used in accordance with the present invention.

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SEQ ID NO: 30 is the amino acid sequence encoded by

SEQ ID NO: 29.

SEQ ID NO: 31 is a nucleotide sequence of a canine influenza virus (FL / 242/03) encoding an HA protein that can be used in accordance with the present invention.

SEQ ID NO: 32 is the amino acid sequence encoded by

SEQ ID NO: 31.

SEQ ID NO: 33 is the mature form of the HA protein shown in SEQ ID NO: 16, where the N10 terminal 16 amino acid signal sequence has been removed.

SEQ ID NO: 34 is the mature form of the HA protein shown in SEQ ID NO: 32, where the 16-amino acid signal sequence Nterminal has been removed.

SEQ ID NO: 35 is an oligonucleotide that can be used in accordance with the present invention.

SEQ ID NO: 36 is an oligonucleotide that can be used in accordance with the present invention.

SEQ ID NO: 37 is an oligonucleotide that can be used in accordance with the present invention.

SEQ ID NO: 38 is an oligonucleotide that can be used in accordance with the present invention.

SEQ ID NO: 39 is an oligonucleotide that can be used in accordance with the present invention.

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SEQ ID NO: 40 is an oligonucleotide that can be used in accordance with the present invention.

SEQ ID NO: 41 is an oligonucleotide that can be used in accordance with the present invention.

SEQ ID NO: 42 is an oligonucleotide that can be used in accordance with the present invention.

SEQ ID NO: 43 is an oligonucleotide that can be used in accordance with the present invention.

SEQ ID NO: 44 is an oligonucleotide that can be used in accordance with the present invention.

SEQ ID NO: 45 is an oligonucleotide that can be used in accordance with the present invention.

SEQ ID NO: 46 is an oligonucleotide that can be used in accordance with the present invention.

SEQ ID NO: 47 is a nucleotide sequence of a canine influenza virus (Miami / 2005) that encodes a PB2 protein that can be used in accordance with the present invention.

SEQ ID NO: 48 is the amino acid sequence encoded by

SEQ ID NO: 47.

SEQ ID NO: 49 is a nucleotide sequence of a canine influenza virus (Miami / 2005) that encodes a PB1 protein that can be used in accordance with the present invention.

SEQ ID NO: 50 is the amino acid sequence encoded by ¹⁴ IMPIí

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SEQ ID NO: 49.

SEQ ID NO: 51 is a nucleotide sequence of a canine influenza virus (Miami / 2005) that encodes a PA protein that can be used in accordance with the present invention.

SEQ ID NO: 52 is the amino acid sequence encoded by

SEQ ID NO: 51.

SEQ ID NO: 53 is a nucleotide sequence of a canine influenza virus (Miami / 2005) encoding an NS protein that can be used in accordance with the present invention.

SEQ ID NO: 54 is the amino acid sequence encoded by

SEQ ID NO: 53.

SEQ ID NO: 55 is a nucleotide sequence of a canine influenza virus (Miami / 2005) that encodes an NP protein that can be used in accordance with the present invention.

SEQ ID NO: 56 is the amino acid sequence encoded by

SEQ ID NO: 55.

SEQ ID NO: 57 is a nucleotide sequence of a canine influenza virus (Miami / 2005) encoding an NA protein that can be used in accordance with the present invention.

SEQ ID NO: 58 is the amino acid sequence encoded by

SEQ ID NO: 57.

SEQ ID NO: 59 is a nucleotide sequence of a canine influenza virus (Miami / 2005) that encodes an MA protein that

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SEQ ID NO: 60 is the amino acid sequence encoded by

SEQ ID NO: 59.

SEQ ID NO: 61 is a nucleotide sequence of a canine influenza virus (Miami / 2005) that encodes an HA protein that can be used in accordance with the present invention.

SEQ ID NO: 62 is the amino acid sequence encoded by

SEQ ID NO: 61.

SEQ ID NO: 63 is a nucleotide sequence of a canine influenza virus (Jacksonville / 2005) that encodes a PB2 protein that can be used in accordance with the present invention.

SEQ ID NO: 64 is the amino acid sequence encoded by

SEQ ID NO: 63.

SEQ ID NO: 65 is a nucleotide sequence of a canine influenza virus (Jacksonville / 2005) that encodes a PB1 protein that can be used in accordance with the present invention.

SEQ ID NO: 66 is the amino acid sequence encoded by

SEQ ID NO: 65.

SEQ ID NO: 67 is a nucleotide sequence of a canine influenza virus (Jacksonville / 2005) that encodes a PA protein that can be used in accordance with the present invention.

SEQ ID NO: 68 is the amino acid sequence encoded by

SEQ ID NO: 67.

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SEQ ID NO: 69 is a nucleotide sequence of a canine influenza virus (Jacksonville / 2005) that encodes an NS protein that can be used in accordance with the present invention.

SEQ ID NO: 70 is the amino acid sequence encoded by

SEQ ID NO: 69.

SEQ ID NO: 71 is a nucleotide sequence of a canine influenza virus (Jacksonville / 2005) that encodes an NP protein that can be used in accordance with the present invention.

SEQ ID NO: 72 is the amino acid sequence encoded by

SEQ ID NO: 71.

SEQ ID NO: 73 is a nucleotide sequence of a canine influenza virus (Jacksonville / 2005) that encodes an NA protein that can be used in accordance with the present invention.

SEQ ID NO: 74 is the amino acid sequence encoded by

SEQ ID NO: 73.

SEQ, D NO: 75 is a nucleotide sequence of a canine influenza virus (Jacksonville / 2005) that encodes an MA protein that can be used in accordance with the present invention.

SEQ ID NO: 76 is the amino acid sequence encoded by

SEQ ID NO: 75.

SEQ ID NO: 77 is a nucleotide sequence of a canine influenza virus (Jacksonville / 2005) that encodes an HA protein that can be used in accordance with the present invention.

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SEQ ID NO: 78 is the amino acid sequence encoded by

SEQ ID NO: 77.

SEQ ID NO: 79 is an oligonucleotide that can be used in accordance with the present invention.

SEQ ID NO: 80 is an oligonucleotide that can be used in accordance with the present invention.

SEQ ID NO: 81 is an oligonucleotide that can be used in accordance with the present invention.

SEQ ID NO: 82 is an oligonucleotide that can be used in accordance with the present invention.

SEQ ID NO: 83 is an oligonucleotide that can be used in accordance with the present invention.

SEQ ID NO: 84 is an oligonucleotide that can be used in accordance with the present invention.

SEQ ID NO: 85 is an oligonucleotide that can be used in accordance with the present invention.

SEQ ID NO: 86 is an oligonucleotide that can be used in accordance with the present invention.

SEQ ID NO: 87 is an oligonucleotide that can be used in accordance with the present invention.

SEQ ID NO: 88 is an oligonucleotide that can be used in accordance with the present invention

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DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated influenza viruses that are capable of infecting canids and causing respiratory disease.

In one embodiment, an influenza virus of the invention comprises a polynucleotide that encodes a protein having an amino acid sequence shown in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78, or a functional and / or immunogenic fragment or variant thereof. In a specific embodiment, the polynucleotide comprises the nucleotide sequence shown in any of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31,47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 or 77, or a fragment or variant thereof. In a specific embodiment, an influenza virus of the present invention is an H3 subtype. The virus can be isolated from infected dogs, and can be cultured in cells or eggs according to methods described herein. In an exemplified embodiment, the influenza virus is an influenza A virus.

The present invention also relates to polynucleotides comprising all or part of a gene or genes or a genomic segment of an influenza virus of the present invention. In one embodiment, a polynucleotide of the invention comprises an influenza hemagglutinin (HA) gene, neuraminidase (NA) gene, nucleoprotein (NP) gene, matrix protein (ΜΑ or M) gene, basic protein gene polymerase (PB) gene

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acid polymerase protein (PA), non-structural protein (NS) gene, or a fragment or functional variant of any of these genes. In a specific embodiment, a polynucleotide of the invention comprises the hemagglutinin (HA) gene, or a fragment or functional variant thereof. In another embodiment, the HA gene encodes a hemagglutinin protein that has one or more of the following; a serine at position 83; a leucine at position 222; a threonine at position 328; and / or a threonine at position 483, against the amino acid sequence of the equine H3 consensus sequence. In one embodiment, the HA gene encodes a polypeptide having an amino acid sequence shown in SEQ ID NOs: 16, 32, 62 or 78, or a functional and / or immunogenic fragment or variant thereof. In a specific embodiment, the HA gene comprises a nucleotide sequence shown in SEQ ID NOs: 15, 31, 61 or 77.

In one embodiment, a polynucleotide of the invention encodes a polypeptide having the amino acid sequence shown in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 , 26, 28, 30, 32, 33,

34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78, or a functional and / or immunogenic fragment or variant thereof. In a specific embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NOs: 2,4,6,9,12,14,14,14,18,20,22,24,26,28,30,

32, 33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78, comprises the nucleotide sequence shown in SEQ ID NOs: 1 , 3, 5, 7,

9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55, 57, 59, 61, 63,

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65, 67, 69, 71, 73, 75 or 77, respectively, or a sequence encoding a functional and / or immunogenic fragment or variant of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50,

52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or 78. Thus, the present invention relates to polynucleotide sequences that comprise the nucleotide sequence shown in either from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55,

57, 59, 61, 63, 65, 67, 69, 71, 73, 75 or 77, or a fragment or variant, including a degenerate variant, of any of SEQ ID NOs: 1, 3, 5, 7,

9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55, 57, 59, 61, 63,

65, 67, 69, 71, 73, 75 or 77. In another specific embodiment, a polynucleotide of the invention may comprise: nucleotides 1-2271 of SEQ ID NO: 3; nucleotides 1-2148 of SEQ ID NO: 5; nucleotides 1-657 of SEQ ID NO: 7;

nucleotides 1-1494 of SEQ ID NO: 9; nucleotides 1-1410 of SEQ ID NO: 11;

nucleotides 1-756 of SEQ ID NO: 13; nucleotides 1-1695 of SEQ ID NO: 15;

nucleotides 1-2271 of SEQ ID NO: 19; nucleotides 1-2148 of SEQ ID NO: 21;

nucleotides 1-657 of SEQ ID NO: 23; nucleotides 1-1494 of SEQ ID NO: 25;

nucleotides 1-756 of SEQ ID NO: 29; nucleotides 1-1695 of SEQ ID NO: 31;

nucleotides 1-2277 of SEQ ID NO: 47; nucleotides 1-2271 of SEQ ID NO: 49;

nucleotides 1-2148 of SEQ ID NO: 51; nucleotides 1-690 of SEQ ID NO: 53;

nucleotides 1-1494 of SEQ ID NO: 55; nucleotides 1-1410 of SEQ ID NO: 57;

nucleotides 1-756 of SEQ ID NO: 59; nucleotides 1-1695 of SEQ ID NO: 61;

nucleotides 1-2277 of SEQ ID NO: 63; nucleotides 1-2271 of SEQ ID NO: 65;

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GIVE THE PROPERTY

INDUSTRIAL nucleotides 1-2148 of SEQ ID NO: 67; nucleotides 1-690 of SEQ ID NO: 69;

nucleotides 1-1494 of SEQ ID NO: 71; nucleotides 1-1410 of SEQ ID NO; 73; nucleotides 1-756 of SEQ ID NO: 75; and nucleotides 1-1695 of SEQ ID NO: 77. The nucleotide and amino acid sequences of the viral polynucleotide and polypeptide sequences contemplated within the scope of the present invention have also been deposited with GenBank under accession numbers DQ124147 at DQ124161 and DQ124190, the disclosure of which is incorporated herein by reference.

The present invention also relates to polynucleotide encoded polypeptides of an influenza virus of the present invention. The present invention also relates to fragments and functional and / or immunogenic variants of the present polypeptides. The contemplated polypeptides include HA protein, NA protein, NS protein, nucleoprotein, basic polymerase protein, acidic polymerase protein and matrix protein of an influenza virus of the invention. In an exemplified embodiment, a polypeptide of the invention has an amino acid sequence shown in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,

16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,

68, 70, 72, 74, 76 or 78, or a functional and / or immunogenic fragment or variant thereof.

The present invention also relates to polynucleotide expression constructs comprising a polynucleotide sequence of the present invention. In one modality, a construction

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MKICANO INSTITUTE

Df LA PRORBpAD

INDUSTRIAL expression of the invention comprises a polynucleotide sequence encoding a polypeptide comprising an amino acid sequence shown in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,

76 or 78, or a functional and / or immunogenic fragment or variant thereof. In a specific embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,

24, 26, 28, 30, 32, 33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78 comprise the nucleotide sequence shown in SEQ ID NOs: 1,

3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55, 57, 59,

61, 63, 65, 67, 69, 71, 73, 75 or 77, respectively, or a sequence encoding a functional and / or immunogenic fragment or variant of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33,

34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78. Thus, the present invention relates to expression constructs comprising a polynucleotide sequence comprising the nucleotide sequence shown in any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17 , 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 or 77, or a fragment or variant, Including a degenerate variant, of any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,

21, 23, 25, 27, 29, 31.47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 or 77. In a preferred embodiment, a Expression Construction of the Present Invention Provides Overexpression of a Bound Polynucleotide

<img file="MX339202B_D0019.tif" />

IMPI

MEXICAN INSTITUTE

OF THE PRORITOAD

INDUSTRIAL operably of the invention.

The expression constructs of the invention generally include regulatory elements that are functional in the desired host cell, in which the expression construct will be expressed. Thus, one skilled in the art can select regulatory elements for use in, for example, human host cells, mammalian host cells, insect host cells, yeast host cells, bacterial host cells, and plant host cells. In one embodiment, regulatory elements are those that are functional in canine cells. Regulatory elements include promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements. As used herein, the term "expression construct" refers to a combination of nucleic acid sequences that provides for the transcription of an operably linked nucleic acid sequence. As used herein, the term "operably linked" refers to a juxtaposition of the described components, where the components are in a relationship that allows them to function in their desired way. In general, the operably linked components are in contiguous relationship.

An expression construct of the invention may comprise a promoter sequence operably linked to a polynucleotide sequence encoding a polypeptide of the invention.

Promoters can be incorporated into a polynucleotide, using techniques

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standards known in the art. Multiple copies of promoters, or multiple promoters, can be used in an expression construct of the invention. In a preferred embodiment, a promoter can be positioned at almost the same distance from the transcription start site in the expression construct, as it is from the transcription start site in its natural genetic environment. Certain variation in this distance is allowed without substantial decrease in promoter activity. A transcription start site is typically included in the expression construct. Preferably, the promoter associated with an expression construct of the invention provides overexpression of an operably linked polynucleotide of the invention.

Promoters for use with an expression construct of the invention in eukaryotic cells can be of viral or cellular origin. Viral promoters include, but are not limited to, promoters of the cytomegalovirus (CMV) gene, early or late promoters of SV40, or promoters of the Rous sarcoma virus (RSV) gene. Promoters of cellular origin include, but are not limited to, the desmin gene promoter and the actin gene promoter. Promoters suitable for use with an expression construct of the invention in yeast cells include, but are not limited to, 3-phosphoglycerate kinase promoter, glyceraldehyde-3 phosphate dehydrogenase promoter, metallothionein promoter, alcohol dehydrogenase-2 promoter, and hexokinase promoter.

If the expression construct is to be provided or entered in

I jM Ρ I

MEXICAN INSTITUTE

OE THE PROPWUAD

INDUSTRIAL a plant cell, then plant viral promoters such as, for example, a cauliflower mosaic virus (CaMV) 35S promoter (Including the enhanced CaMV 35S promoter (see, for example, US Patent No. 5,106,739, and An, 1997)) or a 19S promoter of CaMV. Other promoters that can be used for expression constructs in plants include, for example, proliferation promoter, Ap3 promoter, heat shock promoters, 1 'or 2' promoter of A-T DNA. tumefaciens, polygalacturonase promoter, petunia chalcone synthase A (CHS-A) promoter, tobacco PR-1a promoter, ubiquitin promoter, actin promoter, alcA gene promoter, p¡n2 promoter (Xu et al. , 1993), maize Wipl promoter, maize trpA gene promoter (US Patent No. 5,625,136), maize CDPK gene promoter and RUBISCO SSU promoter (US Patent No. 5,034,322). Root specific promoters may be used, such as any of the promoter sequences described in US Patent No. 6,455,760 or in US Patent No. 6,696,623, or in published US Patent Application Nos.

20040078841; 20040067506; 20040019934; 20030177536; 20030084486; or

20040123349, with an expression construct of the invention. Constitutive promoters (such as promoters of CaMV, ubiquitin, actin or

NOS), developmentally regulated promoters, and inducible promoters (such as those promoters that can be induced by heat, light, hormones, or chemicals), are also contemplated for use with the polynucleotide expression constructs of the invention . They can

IMPI

MMICANO INSTITUTE

DS LA MOntPAD industrial also use tissue-specific promoters, eg, fruit-specific promoters, such as the tomato E8 promoter (accession number:

AF515784; Good et al. (1994)). Seed-specific promoters may also be used, such as the promoter for a β-phaseolin gene (eg, bean) or the promoter for a glycine gene (eg, soybean), and others.

For expression in prokaryotic systems, an expression construct of the invention may comprise promoters such as, for example, alkaline phosphatase promoter, tryptophan (trp) promoter, promoter

Pl lambda, β-lactamase promoter, lactose promoter, pho A promoter, T3 promoter, T7 promoter or tac promoter (de Boer et al., 1983).

The expression constructs of the invention may optionally contain a transcription termination sequence, a translation termination sequence, a sequence encoding a signal peptide and / or enhancer elements. Transcription termination regions can typically be derived from the 3 'untranslated region of a viral or eukaryotic gene sequence. Transcription termination sequences can be positioned towards the 3 'end of a coding sequence, to provide efficient termination. A signal peptide sequence is a short amino acid sequence typically present at the amino terminus of a protein that is responsible for the relocation of a mature polypeptide operably linked to a wide range of post-translational cell destinations,

IMPIí

MIXICAN INSTITUTE 1

SAY THE IRONOAD I

INDUSTRIAL ranging from a specific compartment of organelles to sites of protein action and the extracellular environment. The targeting of gene products for a desired cellular and / or extracellular destination through the use of an operably linked signal peptide sequence is contemplated for use with the polypeptides of the invention. Classical enhancers are elements that act in the cis position that increase gene transcription, and can also be included in the expression construct. Classical enhancer elements are known in the art and include, but are not limited to, the CaMV 35S enhancer element, the cytomegalovirus (CMV) early promoter enhancer element and the SV40 enhancer element. Intron-mediated enhancer elements that enhance gene expression are also known in the art. These elements must be present within the transcribed region, and depend on orientation.

DNA sequences that direct the polyadenylation of the transcribed messenger RNA of the expression construct can also be included in the expression construct and include, but are not limited to, an octopine synthase or nopaline synthase signal.

Expression constructs may also include one or more dominant, cleavable marker genes including, for example, genes encoding for antibiotic resistance and / or herbicide resistance for selection of transformed cells. Antibiotic resistance genes can be provided for resistance to one or more of the following

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antibiotics: hygromycin, kanamycin, bleomycin, G418, streptomycin, paromomycin, neomycin, and spectinomycin. Kanamycin resistance can be provided by the enzyme neomycin phosphotransferase (NPT II). Herbicide resistance genes can be provided for resistance to phosphinothricin acetyltransferase or glyphosate. Other markers used for identification of cell transformation include, but are not limited to, genes encoding β-glucuronidase (GUS), β-galactosidase, luciferase, nopaline synthase, chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP ) or intensified GFP (Yang et al., 1996).

The present invention also relates to polynucleotide vectors comprising a polynucleotide sequence of the invention that encodes a polypeptide of the invention. Unique restriction enzyme sites may be included at the 5 'and 3' ends of an expression or polynucleotide construct of the invention, allowing insertion into a polynucleotide vector. As used herein, the term "vector" refers to any genetic element including, for example, plasmids, cosmids, chromosomes, phages, viruses, and the like, that is capable of replication when associated with suitable control elements, and that it can transfer polynucleotide sequences between cells. Vectors contain a nucleotide sequence that allows the vector to replicate in a selected host cell. Many vectors are available for expression and / or cloning and include, but are not limited to, pBR322, pUC series, M13 series, pGEM series, and pBLUESCRIPT vectors (Stratagene, La

ΙΜΡΙ

<img file="MX339202B_D0022.tif" />

Jolla, CA, and Promega, Madison, Wl).

The present invention also relates to oligonucleotide probes and oligonucleotide primers, such as polymerase chain reaction (PCR) primers, which can hybridize to a coding or non-coding sequence of a polynucleotide of the present invention. Oligonucleotide probes of the invention can be used in methods for detecting nucleic acid sequences of influenza virus. Initiating oligonucleotides of the invention can be used in PCR methods and other methods involving nucleic acid amplification. In a preferred embodiment, a probe or primer of the invention can hybridize to a polynucleotide of the invention under severe conditions. Probes and primers of the invention may optionally comprise a detectable label or reporter molecule, such as fluorescent molecules, enzymes, radioactive moiety, and the like. The probes and primers of the invention can be of any length suitable for the method or test in which they are being used. Typically, the probes and primers of the invention will be 10 to 500 or more nucleotides in length. Probes and primers that are 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 81 to 90, 91 to 100, or 101 or more nucleotides in length are contemplate within the scope of the invention. In one embodiment, the probes and initiators are any of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,

21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The probes and primers of the invention may have sequence identity of

ΙΜΡΙ • MCTiTirrr »

<img file="MX339202B_D0023.tif" />

complete nucleotides (100%) with the sequence of nolinunleotides. or the sequence identity may be less than 100%. For example, the sequence identity between a probe or primer and a sequence can be 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, or any another percentage of sequence identity, as long as the probe or the primer can hybridize under severe conditions with a nucleotide sequence of a polynucleotide of the invention. Exemplified probes and primers of the invention include those having the nucleotide sequence shown in any of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID

NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, or a fragment or functional variant of any of SEQ ID NOs: 35-46.

As used herein, the terms "nucleic acid", "polynucleotide" and "oligonucleotide" refer to a deoxyribonucleotide, ribonucleotide, or a mixed polymer of deoxyribonucleotides and ribonucleotides in single or double stranded form, and less than otherwise limited, they would encompass known analogs of natural nucleotides that can function in a similar way as naturally occurring nucleotides. The polynucleotide sequences include the DNA strand sequence that can be transcribed into RNA, and the RNA strand that can be translated into protein. The complementary sequence of any nucleic acid, polynucleotide, or oligonucleotide of the present invention is also contemplated within the scope of the invention.

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MWICANO INSTITUTE

OF THE PROPERTY

INDUSTRIAL

Polynucleotide sequences also include full length sequences, as well as shorter sequences derived from full length sequences. The present invention also encompasses those polynucleotides that are sequentially complementary to the polynucleotides described herein. The polynucleotides and polypeptides of the invention can be provided in purified or isolated form.

Due to the degeneracy of the genetic code, a variety of different polynucleotide sequences can code for a polypeptide of the present invention. A table showing all possible triplet codons (and where U also means T) and the amino acid encoded by each codon, is described in Lewln (1985). Furthermore, it is well within the skill of one skilled in the art to create alternative polynucleotide sequences encoding the same, or essentially the same, polypeptides of the present invention. These degenerate alternative and variant polynucleotide sequences are within the scope of the present invention. As used herein, references to "essentially the same" sequence refer to sequences encoding amino acid substitutions, deletions, additions, or insertions that do not materially alter the functional and / or immunogenic activity of the polypeptide encoded by the polynucleotides of the present invention.

The present invention also relates to variants of the polynucleotides of the present invention that encode polypeptides of the

ΙΜΡΙ

<img file="MX339202B_D0024.tif" />

invention. Variant sequences include those sequences where one or more nucleotides in the sequence have been substituted, deleted and / or inserted. Nucleotides that can be substituted for natural DNA nucleotides have a base portion that can include, but is not limited to, inosine, 5-fluorouracil, 5-bromouracil, hypoxanthine, 1-methylguanine, 5-methylcytosine, and tritylated bases. The sugar portion of the nucleotide in a sequence can also be modified and includes, but is not limited to, arabinose, xylulose, and hexose. Furthermore, the adenine, cytosine, guanine, thymine and uracil bases of the nucleotides can be modified with acetyl, methyl and / or uncle groups. Sequences containing nucleotide substitutions, deletions, and / or insertions can be prepared and tested using standard techniques known in the art.

Substitution of amino acids minus those specifically exemplified or naturally occurring in a polypeptide of the invention is also contemplated within the scope of the present invention. For example, unnatural amino acids can be replaced by amino acids from a polypeptide, as long as the polypeptide having the substituted amino acids retains substantially the same functional activity as the polypeptide in which the amino acids have not been substituted. Examples of unnatural amino acids include, but are not limited to, ornithine, citrulline, hydroxyproline, homoserine, phenylglycine, taurine, iodothyrosine, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid , γ-aminobutyric acid, ε-aminohexanoic acid, 633 acid

<img file="MX339202B_D0025.tif" />

aminohexanoic, 2-aminoisobutyric acid, 3-aminopropionic acid, norleucine, norvaline, sarcosine, homocitrulline, cysteic acid, τ-butylglycine, τbutylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designed amino acids such as β-methylacids, -methylamino acids, N-methylamino acids, and analogues of amino acids in general. Unnatural amino acids also include amino acids that have derivatized side groups. Furthermore, any of the amino acids in the protein can be of the form D (dextrorotatory) or L (levorotatory). Allelic variants of a protein sequence of a polypeptide of the present invention are also contemplated within the scope of the invention.

Amino acids can generally be classified into the following classes: nonpolar, polar uncharged, basic, and acidic. Conservative substitutions whereby a polypeptide of the present invention having an amino acid of one class is replaced with another amino acid of the same class, are within the scope of the present invention, so long as the polypeptide having the substitution still retains substantially the same functional activity than the polypeptide that does not have the substitution. Polynucleotides encoding a polypeptide having one or more amino acid substitutions in the sequence are contemplated within the scope of the present invention. Table 11 below provides a list of examples of amino acids that belong to each class. Single letter amino acid abbreviations are defined in Table 12.

Flu virus polypeptide fragments and variants

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of the present invention can be generated using standard methods known in the art, and can be tested for the presence of function or immunogenicity using standard techniques known in the art. For example, for the testing of fragments and / or variants of a neuraminidase polypeptide of the invention, enzyme activity can be tested. In this way, the person skilled in the art can easily prepare and test fragments and variants of a polypeptide of the invention, and determine if the fragment or variant retains activity with respect to the full length polypeptide or a non-variant polypeptide.

The polynucleotides and polypeptides contemplated within the scope of the present invention may also be defined in terms of more particular identity and / or similarity scales, with those sequences of the invention specifically exemplified herein. The sequence identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and may be greater than 95%. The identity and / or similarity of a sequence can be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,

68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,

89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, compared to a sequence exemplified herein. Unless otherwise specified, as used herein, the percent identity and / or sequence similarity of two sequences can be determined using the algorithm of Karlin and Altschul (1990), modified as in Karlin and Altschul (1993). Saying³⁵ IMPI

MEXICAN INSTITUTE

DS THE PROPERTY

INDUSTRIAL algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al.

(1990). BLAST searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain sequences with the desired sequence identity percent. To obtain interstitial alignments for comparison purposes, BLAST Gapped can be used as described in Altschul et al. (1997). When using the BLAST and BLAST Gapped programs, the default parameters of the respective programs (NBLAST and XBLAST) can be used. See the NCBI / NIH website.

The present invention also contemplates those polynucleotide molecules that have sequences that are sufficiently homologous with the polynucleotide sequences exemplified herein, to allow hybridization to that sequence under severe standard conditions and standard methods (Maniatis et al., 1982). As used herein, "severe" conditions for hybridization refer to conditions where hybridization typically takes place overnight at 20-25 ° C below the melting temperature (Tm) of the DNA hybrid in 6x SSPE, 5x Denhardt's solution, 0.1% SDS and 0.1 mg / ml denatured DNA. The melting temperature, Tm, is described by the following formula (Beltz et al., 1983):

Tm = 81.5 C + 16.6 Log [Na +] + 0.41 (% G + C) -0.61 (% formamide) 600 / duplex length in base pairs

The washes are typically carried out as follows:

(1) Twice at room temperature for 15 minutes in 1x

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BRÍTITUTO MEXICANO »F LA RROFIID / O

INDUSTRIAL

SSPE, SDS at 0.1% (low severity washing).

(2) Once at Tm-20 ° C for 15 minutes in 0.2x SSPE, SDS at

0.1% (moderate severity wash).

The present invention also relates to viral proteins and peptides encoded by the genes of an influenza virus of the present invention. In one embodiment, the viral protein is a mature HA protein. In a specific embodiment, the mature HA protein comprises one or more of the following: a serine at position 82; a leucine at position 221; a threonine at position 327; and / or a threonine at position 482. In an exemplified embodiment, the mature HA protein has an amino acid sequence shown in SEQ ID NO: 33 or SEQ ID NO: 34, or a functional and / or immunogenic fragment or variant of SEQ ID NO: 33 or SEQ ID NO: 34 In another embodiment, the viral protein is an NA protein, NS protein, PB protein, PA protein, or MA protein. The viral proteins and peptides of the invention can be used to generate antibodies that specifically bind to the protein or peptide. The viral proteins and peptides of the present invention can also be used as immunogens and in vaccine compositions.

The present invention also relates to compositions and methods for inducing an immune response against an influenza virus that is capable of infecting a susceptible host animal and causing respiratory disease. The invention can be used to induce an immune response against an influenza virus of any subtype in a susceptible host animal. For example, the influenza virus can be

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a subtype H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16 of HA, and a subtype N1, N2, N3, N4, N5, N6 , N7, N8 or N9 of NA. In one embodiment, the HA subtype is H3 or H5. In another embodiment, the NA subtype is N7 or N8. In a specific embodiment, an immune response is induced against an H3N8 subtype influenza virus. In one embodiment, the host animal is a canid. Canines include wild, zoo, and domestic canines such as wolves, coyotes, and foxes. Canines also include dogs, in particular domestic dogs such as, for example, cross and / or thoroughbred companion dogs, show dogs, working dogs, sheepdogs, hunting dogs, guard dogs, police dogs, racing dogs and / or laboratory dogs. In a specific embodiment, the host animal is a domesticated dog, such as a greyhound. In one embodiment, an effective amount of an immunogenic composition of the present invention is administered to an animal, sufficient to induce an immune response against an influenza virus of the invention. The immune response may be a humoral and / or cellular immune response. In a specific embodiment, the immune response is a protective immune response that is capable of preventing or minimizing viral infection in the immunized host animal for a period subsequent to immunization. Thus, the present invention also relates to vaccine compositions and methods that can provide a vaccinated animal with a protective immune response to a virus herein.

Invention.

₃₈ IMPI

MBKICANO INSTITUTE

OF THE FRUIT?

INDUSTRIAL

As described herein, the composition, vaccine, or immunogens of the present invention may comprise whole cell-free viruses, including attenuated or inactivated viruses, or portions of the virus, including subvirion particles (Including a "divided vaccine" ", Wherein a vortex is treated to remove all viral lipids, or some of them), viral proteins (including individual proteins and multiple protein macromolecular complexes), polypeptides and peptides, as well as cell lines infected with viruses, or a combination of any of these. Vaccine or immunogenic compositions comprising virus-infected cell lines can comprise multiple cell lines, each infected with a different viral strain.

The vaccine or immunogenic compositions of the present invention also encompass recombinant viral vector based constructs which may comprise, for example, genes encoding HA protein, NA protein, nucleoprotein, basic polymerase protein, acidic polymerase protein and / or matrix protein of an influenza virus of the present invention. Any suitable viral vector that can be used

J to prepare a recombinant virus / vector construct is contemplated for use with the present invention. For example, viral vectors derived from adenovirus, avipox, herpesvirus, vaccinia, canarypox, entomopox, swinepox, West Nile virus, and others known in the art, can be used with the compositions and methods of the present invention.

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MUDCANO INSTITUTE

PROPBD / hD

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Recombinant polynucleotide vectors that encode for and express components can be constructed using standard genetic engineering techniques known in the art. Furthermore, the various vaccine compositions described herein can be used separately and in combination with each other. For example, primary immunizations of an animal can use recombinant vector-based constructs having single or multiple strain components, followed by secondary boosts with vaccine compositions comprising inactivated virus or inactivated virus infected cell lines. Other immunization protocols with the vaccine compositions of the invention are apparent to those skilled in the art, and are contemplated within the scope of the present invention.

The present invention also relates to rearranging viruses comprising at least one gene or genomic segment of an influenza virus of the present invention, and the rest of viral genes or genomic segments of a different influenza virus of the invention, or from an influenza virus minus a virus of the present invention. Rearranging viruses can be produced by genetic rearrangement of nucleic acid from a donor influenza virus of the present invention, with nucleic acid from a recipient influenza virus, and then selecting for rearranging viruses that comprise nucleic acid from the donor virus. Methods for producing and isolating rearranging viruses are well known in the art (Fields et al., 1996). In one embodiment, a rearranging virus of the invention

IMPI

MfcXtCANO INSTITUTE

Dí LA nOFRUAD nDUSTMJAL comprises genes or genomic segments of a da virus. Ha ..iofluanga human, avian, porcine or equine. A rearranging virus of the present invention can include any combination of donor and recipient influenza virus nucleic acid, as long as the rearranging virus comprises at least one gene or genomic segment of a donor influenza virus of the present invention. In one embodiment, a recipient influenza virus can be an equine influenza virus.

Natural, recombinant or synthetic polypeptides of viral proteins, and fragments of peptides thereof, can also be used as vaccine compositions according to the present methods. In one embodiment, the viral polypeptides derived from multiple strains can be combined into a vaccine composition, and used to vaccinate a host animal. For example, polypeptides based on the viral HA protein from at least two different strains of influenza virus of the invention can be combined in the vaccine. The polypeptides can be homologous to a strain, or can comprise hybrid or chimeric polypeptides, the amino acid sequence of which is derived from the binding or binding of polypeptides of at least two different strains. Procedures for preparing viral polypeptides are well known in the art. For example, viral polypeptides and peptides can be synthesized using solid phase synthesis methods (Merrifield, 1963). Viral polypeptides and peptides can also be produced using recombinant DNA techniques, where a polynucleotide molecule encoding a protein or

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Viral peptide is expressed in a host cell, such as bacteria, yeast, or mammalian cell lines, and the expressed protein is purified using standard techniques of the art.

The vaccine compositions of the present invention also include bare nucleic acid compositions. In one embodiment, a nucleic acid can comprise a nucleotide sequence encoding an HA protein and / or a NA protein of an influenza virus of the present invention. Methods for nucleic acid vaccination are known in the art and are described, for example, in US Patent Nos. 6,063,385 and 6,472,375. The nucleic acid can be in the form of a plasmid or a gene expression cassette. In one embodiment, the nucleic acid is provided encapsulated in a liposome that is administered to an animal.

Vaccine and immunogen compositions, such as polypeptides and nucleic acids, that can be used in accordance with the present invention, can be provided with a pharmaceutically acceptable carrier or diluent. The compounds and compositions useful in the present invention can be formulated according to known methods for the preparation of pharmaceutically useful compositions. Formulations are described in detail in many sources that are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science by EW Martin, Easton Pennsylvania, Mack Publishing Company, 19th Edition, 1995, describes

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formulations that can be used in connection with the present invention. In general, the compositions of the present invention will be formulated such that an effective amount of an immunogen is combined with a suitable vehicle to facilitate effective administration of the composition. The compositions used in the present methods can also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, suspensions or liquid solutions, suppositories, injectable and infusible solutions, and sprays. The preferred form depends on the desired mode of administration and therapeutic application. The compositions also preferably include conventional pharmaceutically acceptable carriers and diluents that are known to those skilled in the art. Examples of vehicles or diluents for use with the present compositions include, but are not limited to, water, saline, oils including mineral oil, ethanol, dimethyl sulfoxide, gelatin, cyclodextrans, magnesium stearate, dextrose, cellulose, sugars , calcium carbonate, glycerol, alumina, starch, and equivalent vehicles and diluents, or mixtures of any of these. Formulations of an immunogen of the invention may also comprise suspending agents, protectants, lubricants, pH regulators, preservatives, and stabilizers. To provide the administration of said dosages for the desired therapeutic treatment, the pharmaceutical compositions of the invention will advantageously comprise between approximately 0.1% and 45%, and especially, 1 and

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MEXICAN INSTITUTE

OF THE CURRENCY D

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15% by weight of the immunogen or immunogens based on the weight of the total composition, including vehicle or diluent.

The vaccine and immunogenic compositions of the present invention can be prepared by procedures well known in the art. For example, the vaccine or immunogens are typically prepared as injectable formulations, eg, suspensions or liquid solutions. The vaccine or immunogens are administered in a form that is compatible with the dosage formulation and in such an amount that they are therapeutically effective and immunogenic at the recipient. Optimal dosages and administration patterns for a particular immunogen or vaccine formulation can be readily determined by those skilled in the art.

The peptides and / or polypeptides of the present invention can also be provided in the form of a multiple antigenic peptide (MAP) construct. The preparation of MAP constructs has been described in Tam (1988). MAP constructs use a central matrix of Usin residues in which multiple copies of an immunogen are synthesized (Posnett et al., 1988). Multiple MAP constructs, each containing the same different immunogens or immunogens, can be prepared and administered in a vaccine composition in accordance with the methods of the present invention. In one embodiment, a MAP construct is provided and / or administered with one or more adjuvants. Influenza polypeptides of the invention can also be produced and administered as structures

IMPIí

INSTfTUT MEXICANO '

FROM THE rWOPIOAD

MDUSTR1AL of macromolecular proteins comprising one or more polypeptides. Published US patent application US2005 / 0009008 describes methods for producing virus-like particles as a vaccine for influenza virus.

In accordance with the methods of the present invention, the immunogenic and vaccine compositions described herein are administered to susceptible hosts, typically canines, and more typically domesticated dogs, in an effective amount and form, to induce protective immunity against challenge or subsequent infection of the host by the virus. In one embodiment, the host animal is a canid. Canines include wild, zoo, and domestic canines such as wolves, coyotes, and foxes. Canines also include dogs, in particular domestic dogs such as, for example, cross and / or thoroughbred companion dogs, show dogs, working dogs, sheepdogs, hunting dogs, guard dogs, police dogs, racing dogs and / or laboratory dogs. In a specific embodiment, the host animal is a domesticated dog, such as a greyhound. Vaccines or immunogens are typically administered parenterally, by injection, for example, either subcutaneously, intraperltoneally, or intramuscularly. Other suitable modes of administration include oral or nasal administration.

Usually, vaccines or immunogens are administered to an animal at least twice, with an interval of one or more weeks between each administration. However, other regimes are contemplated for

<img file="MX339202B_D0030.tif" />

They may depend on the judgment of the medical professional and the particular host animal being treated.

Viruses and virus infected cells in a vaccine formulation can be inactivated or attenuated using methods known in the art. For example, whole viruses and infected cells can be inactivated or attenuated by exposure to paraformaldehyde, formalin, phenol, ultraviolet light, elevated temperature, and the like. The amount of whole cell-free virus in a vaccine dose will usually be on the scale of about 0.1 mg to about 5 mg, and more usually being from about 0.2 mg to about 2 mg. The dosage for vaccine formulations comprising virus infected cell lines will usually contain from about 10® to about 10® cells per dose, and more usually from about 5 x 10® to about 7.5 χ 10⁷ cells per dose. The amount of immunogenic protein or peptide in one dose for an animal can range from about 0.1 pg to 10,000 pg, or about 1 pg to 5,000 pg, or about 10 pg to 1,000 pg, or about 25 pg to 750 pg, or about 50 pg to 500 pg, or 100 pg to 250 pg, depending on the size, age, etc., of the animal receiving the dose.

A vaccine or immunogenic composition of the invention, such as viruses or cells infected with viruses or viral proteins or peptides, can be combined with an adjuvant, typically shortly before administration.

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Adjuvants contemplated for use in vaccine formulations include threonyl muramyl dipeptide (MDP) (Byars et al., 1987), saponin, Corynebacterium parvum, Freund's complete and incomplete adjuvants, alumina, or a mixture of any of these. A variety of other adjuvants suitable for use with the methods and vaccines of the present invention, such as alum, are well known in the art, and are contemplated for use with the present invention.

The present invention also relates to antibodies that specifically bind to a protein or peptide of the present invention. The antibodies of the present invention include monoclonal and polyclonal antibody compositions. Preferably, the antibodies of the present invention are monoclonal antibodies. Whole antibodies and antigen-binding fragments thereof are contemplated by the present invention. Thus, for example, suitable antigen-binding fragments include Fab2, Fab and Fv antibody fragments. The antibodies of the invention can be labeled with a detectable moiety, such as a fluorescent molecule (eg, fluorescein or an enzyme).

The present invention also relates to methods and compositions for the detection and identification of an influenza virus of the invention, and for the diagnosis of infection of an animal with an influenza virus of the present invention. The methods of the invention include detecting the presence of canine influenza in a biological sample from an animal. Detection of canine influenza in a sample is useful for

ΙΜΡΙ

MEXICAN INSTITUTE

Dt THE PROPERTY

INDUSTRIAL diagnose canine influenza in an animal. In turn, this information can provide the ability to determine the prognosis of an animal based on the distinction of the levels of canine influenza present over time, and can assist in the selection of agents and therapeutic treatments for the animal, and assist in monitoring therapy. The method also provides the ability to establish the absence of canine influenza in a tested animal.

The ability to detect canine influenza in an animal allows evaluation of outbreaks of canine influenza at different geographic sites. This information also allows for early detection, so that infected animals can be isolated to limit the spread of the disease, and allows early intervention for treatment options. In addition, by having this information available, you can provide guidance to medical personnel in preparing to treat large numbers of sick animals, including assembling medical supplies and, if available, vaccines.

In one embodiment, a method of the present invention involves collecting a biological sample from a test animal, such as a canine. The biological sample can be any biological material, including cells, tissue, hair, whole blood, serum, plasma, nipple aspirate, lung lavage, cerebrospinal fluid, saliva, sweat, and tears.

The animal's test sample may come from an animal suspected of having the canine influenza virus, whether the animal

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ΙΜΡΙ

MKICANO INSTITUTE

Df LA FROFItOAC

INDUSTRIAL does or does not show symptoms of the disease. Control samples from animals known to be free of canine influenza may also be provided or collected. Additional controls may be provided, for example, to reduce false positive and false negative results, and to verify that the reagents in the test are actively detecting the canine influenza virus.

In addition to detecting the presence or absence of canine influenza in a biological sample, the detection methods used in the invention can detect mutations in the canine influenza virus, such as changes in the nucleic acid sequence, that can result from the environment. , drug treatment, genetic manipulations or mutations, injury, change in diet, aging, and any other characteristic of an animal. Mutations can also cause canine influenza to become resistant to a drug that was first effective, or they can allow the virus to infect and spread in a different species of animal, or human. For example, avian influenza A virus has been shown to infect other animals and humans.

In a modality for detecting an influenza virus in an animal, diagnosis is facilitated by collecting high-quality samples, promptly transporting them to a test facility, and adequate storage, prior to conducting laboratory tests. The virus is best detected in samples containing infected cells and secretions. In one embodiment, samples for direct detection of viral antigens

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XSTITVTO MSXICANO »» s LA nOHIUM) Ϊ

INDUSTRIAL 'and / or for nucleic acids and / or virus isolation in cell cultures, are taken during the first 3 days after the onset of clinical symptoms. Many types of specimens are suitable for diagnosing viral upper respiratory tract infections and include, but are not limited to, nasal swab, nasopharyngeal swab, nasopharyngeal aspirate, nasal wash, and throat swabs. In addition to swabs, tissue or serum samples can be taken, and invasive procedures can also be performed.

In one embodiment, respiratory samples are collected and transported in 1 to 5 ml of virus transport medium. Many means that are satisfactory for the recovery of a wide variety of viruses are commercially available. Clinical samples are added to the transport medium. Nasal or nasopharyngeal swabs can also be transported in the virus transport medium. An example of a means of transport is 10 g of beef infusion broth and 2 g of bovine albumin fraction V, added to sterile distilled water up to 400 m. Antibiotics such as 0.8 ml of gentamicin sulfate solution (50 mg / ml) and 3.2 ml of amphotericin B (250 pg / ml) can also be added. The medium is preferably filter sterilized. Nasal lavages, such as sterile saline (0.85% NaCl), can also be used to collect samples of respiratory viruses.

In one embodiment, sera are collected in an amount of 1 to 5 ml of whole blood from an acute phase animal, soon after the onset of clinical symptoms, and preferably no later than 7 days. Can⁵⁰ IMPI sSTrfOTO MIXJCANO

DC LA FRONCOAD

INDUSTRIAL also collect a serum sample in convalescent phase, for example, approximately 14 days after the onset of symptoms. Serum samples can be useful to detect antibodies against respiratory viruses in a neutralization test.

In some cases, samples can be collected from individual animals over a period of time (for example, once a day, once a week, once a month, manually, or annually). Obtaining numerous samples from an individual animal, over a period, can be used to verify the results of the first detections, and / or to identify the response or resistance to a specific treatment, for example, a selected therapeutic drug.

The methods of the present invention can be used to detect the presence of one or more pathological agents in a test sample of an animal, and the level of each pathological agent. Any method can be used to detect the pathological agent including, but not limited to, antibody tests, including enzyme-linked immunosorbent tests (ELISAs), indirect fluorescent antibody (IFA) tests, hemagglutination, and inhibition of hemagglutination tests (Hl ), and Western Blot. Known methods can also be used for cell culture. Positive cultures can also be identified using the immunofluorescence of cell cultures or the Hl test of the cell culture medium (supernatant).

In addition, methods for detecting nucleic acids can be used.

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(DNA or RNA) or proteins. Such methods include, but are not limited to, polymerase chain reaction (PCR) and Reverse transcriptase (RT) -PCR tests and real-time tests, and quantitative nuclease protection tests. Commercially available test kits are available to perform these tests. For example, QIAGEN (Valencia, CA) sells a one-step RT-PCR kit, and a viral RNA extraction kit.

In one embodiment, the method uses an antibody specific for a virus or viral protein of the invention. In a specific embodiment, an antibody specific for an HA protein of a virus of the invention is used. In another embodiment, an antibody specific for an NP protein of a virus of the invention is used. A suitable sample, such as from the nasal or nasopharyngeal region, is obtained from an animal, and a virus or viral protein is isolated therefrom. Viral components are then identified by binding of a specific antibody to a protein, such as HA or NP, of a virus of the invention. In another embodiment, a serum sample (or other antibody-containing sample) is obtained from an animal, and the serum is identified for the presence of antibodies that bind to a protein of a virus of the invention. For example, an ELISA test can be performed, where the plaque walls have HA and / or NP protein, or a fragment of peptides thereof, attached to the wall. The plate wall is then contacted with serum or antibody from a test animal. The presence of antibodies in the animal that specifically bind to the HA protein and / or

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MEXICAN INSTITUTE

DS THE PROP «DAD

1NDUST1IAL

NP, is indicative that the test animal is infecting it or has been infected with an influenza virus of the present invention.

In one embodiment, the presence of a pathological agent is detected by determining the presence or absence of antibodies against the agent, in a biological sample. It may take some time (for example, months) after an animal is infected, before antibodies can be detected in a blood test. Once formed, the antibodies usually persist for many years, even after successful treatment of the disease. The finding of antibodies to canine influenza A may not indicate whether the infection was recent, or ever in the past.

Antibody testing can also be done in fluids. Antibody tests include enzyme-linked immunosorbent tests (ELISAs), indirect fluorescent antibody tests (IFA), and

Western Blot. Preferably, antibody testing is done using multiple tests, eg, ELISA or IFA, followed by Western blotting. Antibody tests can be done in a two-step procedure, using an ELISA or IFA test, followed by a Western blot test. ELISA is considered a more reliable and accurate test than

IFA, but IFA can be used if ELISA is not available. The test of

Western blotting (which is a more specific test) can also be done in all animals, particularly those that have been shown to be positive or borderline I positive (equivocal) in an ELISA or IFA test.

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Other antibody-based tests that can be used to detect influenza virus, include hemagglutination inhibition tests. Hemagglutination activity can be detected in a biological sample from an animal, using chicken or turkey erythrocytes, as described (Burleson et al., 1992, and Kendal et al., 1982). In one embodiment, an influenza protein or peptide or an HA protein or peptide of the invention is contacted with a test sample containing serum or antibody. Erythrocytes (RBCs) from an animal, such as a bird, are then added. If the HA antibody is present, then the RBCs will not clump together. If the antibody to HA is not present, the RBCs will clump together in the presence of HA. Variations and modifications to standard hemagglutination inhibition tests are known in the art, and are contemplated within the scope of the present invention.

Infection of an animal can also be determined by isolating the virus from a sample, such as a nasal or nasopharyngeal swab. Virus isolation can be performed using standard methods, including cell culture and egg inoculation.

In another embodiment, a nucleic acid based test can be used for detection of a virus of the present invention. In one embodiment, a nucleic acid sample is obtained from an animal, and the nucleic acid is subjected to PCR using primers that generate an amplification product, if the nucleic acid contains a sequence specific for an influenza virus of the present invention. . In a specific modality,⁵⁴ IMPI

MMICANO INSTITUTE

M LA PROTODAD

INDUSTRIAL RT-PCR is used in a test for the present virus. In an exemplified embodiment, real-time RT-PCR is used to test an influenza virus of the invention. PCR, RT-PCR, and real-time PCR methods are known in the art, and have been described in US Patent Nos.

4,683,202; 4,683,195; 4,800,159; 4,965,188; 5,994,056; and 6,814,934; and in

Saiki et al. (1985); Sambrook et al. (1989); Lee et al. (1993); and Livak et al. (nineteen ninety five). In one embodiment, the PCR test uses oligonucleotides specific for a matrix protein (MA) gene and / or influenza HA gene. The amplification product can also be sequenced to determine if the product has a sequence of an influenza virus of the present invention. Other nucleic acid-based tests can be used for the detection and diagnosis of viral infection by a virus of the invention, and such tests are contemplated within the scope of the present invention. In one embodiment, a nucleic acid containing sample is subjected to PCR-based amplification using forward and reverse primers, where the primers are specific for a viral gene or polynucleotide sequence. If the nucleic acid in the sample is RNA, then RT-PCR can be performed. For real-time PCR, a probe detectable with primers is used.

Series of primers specific for the hemagglutinin (HA) gene of many of the influenza viruses in circulation are known and are continually being developed. The genome of the influenza virus is

Single stranded RNA, and a copy of DNA (cDNA) must be obtained using

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a reverse transcriptase polymerase (RT). Amplification of the RNA genome, for example, using RT-PCR, requires a pair of primer oligonucleotides, typically designed based on the known HA sequence of influenza A subtypes and neuraminedase (NM) -1. The primers can be selected so that they specifically amplify RNA from only one virus subtype. DNA molecules generated using subtype-specific primers can be further analyzed by molecular genetic techniques such as sequencing. The test is preferably carried out with a positive control, or products are confirmed by sequencing and comparison with known sequences. The absence of objective PCR products (ie, a "negative" result) may not exclude the presence of the virus. The results can then be made available within a few hours, from clinical swabs or cultures of infected cells. PCR and RT-PCR tests for influenza A virus are described in Fouchier et al., 2000 and Maertzdorf et al., 2004.

The present invention also relates to methods for identifying compounds or drugs that have antiviral activity against a virus of the present invention. In one embodiment, cells infected with a virus of the invention are contacted with a drug or test compound. The amount of virus or viral activity after contact is then determined. Those compounds or drugs that exhibit antiviral activity can be selected for further evaluation.

IMPIAS

MSXICAN INSTITUTE

SAY THE PROMOTION

INDUSTRIAL

The present invention also relates to isolated cells infected with an influenza virus of the present invention. In one embodiment, the cell is a canine cell, such as canine kidney epithelial cells.

The present invention also relates to cells transformed with a polynucleotide of the present invention that encodes a polypeptide of the invention. Preferably, the polynucleotide sequence is provided in an expression construct of the invention. More preferably, the expression construct provides for overexpression in the cell of an operably linked polynucleotide of the invention. In one embodiment, the cell is transformed with a polynucleotide sequence comprising a sequence encoding the amino acid sequence shown in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,

16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,

68, 70, 72, 74, 76 or 78, or a fragment or functional variant thereof. In a specific embodiment, the cell that is transformed with a polynucleotide that codes for the amino acid sequence shown in SEQ ID NOs: 2,

4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52, 54, 56,

58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78, comprises the nucleotide sequence shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17 , 19, 21, 23,

25, 27, 29, 31, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 or 77, respectively, or a sequence encoding a fragment or functional variant of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, ⁵⁷ IMPI

MIKICANO INSTITUTE

DS LA MO »E» AD

INBUSTRIAL

24, 26, 28, 30, 32, 33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78. In this way, The present invention relates to cells transformed with a polynucleotide sequence comprising the nucleotide sequence shown in any of SEQ ID NOs: 1, 3, 5, 7,

9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55, 57, 59, 61, 63,

65, 67, 69, 71, 73, 75 or 77, or a fragment or variant, including a degenerate variant, of any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,

21, 23, 25, 27, 29, 31.47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 or 77.

The transformed cell can be a eukaryotic cell, for example, a plant cell, including protoplasts, or the transformed cell can be a prokaryotic cell, for example, a bacterial cell such as E. coli or phi. subtilis. Animal cells include human cells, mammalian cells, partially canine cells, bird cells, and insect cells.

Plant cells include, but are not limited to, dicot, monocot, and conifer cells.

The present invention also relates to plants, including transgenic plants that express and produce a viral protein or polypeptide of the present invention. Plants, plant tissues, and plant cells transformed with or reproduced to contain the polynucleotide of the invention are contemplated by the present invention. Preferably, the polynucleotide of the invention is overexpressed in the plant, plant tissue, or plant cell. Plants can be used to produce compositions

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ΙΝΪΤΠνΤΟ MfUCANO ¡k la raomvAD

INDUSTRIAL influenza vaccine of the present Invention, and vaccines can be administered through plant consumption (see, eg, US Patent Nos. 5,484,719 and 6,136,320).

The present invention also relates to kits for detecting a virus or for diagnosing an infection with a virus of the present invention. In one embodiment, a kit comprises an antibody of the invention that specifically binds to an influenza virus of the present invention, or an antigenic portion thereof. In another embodiment, a kit comprises one or more polypeptides or peptides of the present invention. In a specific embodiment, the polypeptides have an amino acid sequence shown in any of SEQ ID NOs. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or 78, or a functional and / or immunogenic fragment or variant thereof. In another embodiment, a kit comprises one or more polynucleotides or oligonucleotides of the present invention. In a specific embodiment, the polynucleotides have a nucleotide sequence shown in any of SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 or 77, or a fragment or variant thereof. A kit may optionally comprise one or more of control antibody, control peptide or polypeptide and / or control polynucleotide or oligonucleotide. The kit antibody, polypeptides, peptides, polynucleotides and / or oligonucleotides can be provided in a suitable container or packaging.

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MEXICAN INSTITUTE '

0C THE PltOnBPAD

INWISTMIAL

The present application also relates to the use of crossbred dogs as a model for influenza virus infection and pathogenesis. In one embodiment, a crossbreed dog is inoculated with an influenza virus, such as the Canine Influenza virus of the present invention.

Optionally, therapeutic agents subsequent to inoculation may be administered to the dog. The dog may also be given a composition to generate an immune response against an influenza virus prior to inoculation with the virus. Tissue, blood, serum, and other biological samples can be obtained before and / or after inoculation, and can be examined for the presence of virus and tissue pathogenesis using methods known in the art including, but not limited to, PCR, RT-PCR, nucleic acid sequencing and immunohistochemistry.

Any element of any embodiment described herein may be combined with any other element or embodiment described herein, and such combinations are specifically contemplated within the scope of the present invention.

All patents, patent applications, provisional applications, and publications, referred to or cited herein, are incorporated herein in their entirety by reference, including all figures and tables, to the extent that they are not inconsistent with the explicit teachings of this specification.

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Materials and methods for examples 1 to 6

Collection of nasal swabs and greyhound blood

Convalescent and acute blood samples were collected by jugular venipuncture, from normal or clinically ill greyhounds in race dog kennels that experienced outbreaks of respiratory disease. Convalescent samples were collected 4 to 12 weeks after the acute sample. Serum was harvested, and stored at -80 ° C. Nasal swabs were collected and placed in Amies transport medium, pending presentation to charcoal (Becton Dickinson Biosciences), for the isolation of bacteria.

Greyhound post mortem examination

Full postmortem exams were performed by the Anatomic Pathology Service at the University of Florida College of Veterinary Medicine (UF CVM) on 5 of the 8 greyhounds that died in the January 2004 outbreak on a Florida track. Post mortem examination of another dog was performed in a private veterinary clinic through tissue submission to the UF CMV for histopathological diagnosis. Tissues were fixed in 10% neutral pH-regulated formalin, embedded in paraffin, and 5 pm sections were stained with hematoxylin and eosin for histopathological diagnosis, or were processed for immunohistochemistry as described below. Unbound tissues were referred for bacterial culture, and stored

IMPI 'NSTITUT MMICANO DI LA rWDNKMD INDUSTRIAL also at -80 ° C.

Serological tests for canine viral respiratory pathogens

Paired convalescent and acute serum samples were referred to the Animal Health Diagnostic Laboratory (AHDL) at Cornell University College of Veterinary Medicine for serum neutralization tests against distemper virus, adenovirus type 2 and canine parainfluenza virus. Antibody titers were expressed as the last serum dilution that inhibited viral infection of the cell cultures. Seroconversion, defined as a&gt; 4-fold increase in antibody titer between convalescent and acute sample, indicated viral infection. Seroconversions to these viral pathogens were not detected.

Microbial tests for canine bacterial respiratory pathogens

Post mortem tissues and paired nasal swabs were referred to the Diagnostic Clinical Microbiology / Parasitology / Serology Service at the UF CVM for the isolation and identification of bacteria. Samples were grown in non-selective media, as well as selective media for Bordetella species (Regan-Lowe; Remel) and Mycoplasma species (Remel). All cultures were maintained for 21 days before any growth was reported. The nasal swabs of some of the greyhounds⁶² ΙΜΡΙ

MWICANO INSTITUTE

M The industrial ra * eiKMo were also referred to the Department of Diagnostic Medicine / Pathobiology at the Kansas State University College of Veterinary Medicine for bacterial culture. Of 70 clinically ill dogs tested, Bordetella bronchiseptica was isolated from the nasal cavity of 1 dog, while Mycoplasma spp was recovered. from the nasal cavity of 33 dogs. Pasteurella multocida was commonly recovered from the nasal cavity of dogs with purulent nasal secretions. Two of the dogs that died in the January 2004 outbreak had poor growth of Escherichia coli in the lungs post mortem, one dog had poor growth of E. coli and

Streptococcus canis, and another had poor growth of Pseudomonas aeruginosa and one yeast. Neither Bordetella bronchiseptica nor Mycoplasma were isolated from the trachea or lungs of the dogs that died.

Isolation of viruses from post mortem tissues

Frozen tissues were thawed, and homogenized in 10 volumes of minimal essential medium (MEM) supplemented with 0.5% bovine serum albumin (BSA) and antibiotics. Solid debris was removed by centrifugation, and supernatants were inoculated into cultured cells or 10-day embryonated chicken eggs. Tissue homogenates from dying greyhounds were inoculated into various cell cultures that supported the replication of a wide range of viral pathogens. Cell cultures included Vero cells (African green monkey kidney epithelial cells, ATCC No. CCL-81), A-72 (tumor fibroblasts ”IMPL ·

MKICANO INSTITUTE 1

Dt LA PtOPIBDAD

INDUSTRIAL canine, CRL-1542), HRT-18 (human rectal epithelial cells, CRL11663), MDCK (canine kidney epithelial cells, CCL-34), canine kidney primary epithelial cells (AHDL, Cornell University), cells primary canine lung epithelial cells (AHDL), and primary bovine testicular cells (AHDL). MDCK and HRT cells were grown in MEM supplemented with 2.5 ug / mL trypsin treated with TPCK (Sigma); the remaining cell lines were cultured in MEM supplemented with 10% fetal calf serum and antibiotics. Cells were grown in 25 cm flasks² at 37 ° C in a humidified atmosphere containing CO₂ to 5%. A control culture was inoculated with the supplemented MEM. Cultures were observed daily for morphological changes, and were harvested 5 days post-inoculation. Harvested fluids and cells were clarified by centrifugation, and inoculated into fresh cells as described for the initial inoculation; two simulated passages were made. Hemagglutination activity in clarified supernatants was determined using chicken or turkey erythrocytes as described (Burleson et al., 1992; Kendal et al., 1982). For virus isolation in chicken embryos, 0.1 mL of tissue homogenate was inoculated into the allantoic sac, and incubated for 48 hours at 35 ° C. After two simulated passages, hemagglutination activity in allantoic fluids was determined as described (Burleson et al., 1992; Kendal et al., 1982).

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RT-PCR, nucleotide sequencing and phylogenetic analysis

Total RNA was extracted from the tissue culture supernatant or allantoic fluid using the RNeasy kit (Qiagen, Valencia, CA), according to the manufacturer's instructions. Total RNA (10 ng) was reverse transcribed to cDNA, using a one-step RT-PCR kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. PCR amplification of the coding region of the 8 viral influenza genes in the cDNA was performed as previously described (Klimov et al., 1992a), using primer sets specific for universal genes. The resulting DNA amplicons were used as templates for automated sequencing on an Applied Biosystems 3100 automated DNA sequencer, using cyclic sequencing dye terminator (ABI) chemistry. Nucleotide sequences were analyzed using the GCG® package, version 10.0 (Accelyrs) (Womble, 2000). The Phylogeny Inferen ce® version 3.5 package was used to estimate the phylogenies and to calculate the initiation values ​​from the nucleotide sequences (Felsenstein, 1989). Phylogenetic trees were compared with those generated by the neighbor binding analysis with the Tamura-Nei gamma model implemented in the MEGA® program (Kumar et al., 2004), and confirmed by the PAUP® 4.0 Beta program (Slnauer Associates).

Experimental inoculation of dogs

Four specific pathogen-free hounds of 6 were used

IMPIí

INSTITUTO MRXICANO 'Di LA PHOttlDA »

INDUSTRIAL months [(2 males and 2 females (Liberty Research)]. Physical examination and blood tests at baseline, including complete / differential blood cell count, serum chemistry panel, and urinalysis, determined that animals were The animals were housed together in an upgraded BSL 2 facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Rectal temperatures at baseline were recorded twice daily for 7 days. Dogs were anesthetized by intravenous injection of propofol (Diprivan®, Zeneca Pharmaceuticals, 0.4 mg / kg body weight for effect) by intubation with endotracheal tubes. Each dog was inoculated with a total dose of 10⁶⁶infectious tissue culture mean doses (TCID50) of virus A / canine / Florida / 43/2004 (canine / FL / 04) (H3N8), with half the dose administered in the distal trachea through the endotracheal tube, and the other half delivered into the deep nasal passage through a catheter. Rectal temperature records and physical examinations were made twice daily for 14 days post-inoculation (pi). Blood samples (4 mL) were collected by jugular venipuncture on days 0, 3, 5, 7, 10, and 14 pi Nasal and oropharyngeal samples were collected with polyester swabs (Fisher Scientific) from each dog on days 0 to 5 , 7, 10 and 14 pi The swabs were placed in viral transport medium (Remel) and stored at -80 ° C.

Two dogs (1 male and 1 female) were euthanized by intravenous inoculation of Beuthanasia-D® solution (1 mL / 5 kg of body weight; Schering-Plow Animal Health Corp) on day 5 pi, and the 2 dogs

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1N »USTRÍAI remaining on day 14 for post mortem examination. Tissues under histological analysis were processed as described. Tissues for virus culture were stored at -80 ° C. This study was approved by the University of Florida Institutional Animal Care and Use Commlttee.

Spread of virus from experimentally inoculated dogs

Serial dilutions of lung homogenates and swab extracts, prepared by clarifying the swab transport medium by centrifugation, were performed in MEM supplemented with 0.5% BSA and antibiotics. Plaque tests were performed, as described (Burleson et al., 1992), using MDCK cell monolayers in 6-well tissue culture plates. Monolayers of inoculated cells were spread with supplemented MEM containing 0.8% agarose and 1.5 ug / mL TPCKtripsin. Cells were grown for 72 hours at 37 ° C in a humidified atmosphere containing CO₂ at 5% before fixation and staining with violet crystal. Virus concentration was expressed as plaque forming units (PFU) per gram of tissue or per swab.

Immunohistochemistry

Rehydrated and deparaffinized 5 pm lung tissue sections of greyhounds and hounds were mounted on slides.

Bond-Rite ™ (Richard-Allan Scientific, Kalamazoo, MI), and subsequently treated with proteinase K (DakoCytomation, Carpenteria, CA),

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W IA NtOmpAD MEXICAN INSTITUTE «• ustrjal

<img file="MX339202B_D0039.tif" />

followed by peroxidase blocking reagent (Dako® EnVision ™ peroxidase kit, Dako Corp.). Sections were incubated with 1: 500 dilutions of monoclonal antibodies to canine distemper virus (VMRD, Inc.), canine adenovirus type 2 (VMRD, Inc.), canine paralnfluenza virus (VMRD, Inc.), or H3 from influenza A (Chemicon International, Inc.), for 2 hours at room temperature. Controls included incubation of the same sections with mouse IgG (1 mg / mL, Serotec, Inc.), and incubation of the monoclonal antibodies with sections of normal canine lung. After treatment with the primary antibodies, the sections were incubated with secondary immunoperoxidase and peroxidase substrate reagents (Dako® EnVision ™ peroxidase kit, Dako Corp.), according to the manufacturer's Instructions. Sections were counterstained with hematoxylin, treated with clarifier # 2 and indigo reagent (Richard-Allan Scientific, Kalamazoo, MI), dehydrated, and coverslips were applied with Permount (ProSciTech).

Hemagglutination Inhibition Test (Hl)

Serum samples were incubated with receptor-destroying enzyme (RDE, Denka) (1 part serum: 3 parts RDE) for 16 hours at 37 ° C, before heat inactivation for 60 minutes at 50 ° C. Influenza A / canlno / FL / 04 (H3N8) virus developed in MDCK cells for 36-48 hours at 37 ° C. The virus culture supernatants were harvested, clarified by centrifugation, and stored at -80 ° C. The Hl test was performed

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MMICANO V INSTITUTE

D £ LA FROMOAD

INDUSTRIAL as previously described (Kendal et al., 1982). Briefly, 4 units of hemagglutination virus in 25 µΙ were added to an equal volume of serially diluted serum in microtiter wells, and incubated at room temperature for 30 minutes. An equal volume of turkey erythrocytes was added at 0.5% v / v, and hemagglutination titers were visually estimated after 30 minutes. Titer of extreme HI was defined as the last dilution of serum that completely inhibited hemagglutination. Seroconversion was defined as the&gt; 4-fold increase in HI titer between paired convalescent and acute samples. The seropositivity of an individual sample was defined as an HI antibody titer&gt; 1:32.

Microneutralization test (MN)

Neutralizing serum antibody responses to A / canine / FL / 04 (H3N8) were detected by an MM test as previously described (Rowe et al., 1999), except that the canine sera were treated with RDE as described earlier, before the test. Extreme titer was defined as the highest serum dilution that gave 50% neutralization of 100 virus TCID50. Seroconversion was defined as a&gt; 4-fold increase in MN titre between paired convalescent and acute samples. The seropositivity of an individual sample was defined as a MN titer&gt; 1:80.

Examples given below illustrate procedures for ⁶⁹ IMPI

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D »LA RROP« »AD industrial practice the invention. These examples are not to be considered. limiting. All percentages are by weight, and all proportions of the solvent mixture are by volume, unless otherwise indicated.

EXAMPL01

In January 2004, an outbreak of respiratory illness occurred in 22 racing greyhounds housed in 2 kennels on a Florida track and the local farm that supplied these kennels with dogs. There were approximately

60 dogs in each kennel and 300 dogs on the farm. The outbreak occurred over a 6-day period, after which no new cases were identified. Fourteen of the 22 dogs showed fevers of 39.5 to 41.5 ° C, a mild cough that caused nausea for 10 to 14 days, and final recovery. Of the remaining 8 dogs, 6 apparently healthy dogs died unexpectedly with bleeding from the mouth and nose. Two other dogs were euthanized within 24 hours of bleeding from the nose and mouth due to rapid deterioration. These dogs had fevers of 41 ° C. Four of the 8 deaths occurred in the kennels, and 4 occurred on the farm. Fifty percent of the deaths occurred on day 3 of the outbreak. The 22 dogs ranged in age from 17 months to 4 years, but 73% were 17 to 33 months.

Two clinical syndromes were evident: a milder disease characterized by initial fever and then cough for 10-14 days (14 dogs) with subsequent recovery, or a perennial death associated with ⁷⁰ IMPI

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INDUSTM At bleeding in the respiratory tract (8 dogs for a mortality rate of

36%). Post mortem examinations were performed in 6 of the 8 fatal cases.

All dogs had extensive bleeding in the lungs, mediastinum, and pleural cavity. Histological examination of the respiratory tract revealed that in addition to pulmonary hemorrhage, all dogs had tracheitis, bronchitis, bronchiolitis, and suppurative bronchopneumonia (Figures 3A-3B). The epithelial lining and airway lumens in these tissues were infiltrated by neutrophils and macrophages. Lung homogenates prepared from these dogs were inoculated into a variety of monkey, human, bovine, and canine cell lines for virus culture. The homogenate of a dog's lung caused cytopathic effects in Madin-Darby canine kidney epithelial cells (MDCK) cultured in the presence of trypsin, and the cell culture supernatant agglutinated chicken erythrocytes. Preliminary evidence of a type A influenza virus was provided by a

Commercial ELISA for detection of nucleoprotein of influenza A and B viruses, and by PCR analysis using primers specific for the influenza A virus matrix protein gene. In addition, hemagglutination activity was inhibited by antisera. of reference for the H3 subtype of equine influenza A, but not for specific antisera for the H1-H11 and H13 subtypes (Table 3) of human influenza A. To characterize the molecular properties of the virus, the present inventors determined the nucleotide sequences of the 8 RNA segments of the viral genome. Sequence comparisons with virus genesⁿ IMPI

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Dt THE known industrial PROPKiMD influenza and phylogenetic analyzes indicated that the 8 genes of the canine isolate were more similar to those of contemporary equine influenza A (H3N8) viruses, with which they shared&gt; 96-97% sequence identity (Figure 1A, Table 4). In contrast, representative genes from avian, porcine, and human influenza A isolates had &lt;94% identity with the canine isolate (Table 4). These data identified the isolate from canine A / canine / Florida / 43/04 (canine / FL / 04), as an influenza A H3N8 virus closely related to contemporary lineages of equine influenza viruses. Since all of the genes in the canine isolate originated from equine influenza viruses, the present inventors concluded that the entire genome of an equine influenza virus had been transmitted to the dog.

EXAMPLE 2

To investigate the role of canine virus / FL / 04 in clinical and pathological observations in greyhounds, the present inventors performed immunohistochemical (IHC) staining in lung tissues, using a monoclonal antibody for influenza A3 H3. Viral H3 antigen was consistently detected in the cytoplasm of bronchial and bronchiolar epithelial cells, bronchial gland epithelial cells, and macrophages in lumens and alveolar airway spaces (Figures 2A). These data support a diagnosis of infection ”IMPI

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INDUSTRIAL pulmonary with Influenza virus subtype H3 in multiple dogs.

EXAMPLE 3

To determine the complication of a canine type virus / FL / 04 in the etiology of the outbreak of respiratory disease, the present inventors analyzed convalescent and paired acute sera from 11 sick dogs and 16 asymptomatic contacts, by inhibiting hemagglutination (Hl) and microneutralization (MN). Seroconversion, defined as a&gt; 4-fold increase in antibody titer for canine / FL / 04 from the acute phase to the convalescent phase, occurred in 8 of 11 (73%) sick dogs in both tests (Table 1). Seroconversion occurred in 6 of 16 (38%) asymptomatic contacts in the Hl test, while seroconversion occurred in 8 of 16 (50%) in the MN test (Table 1). The seroconversion data demonstrated infection of the dogs with a canine / FL / 04 virus that temporarily coincided with the onset of respiratory disease in most animals.

Individual serum samples were collected 3 months after the outbreak in 46 additional asymptomatic dogs housed with the sick dogs. Of these, 43 (93%) were HIV positive in both tests. For the total population of 73 dogs tested, 93% were seropositive in both tests, including 82% (9/11) of sick dogs and 95% (59/62) of healthy contacts. High seroprevalence in dogs without a history of 'IMPI

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INDUSTRIAL respiratory disease indicates that the majority of infections with the Canine Influenza virus are subclinical, and suggest efficient spread of the virus among dogs. It is not known if Subclinical Infections contribute to the spread of the virus.

EXAMPLE 4

To better understand the ability of canine virus / FL / 04 to infect dogs, 4 hounds for procreation purposes of 6 months were each inoculated with 10⁶⁶ Infectious Dose Tissue Culture Media (TCID)₅₀), through the intratracheal and intranasal routes. All dogs developed a fever (rectal temperature&gt; 39 ° C) during the first 2 days post-inoculation (pL), but none exhibited respiratory symptoms such as cough or runny nose during a 14-day observation period. Virus spread was examined by quantifying the virus on nasal and oropharyngeal swabs. Only 2 of the 4 dogs spread detectable amounts of virus. One dog spread virus on days 1 and 2 pl (1.0-2.5 log PFU per swab), while the other dog spread virus for 4 consecutive days after inoculation (1.4-4.5 log-ιο PFU per swab). Postmortem examination of 2 dogs on day 5 pl revealed necrotizing and hyperplastic tracheitis, bronchitis, and bronchiolitis similar to that found in spontaneous greyhound disease, but there was no pulmonary hemorrhage or bronchopneumonia. Viral H3 antigen was detected in the cytoplasm of⁷⁴ IMPI

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INDUSTWAL epithelial cells from bronchi, bronchioles, and bronchial glands using IHC (Figure 2B). Infectious virus was recovered from the lung tissue of one of the dogs. Post mortem examination of the remaining 2 dogs on day 14 pi, showed minimal histological changes in the respiratory tissues, without viral H3 antigen by IHC, and without recovery of the virus from the lung homogenates. Seroconversion was detected in these last 2 dogs in MN tests around day 7 pi, with a further 2 to 3-fold increase in antibody titers around day 14. These results established the dogs' susceptibility to infection with canine / FL / 04, as evidenced by the febrile response, presence of viral antigen and infectious virus in the lung parenchyma, typical histopathological findings for influenza, and seroconversion. The inability to reproduce severe disease and death in experimentally inoculated hounds is not surprising, since a large proportion of the naturally infected greyhounds were asymptomatic.

EXAMPLE 5

To investigate whether a canine influenza / FL / 04 virus had circulated among Greyhound populations in Florida prior to the January 2004 outbreak, archival sera from 65 racing greyhounds were tested for the presence of canine antibodies. / FL / 04, using the HI and MN tests. There were no detectable antibodies in 33 dogs sampled from 1996 to

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<img file="MX339202B_D0040.tif" />

1999. Of 32 dogs sampled between 2000 and 2003, 9 were seropositive in both tests -1 in 2000, 2 in 2002 and 6 in 2003 (Table 5). The seropositive dogs were located on Florida tracks implicated in outbreaks of respiratory disease of unknown etiology from 1999 to 2003, suggesting that a canine / FL / 04 virus may have been the causative agent of those outbreaks. To further investigate this possibility, the present inventors examined archival tissues of greyhounds that died of hemorrhagic bronchopneumonia in March 2003. Lung homogenates inoculated into MDCK cells and chicken embryos from a dog gave H3N8 influenza virus, named A / canine / Florida / 242/2003 (canine / FL / 03). Sequence analysis of the complete canine / FL / 03 genome revealed&gt; 99% identity with canine / FL / 04 (Table 4), indicating that canine / FL / 04 viruses had infected greyhounds before 2004.

EXAMPLE 6

From June to August 2004, outbreaks of respiratory illness occurred in thousands of racing greyhounds on 14 tracks in Florida, Texas, Alabama, Arkansas, West Virginia, and Kansas.

Officials on some of these tracks, estimated that at least 80% of their dog population had clinical disease. Most dogs had clinical signs of fever (&gt; 39 ° C) and cough similar to that of dogs in the January 2004 outbreak, but many dogs also had⁷⁶ IMPI

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INDUSTRIAL a mucopurulent nasal discharge. Multiple deaths were reported, but an accurate mortality rate could not be determined.

The present inventors collected paired acute and convalescent sera from 94 dogs located on 4 Florida tracks: 56% of these dogs had&gt; 4-fold increases in canine / FL / 04 antibody titers, and 100% were seropositive (Table 6 ). Convalescent sera from 29 dogs in West Virginia and Kansas also had canine / FL / 04 antibodies. The present inventors isolated the influenza A (H3N8) virus from the lungs of a greyhound that died of hemorrhagic broconeumonia on a track in Texas. Sequence analysis of the entire genome of this isolate, named A / canino / Texas / 1/2004 (canine / TX / 04), revealed&gt; 99% identity with canine / FL / 04 (Table 4). Isolation of three closely related influenza viruses, from fatal cases in canines over a 13-month period and from different geographic sites, in conjunction with substantial serological evidence of widespread infection among racing greyhounds, suggested sustained circulation of a type virus canine / FL / 04 in the dog population.

Phylogenetic analysis of HA genes from canine / FL / 03, canine / FL / 04 and canine / TX / 04, showed that they constitute a monophyletic group with vigorous initiation support that was clearly distinct from the genes of

H3 contemporaries of equine viruses isolated in 2002 and 2003 (Figure 1B).

Phylogenetic analysis and pairwise nucleotide sequence comparisons of the other 7 genomic segments supported the segregation of the

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INDUSTRIAL canine genes as a distinct sublineage more closely related to the equine virus lineage (data not shown and Table 4). Clustering of canine influenza genes as a separate monophyletic group from equine influenza is also supported by the presence of 4 identifying amino acid changes in HA (Table 2). Together with the serological results of 2003 and 2004, these data are consistent with the event of individual virus transmission from horses to dogs with subsequent horizontal spread of the virus in the greyhound population. However, repeated introductions of this unique lineage of influenza virus from an unidentified reservoir species cannot be formally excluded, unlikely as it may be.

Viral HA is a critical determinant of the specificity of the influenza virus by the host species (Suzuki et al., 2000). To identify residues within HA that may be associated with adaptation to the canine host, the present inventors compared the deduced amino acid sequence of canine HAs with that of contemporary equine viruses. Four amino acid changes differentiate the consensus amino acid sequences of mature canine and equine HA: N83S, W222L, I328T, and N483T (see Table 2). Canine viruses have an amino acid deletion when compared to equine consensus sequences. Therefore, amino acid position 7 in the equine HA sequence is position 6 in the canine HA sequence, amino acid position in the equine HA sequence is position 28 in the

IMPI

XSTmTO MWCANO OF THE PRQRKDAC INDUSTRIAL canine HA sequence, position 83 amino acid in the equine HA sequence is position 82 in the canine HA sequence, etc. Thus, the four substituted amino acids are at position 82, 221, 327 and

482 of the amino acid sequence shown in SEQ ID NO: 33 and SEQ ID NO: 34. The substitution of serine for serine at position 83 of the consensus sequence is an unknown functional change in meaning, since several polar residues are found in the H3 molecules from other species. Isoleucine, strictly conserved at position 328 of the consensus sequence near the HA H3 digestion site, has been replaced by threonine. The essential role of HA digestion by host proteases in pathogenesis suggests that this change warrants further study. The replacement of tryptophan with leucine at position 222 of the consensus sequence is quite remarkable, because it represents a non-conservative change adjacent to the sialic acid binding cavity that could modulate receptor function (Weis et al., 1988) . Interestingly, leucine at position 222 is not unique to canine HA H3, as it is typically found in HA subtypes H4, H8, H9, and H12 (Nobusawa et al., 1991; Kovacova et al. , 2002). Leucine substitution may be more compatible with the specificity of the virus by host mammals, since infections have been reported in pigs with the H4 subtype (Karasin et al., 2000) and in humans and pigs with the H9 subtype (Peiris et al., 1999). Replacement of asparagine with threonine in position

483 of the consensus sequence, resulted in the loss of a

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<img file="MX339202B_D0041.tif" />

glycosylation in the HA2 subunit that is conserved in all HA subtypes (Wagner et al., 2002). Although the importance of these amino acid changes in HA for the adaptation of an equine virus to dogs remains to be determined, similar amino acid changes have previously been observed in association with inter-species transfer (Vines et al., 1998; Matrosovich et al., 2000). The amino acid differences between other influenza viral proteins of the invention and the equine consensus sequence are shown in Tables 19 to 25.

The source of the equine influenza virus that initially infected racing greyhounds continues to be speculative. Kennels on greyhound racing tracks are not located near horses or horse racing tracks, suggesting that contact between greyhounds and stray horses is not a sufficient explanation for multiple outbreaks in different states in 2004. A source of potential exposure to the equine virus is the provision of horse meat to greyhounds, whose diet is supplemented by raw meat provided by carcasses that produce carcasses, including horses that could carry influenza. The precedents for this mode of infection include reports of the transmission, between species, of the H5N1 avian influenza virus to pigs and zoo felidae fed on infected chicken carcasses (Webster, 1998; Keawcharoen et al., 2004; Kuiken et al., 2004). Although this is a plausible route for the initial introduction of equine influenza in dogs, it does not explain recent multiple influenza outbreaks in thousands of dogs in different states. The inoculation study⁸⁰ IMPIí iNjTrruTo amkicano f ^D«The ntcnvMc r

INDUSTRIAL 'experimental of the present inventors, showed the prese-eia-de_xdü4S-fiD the nasal passages and oropharynx of dogs, although to modest titles. However, these results indicate that virus spread is possible, and that transmission of the virus from dog to dog via large droplet aerosols, fomites, or direct contact with mucosa could play a role in the epizootology of the disease.

Inter-species transfer of an entire mammalian influenza virus to an unrelated mammalian species is a rare event. Previous studies have provided limited, but not both, serological or virological evidence of transient infection of dogs with human influenza A (H3N2) virus (Nikitin et al., 1972, Kilbourne, et al., 1975; Chang et al., 1976; Houser et al., 1980). However, there was no evidence of sustained circulation in the canine host. Although the direct transfer of swine influenza virus from pigs to humans is well documented (Dacso et al., 1984; Kimura et al., 1998; Patriarca et al., 1984; Top et al., 1977), no There is evidence of adaptation of pig viruses in human hosts. In this report, the present inventors provide virological, serological, and molecular evidence for the transmission, between species, of an entire equine influenza virus (H3N8) to another mammalian species, the dog. Unique amino acid substitutions in HA of canine viruses, coupled with serological confirmation of infection of dogs in multiple states in the United States, suggest adaptation of the virus to the canine host. Since dogs are a companion animal

IMPI

<img file="MX339202B_D0042.tif" />

in humans, these findings have implications for public health; Dogs can provide a new source of transmission of novel Influenza A viruses to humans.

TABLE 1

Antibody response to A / canine / Florida / 43/04 ÍH3N8)

<td></td><td colspan="2">Sick dogs (11)^to</td><td colspan="2">Healthy Contacts (16)^b</td>

<td>Answer</td><td>Hl^c</td><td>YN^d</td><td>HI</td><td>YN</td>

<td>Seroconversion (%)^and</td><td> 73</td><td> 73</td><td> 38</td><td> 50</td>

<td>Seropositive (%)^T</td><td> 82</td><td> 82</td><td> 100</td><td> 100</td>

<td>Geometric mean title⁹</td><td> 329</td><td> 424</td><td> 268</td><td> 431</td>

^to Number of dogs with clinical signs of disease.

^b Number of asymptomatic dogs housed in contact with clinically ill dogs.

^c Hemagglutinclone (HI) inhibition test using virus

A / canine / Florida / 43/04.

^d Mlcroneutrallzaclón (MN) test using virus A / canine / Florida / 43/04.

^and Percentage of dogs with at least a 4-fold Increase in antibody titre in paired convalescent and acute sera.

^F Percentage of dogs with positive antibody titer (HI titer&gt; 32: MN titer&gt; 80) in convalescent sera.

⁹ Antibody titer of geometric mean for convalescent sera.

⁸² IMPI

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OF THE MOTOT

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TABLE 2 __

Amino Acid Differences Between Canine and Equine H3 Hemagglutinins

<td>Equine H3 Consensus</td><td>Canine / FL / 03</td><td>Canine / FL / 04</td><td>Canine / TX / 04</td><td>Potential functional meaning</td>

<td>G7 *</td><td>D</td><td></td><td> -</td><td>D is also found in HA H3 of duck and human</td>

<td> 129</td><td> -</td><td>M</td><td>M</td><td>I is conserved in HAs H3 of all species</td>

<td>N83</td><td>S</td><td>S</td><td>S</td><td>Various polar amino acids present in this position in the HA3 Hs of other species</td>

<td>S92</td><td> -</td><td>N</td><td> -</td><td>N is present in some duck H3 HAs</td>

<td>L118</td><td> -</td><td> -</td><td>V</td><td>L is preserved in all HAs H3</td>

<td>W222</td><td>L</td><td>L</td><td>L</td><td>W is conserved in the majority of HAs H3 of all species; located near the receptor binding site</td>

<td>A272</td><td>V</td><td>TO</td><td>V</td><td>V is present in some recent isolates of equine</td>

<td>I328</td><td>T</td><td>T</td><td>T</td><td>You are strictly preserved in all H3 Hs of birds, pigs or humans</td>

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TABLE 2 (CONTINUED)

<td>N483</td><td>T</td><td>T</td><td>T</td><td>N occurs in all H3 subtypes and other HA subtypes. Replacement results in loss of a glycosylation site</td>

<td>K541</td><td> -</td><td>R</td><td> -</td><td>Conservative change of basic amino acid</td>

* Amino acid residue (single letter code) and position in HA

H3 matures. The amino acid code is: A = alanine, D = aspartic acid, G = glycine, l = isoleucine, K = lysine, L = leucine, M = methionine, N = asparagine,

R = arginine, S = serine, T = threonine, V = valine, W = tryptophan.

^F Denotes no change from the equine consensus HA3 Hs.

TABLE 3

Inhibition of hemagglutination of a virus isolate by reference antisera for different HA subtypes

<td>Reference antisera</td><td>Specificity of HA</td><td>Title of the Hl^to</td>

<td>Puerto Rico / 8/34</td><td>H1</td><td> 5</td>

<td>Pig / lowal 5/30</td><td>H1</td><td> 5</td>

<td>Singapore / 01/57</td><td>H2</td><td> 5</td>

<td>Shanghai / 11/87</td><td>H3^b</td><td> 5</td>

<td>Equine / Miami / 1/63</td><td>H3</td><td> 160</td>

<td>Duck / Czechoslovakia / 56</td><td>H4</td><td> 5</td>

<td>Tern / South Africa / 61</td><td>H5</td><td> 5</td>

<td>Turkey / Massachusetts / 65</td><td>H6</td><td> 5</td>

<td>Poultry Plague / Netherlands / 27</td><td>H7</td><td> 5</td>

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TABLE 3 (CONTINUED) _

<td>Poultry Plague / Rostock / 34</td><td>H7</td><td> 5</td>

<td>Equine / Prague / 1/56</td><td>H7</td><td> 5</td>

<td>Turkey / O ntario / 6118/68</td><td>H8</td><td> 5</td>

<td>Quail / Hong Kong / G1 / 97</td><td>H9^b</td><td> 5</td>

<td>Chicken / Hong Kong / G9 / 97</td><td>H9^b</td><td> 5</td>

<td>Chicken / Germany / 49</td><td>H10</td><td> 5</td>

<td>Duck / england / 56</td><td>H11</td><td> 5</td>

<td>Seagull / Maryland / 704/77</td><td>H13</td><td> 5</td>

<td>Normal sheep serum</td><td> -</td><td> 5</td>

<td>Ferret Normal Serum</td><td> -</td><td> 5</td>

^to Hemagglutination inhibition titer for dog virus isolate # 43.

^b The polyclonal antisera were produced in ferrets, while the other antisera were produced in sheep or goats.

IMPI

<img file="MX339202B_D0043.tif" />

A / canine / Fiorida / 43/04 (H3N8) gene sequence homology for strains of avian, equine influenza A.

<img file="MX339202B_D0044.tif" />

<img file="MX339202B_D0045.tif" />

TABLE 4 (CONTINUED)

<td>οσ&gt; c ™ £ co ϊο S £ £ r- cn t. 5? mm '-'oo) oo m lo h- t- C CO CM CM⁰⁰ ζ ϊ. And it is,</td><td>JO ro c</td><td>91.2 (95.4) WSN / 33 (Hong Kong/ 1073/99) J02177 (AJ278646)</td><td>91.4 (90.0) Brevig M¡ss¡on / 1/18 AF333238</td>

<td>85.4 (93.5) Pig / Ontario / 42729a / 01 (Pig / Fujian / 1/2003) AY619974 (AY747611)</td><td>XI ro c</td><td>93.7 (93.5) Pig / Saskatchewan / 18789/02 M63527</td><td>91.1 (89.1) Pig / Chlna / 8/78 (Pig / Korea / s452 / 04) M80968 (AY790309)</td>

<td>87.9 (95.1) Chicken / Chile / 176822/0 2 AY303658</td><td>84.0 (85.2) Pato / NJ / 2000 L06583</td><td>94.1 (94.0) Turkey / Mn / 833/80 AF001683</td><td>92.0 (90.4) Duck Sylvestre / NY / 6750/78 M80945</td>

<td>96.6 (97.9) Equino / Tennesee / 5/8 6 M30758</td><td>96.8 (97.0) Equino / Tennesee / 5/8 6 L06583</td><td>97.9 (95.7) Equino / Tennesee / 5/8 6 (Equine / Kentucky / 92) M63529 (AF001683)</td><td>97.5 (95.7) Equine / Tn / 5/86 (Equine / Kentucky / 92) M80973 (AF001671)</td>

<td>NP DQ124150</td><td>NA (N8) DQ124151</td><td>M DQ124152</td><td>NS DQ124153</td>

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TABLE 5

Antibody titers for A / canine / Florida / 43/04 ÍH3N8) in greyhound serum collected from 1996 to 2003

<td rowspan="2"></td><td colspan="6">Year³</td>

<td> 1996</td><td> 1997</td><td> 1998</td><td> 2000</td><td> 2002</td><td> 2003</td>

<td>Number of dogs tested</td><td> 8</td><td> 6</td><td> 19</td><td> 4</td><td> 6</td><td> 22</td>

<td>Number of HIV positive dogs</td><td> 0</td><td> 0</td><td> 0</td><td> 1</td><td> 2</td><td> 6</td>

<td>Antibody Titers¹*</td><td></td><td></td><td></td><td> 512</td><td> 232, 524</td><td> 280- 2242</td>

³ Year of the collection of serum samples from racing greyhounds in

Florida.

^b Antibody titers of the microneutraisation test for seropositive dogs, including the scale for the 6 seropositive dogs of

2003.

PICTURE

Antibody response to A / canine / Florida / 43/04 (H3N8) in racing greyhounds on 4 Florida tracks in June 2004

<td>Answer</td><td>Track A</td><td>Track B</td><td>Track C</td><td>Track D</td>

<td>Number of dogs tested³</td><td> 37</td><td> 10</td><td> 22</td><td> 25</td>

<td>Seroconversion (%)^b</td><td> 46</td><td> 90</td><td> 100</td><td> 64</td>

<td>Seropositive (%)^c</td><td> 100</td><td> 100</td><td> 100</td><td> 100</td>

⁸⁸ IMPI

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TABLE 6 (CONTINUED) _______

<td>Media Title</td><td> 401</td><td> 512</td><td> 290</td><td> 446</td>

<td>geometric</td><td></td><td></td><td></td><td></td>

^to Number of clinically ill dogs tested by

Hl using A / canine / Florida / 43/04 (H3N8).

^b Percentage of dogs with&gt; 4-fold increase in antibody titer between acute and convalescent sera.

^c Percentage of dogs with positive antibody titer (Hl titer&gt; 16) in convalescent sera.

^d Antibody titer of geometric mean for convalescent sera.

Materials and methods for examples 7 to 11

Canine tissues

Post mortem examinations were performed by the Anatomic Pathology Service at the University of Florida College of Veterinary Medicine on 6 mixed breed dogs that died in an April / May 2005 outbreak of influenza at a shelter in North Florida, and in a Pet dog

Yorkshire Terrier who died in May 2005 during an influenza outbreak at a veterinary clinic in Southeast Florida. Tissues were fixed in 10% neutral pH regulated formalin, embedded in paraffin, and 5 pm sections were stained with hematoxylin and eosin for diagnosis⁸⁹ IMPI

MEXICAN INSTITUTE κ la prorioad

Histopathological INtXISTRIAL. Unbound tissues were stored at -ftn ° r. for pending virological analyzes.

Extraction of RNA from canine tissue samples

Is of scongelaron lung tissues frozen from each of the 7 dogs, and homogenized in minimum essential medium (MEM) supplemented with serum albumin bovine (BSA) at 0.5% and antibiotics (gentamycin and ciprofloxacin) using a mill disposable tissues (Kendall, Lifeline Medical Inc., Danbury, CT). Total RNA was extracted using a commercial kit (mlniequlpo RNeasy®, QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions, and eluted in a final volume of 60 pL of pH regulator. Total RNA was also extracted from lung tissue collected from dogs without respiratory disease.

RT-PCR in real time

One-step quantitative real-time RT-PCR was performed on total RNA extracted from canine tissue samples using the QuantiTect® probe RT-PCR kit containing ROX as a passive reference dye (QIAGEN Inc., Valencia , CA). In summary, two sets of primer-probe were used for the detection of influenza A sequences in each sample (Table 7). One primer-probe series was selective for canine hemagglutinin (H3) gene sequences. The other primer-probe series targeted a highly conserved region of the gene.

IMPI iwrrrvro mjuécano mu ntOWRMD ifwusnuAi matrix protein (M) of influenza virus type A. For each real-time RT-PCR reaction, 5 µΙ_ of extracted total RNA was added to a reaction mix containing 12.5 µΙ_ of 2X RTi-PCR master mix with QuantiTech® probe, 0.25 µΙ_ RT QuantiTech® mix, forward and reverse initiators (final concentration of 0.4 μΜ for each), probe (final concentration of 0.1 μΜ) and ribonuclease-free water in a final volume of 25 μΙ_. TaqMan® ribosomal RNA control reagents (Applied Biosystems, Foster City, CA), were used according to the manufacturer's instructions for the detection of 18S rRNA as an endogenous internal control for the presence of RNA extracted from tissue samples of canine.

Quantitative one-step real-time RT-PCR was performed on the reaction mixtures in a QPCR Mx3000P® system (Stratagene, La Jolla, CA). Cyclic conditions included a reverse transcription step at 50 ° C for 30 minutes, an initial denaturation step at 95 ° C by

fifteen minutes to activate Thermus aquaticus HotStar® DNA polymerase, and amplification for 40 cycles. Each cycle of amplification included denaturation at 94 ° C for 15 seconds, followed by binding / extension at 60 ° C for 1 minute. Fluorescent FAM (518 nm emission wavelength) and VIC (554 nm emission wavelength) signals were recorded at the end of each cycle. The threshold cycle (Ct) was determined by adjusting the threshold fluorescence (dR) to 1000 in each individual experiment. The software program was used

Mx3000P® version 2.0 (Stratagene, La Jolla, CA) for data acquisition and analysis. The samples were considered positive for the⁹¹ IMPI

INSTITUTO MSXICANO n LA FROFItOAD

INDUSTRIAL influenza virus when the threshold cycle (Ct) for the H3 or M gene was 3 units smaller than the Ct for lung tissues of dogs without respiratory disease. The positive control consisted of amplification of RNA extracted from A / canine / FL / 242/03 virus (H3N8).

Virus isolation in MDCK cells

Frozen lung tissues from each of the 7 dogs were thawed and homogenized in 10 volumes of Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 0.5% bovine serum albumin (BSA) and antibiotics (gentamicin and ciprofloxacin). . Solid debris was removed by centrifugation, and supernatants were inoculated into DME-grown Madin-Darby canine kidney cells (MDCK) supplemented with 1 gg / mL trypsin treated with TPCK (SigmaAldrich Corp., St. Louis, MO) and antibiotics (gentamicin and ciprofloxacin). Cells were grown in 25 cm flasks² at 37 ° C in a humidified atmosphere containing CO₂ to 5%. Cultures were observed daily for morphological changes, and were harvested 5 days post-inoculation. The harvested cultures were clarified by centrifugation, and the supernatants were inoculated into fresh MDCK cells as described for the initial inoculation; Two additional passages were performed for samples that showed no evidence of influenza virus by hemagglutination or RT-PCR. Hemagglutination activity in clarified supernatants was determined using 0.5% turkey erythrocytes ⁹² ΙΜΡΙ

MEXICAN INSTITUTE

W THE FROMEIAD

INDUSTRIAL as previously described (Burleson, F. et al., 1992; Kendal, P. et al., 1982). RT-PCR was performed as described below.

Virus isolation in embryonated chicken eggs

Frozen lung tissue homogenates were prepared as described above for inoculation of MDCK cells. Homogenates (0.2 mL) were inoculated into the allantoic sac of 10-day-old embryonated chicken eggs. After 48 hours of inoculation at 35 ° C, the eggs were cooled to 4 ° C overnight before harvesting the allantoic fluid. Hemagglutination activity in clarified supernatants was determined using 0.5% turkey erythrocytes as previously described (Burleson, F. et al., 1992; Kendal, P. et al., 1982). RTPCR was performed as described below. Two additional passages were made in embryonated eggs for samples that showed no evidence of influenza virus after initial inoculation.

RT-PCR, nucleotide sequencing and phylogenetic analysis

Viral RNA was extracted from the MDCK cell supernatant or allantoic fluid using the QlAamp® viral RNA mini-kit (QIAGEN Inc.,

Valencia, CA), according to the manufacturer's instructions. Viral RNA was reverse transcribed to cDNA using the QIAGEN® One-Step RT-PCR Kit (QIAGEN Inc., Valencia, CA), according to the manufacturer's instructions. PCR amplification of the coding region of the

IMPI

MEXICAN INSTITUTE OF LA roOPIIDAD

INDUSTRIAL influenza viral genes in cDNA as previously described (Klimov, A. et al., 1992b), using universal gene specific primer sets (primer sequences available upon request). The resulting DNA amplicons were used as templates for automated sequencing in the ABI PRISM® 3100 automated DNA sequencer using cyclic sequencing dye terminator chemistry (Applied Biosystems, Foster City, CA). Nucleotide sequences were analyzed using the Lasergene 6® package (DNASTAR, Inc., Madison, Wl). The PHYLIP version 3.5® software program was used to estimate the phylogenies and calculate the initiation values ​​of the nucleotide sequences (Felsenstein, J., 1989). The phylogenetic trees were compared with those generated by the neighbor junction analysis with the TamuraNei gamma model implemented in the MEGA® program (Kumar, S. et al., 2004) and confirmed by the PAUP® 4.0 Beta program (Sinauer Associates, Inc., Sunderland, MA).

Hemaqlutlination inhibition test (Hl)

Serum samples were incubated with receptor-destroying enzyme (RDE, DENKA SEIKEN Co., Ltd., Tokyo, Japan) (1 part serum: 20 3 parts RDE) for 16 hours at 37 ° C before heat inactivation by 30 minutes at 56 ° C. Influenza A / canine / Jacksonville / 05 (H3N8) virus developed in MDCK cells for 72 hours at 37 ° C in CO₂ to 5%. The virus culture supernatants were harvested, clarified by

IMPI

MEXICAN INSTITUTE Dt LA INDUSTRIAL property

<img file="MX339202B_D0047.tif" />

centrifugation and stored at -80 ° C. All other viruses used in the Hl test developed in 10-day embryonated chicken eggs from which the allantoic fluid was collected and stored at -80 ° C. The Hl test was performed as previously described (Kendal, P. et ai, 1982). Briefly, 4 units of virus hemagglutination in 25 µΙ were added to an equal volume of serially diluted serum in 96 well plastic plates, and incubated at room temperature for 30 minutes. An equal volume of turkey red blood cells was added at 0.5%, and hemagglutination titers were visually estimated after 30 minutes. Extreme Hl titer was defined as the last serum dilution that completely Inhibited hemagglutination.

EXAMPLE 7

Clinical cases

In April and May 2005, a previously described outbreak of respiratory disease (Crawford, PC et al., 2005) occurred in dogs housed at a shelter in Northeast Florida. The outbreak included at least 58 dogs ranging in age from 3 months to 9 years, and included thoroughbred dogs as well as mixed breeds. The most common clinical signs were purulent runny nose and a cough for 7 to 21 days. Of the 43 dogs that had clinical disease for&gt; 7 days, 41 had Hl antibody titers for canlno / FL / 04 (H3N8) ranging from 32 to&gt; 1024. At least 10 dogs⁹⁵ IMPIY Mexican Institute t

FROM FAOftOM? \

INIMJSTRIAL progressed to pneumonia, of which 6 were euthanized.

These 6 mixed breed dogs included 3 males and 3 females ranging in age from 4 months to 3 years. The duration of clinical signs varied from 2 to days at the time of euthanasia. On post mortem examination, these dogs had pulmonary congestion and edema. Histological examination of the respiratory tract revealed rhinitis, tracheitis, bronchitis, bronchiolitis, and suppurative bronchopneumonia. There was necrosis of epithelial cells and erosion in the trachea, bronchi, bronchioles, and bronchial glands. The respiratory tissues were infiltrated by neutrophils and macrophages.

In May 2005, an outbreak of respiratory illness occurred in 40 pet dogs at a veterinary clinic in Southeast Florida. The most common clinical signs were purulent runny nose and a cough for 10 to 30 days. Of the 40 dogs, 17 were seropositive for canine / FL / 04 (H3N8) with Hl antibody titers ranging from 32 to&gt; 1024. Seroconversion occurred in 10 dogs for which matched convalescent and acute sera were available. Three dogs progressed to pneumonia. One of these dogs, a 9-year-old male Yorkshire Terrier, died 3 days after the onset of clinical signs. This dog had tracheobronchitis, edema and pulmonary congestion, and severe bronchopneumonia. Similar to the 6 dogs in the shelter, there was epithelial cell necrosis and erosion of the airways and infiltrates of neutrophils in the tissues.

⁹⁶ IMPI niJWVTO MWICANO IWBfomiAl

EXAMPLE 8 _

Real-time RT-PCR and virus isolation

Lung tissues from the 7 dogs were analyzed using quantitative real-time RT-PCR tests that detect the M gene of influenza virus type A and the H3 gene of canine influenza A virus H3N8. The lungs of the 7 dogs were positive for the influenza A M gene and the canine influenza H3 gene (Table 8). After 3 passages in MDCK cells, influenza A subtype H3N8 virus was isolated from the lungs of a shelter dog that died after 3 days of pneumonia. The virus was named A / canine / Jacksonville / 05 (H3N8) (canine / Jax / 05). After 2 passages in embryonated chicken eggs, Influenza A subtype H3N8 virus was recovered from the lungs of the pet dog that also died after 3 days of pneumonia. This virus was named A / canlno / M¡ami // 05 (H3N8) (can¡no / M¡am¡ / 05).

EXAMPLE 9

Genetic analysis of H3N8 isolates from canine influenza A

Sequence analyzes of canine / Jax / 05 and canine / Miami / 05 revealed that their hemagglutinin (HA) genes were 98% Identical to isolates of canino / FL / 04, canino / TX / 04 and canlno / lowa / 05 recovered from racing greyhound lungs that died of pneumonia during outbreaks

IMPI

<img file="MX339202B_D0048.tif" />

Influenza on the Runways in 2004 and 2005 (Crawford, PC ^{to the}, Vnnn KY. et al., 2005). Furthermore, the canine / Jax / 05 and canine / Miami / 05 HA genes were 98% identical to contemporary equine influenza viruses isolated after 2000. Phylogenetic comparisons of the HA genes showed that canine / Jax / 05 and canine / Miami / 05 viruses were grouped with canine / FL / 04, canine / TX / 04 and canine / lowa / 05 greyhound isolates and Contemporary equines, forming a distinct group of the longest equine viruses isolated in the early 1990s (Figures 4A and 4B). Furthermore, isolates from canine / Jax / 05, canine / Miami / 05 and canine / lowa / 05 were more closely related to canine / Tx / 04 than to canine / FL / 04 or canine / FL / 03. Isolates from 2005 formed a subset that appears to branch off from the earliest canine viruses of 2003 and 2004 with differences at approximately 10 informative parsimony sites. These differences support the hypothesis that the canine influenza virus is being transmitted horizontally from dog to dog, as opposed to being periodically reintroduced from an external source. The accumulation of mutations from 2003 to 2005 illustrates the ongoing adaptation process that the virus must undergo after it is transmitted to a new host, as is expected to have occurred for canine influenza viruses.

⁹⁸ IMPI

INÍTÍTUTO MiXICANO DS LA ntOWftPO

INDUSTRIAL

EXAMPLE010 _

Amino Acid Analysis of H3N8 Isolates from Canine Influenza

There were conserved amino acid substitutions in the 6 canine isolates that differed from contemporary equine influenza viruses (Table 9). These conserved substitutions were 115M, N83S, W222L, I328T, and N483T. Phylogenetic comparisons of the mature HA protein showed that the canine / Jax / 05, canine / Mami / 05 and canine / lowa / 05 viruses formed a subgroup with the canine / TX / 04 isolate (Figures

4A and 4B). There were 3 amino acid changes (L118V, K261N, and G479E) that differentiated this subgroup from the other canine viruses (Table 9). There were two amino acid changes (F79L and G218E) that differentiated the 2005 isolates from their canine root / TX / 04. In addition, the 2005 isolates from non-greyhound dogs, canine / Jax / 05 and canine / Miami / 05, differed from the canine / lowa / 05 greyhound isolate by an amino acid change, R492K. Finally, canine / Jax / 05 differed from canine / Miami / 05 in a single amino acid, S107P. In all other equine and canine viruses H3N8, S is conserved at position 107, except for A / equino / Jlln / 1/89, which has a T (Guo Y. et al., 1992 ).

IMPI

<img file="MX339202B_D0049.tif" />

EXAMPLE 11

Antigenic analysis of H3N8 isolates from canine influenza

Hemagglutination inhibition (HI) tests were performed using a panel of contemporary and older equine influenza virus antigens and canine influenza viruses, and serum was collected in 2005 from horses and dogs that had been infected with the influenza virus (Table 10). Serum from ferrets immunized against canlno / FL / 04 was also included in the analyzes. Equine serum H1 antibody titers were 8 to 16 times higher when tested with contemporary equine viruses compared to longer isolates, but decreased by at least 4 times when tested with canine virus. Canine serum was nonreactive with the longest equine viruses, but antibody titers were increased 4-fold when tested with contemporary equine isolates and canine isolates. This was also observed for the serum of ferrets immunized against the canine influenza virus. These patterns of seroreactivity demonstrated antigenic similarity between contemporary canine influenza viruses and contemporary equine influenza viruses, and were consistent with phylogenetic analyzes. Antibody titers in equine, canine, and ferret sera for the canine isolate / Miami / 05 were similar to those for the 2003 and 2004 canine isolates. However, the titers were 2 to 4 times lower for the canine isolate / Jax / 05. This

100

IMPI institute mwiCamo ut la ruorkjad INDUSTRIAL suggests that canine / Jax / 05 is antigenically distinct from the other canine isolates, which may be related in part to the individual amino acid change at position 107 in mature HA.

101

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<td></td><td></td><td>A / Equine / KY / 5/02</td><td>A / Equine / MA / 213/03</td><td>A / Equine / OH / 1/03</td><td>A / canine / FL / 242/03</td><td>A / canine / FL / 43/04</td><td>A / canine / TX / 1/04</td><td>A / canino / lowa / 05</td><td>A / canine / Miami / 05</td><td>To / canine / Jacksonville / 05</td>

IMPI

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TABLE 10

Antibody Titres in Equine, Canine and Ferret Serum to Contemporary and Longest Equine Influenza Viru and Canine Influenza Viruses

<td></td><td colspan="3">Serum antibody titers³</td>

<td>Antigens</td><td>Equine</td><td>Canine</td><td>Ferret*³</td>

<td>Equine / Miami / 63</td><td> 40</td><td> &lt;10</td><td> 16</td>

<td>equine / Ky / 86</td><td> 40</td><td> 40</td><td> 32</td>

<td>equine / KY / 92</td><td> 40</td><td> &lt;10</td><td> 32</td>

<td>equine / NY / 99</td><td> 320</td><td> 40</td><td> 128</td>

<td>equine / KY / 05/02</td><td> 320</td><td> 160</td><td> 256</td>

<td>equine / MA / 213/03</td><td> 640</td><td> 160</td><td> 512</td>

<td>equine / OH / 01/03</td><td> 640</td><td> 160</td><td> 512</td>

<td>canlno / FL / 03</td><td> 160</td><td> 160</td><td> 512</td>

<td>canlno / FL / 04</td><td> 160</td><td> 80</td><td> 512</td>

<td>canlno / Tx / 04</td><td> 160</td><td> 160</td><td> 512</td>

<td>canlno / Miami / 05</td><td> 160</td><td> 80</td><td> 256</td>

<td>canine / Jax / 05</td><td> 40</td><td> 40</td><td> 128</td>

^to Antibody titers were determined in a hemagglutinclone inhibition test performed with serial dilutions of equine serum, canine ferret, and viruses listed on the antigen column.

^b Serum of ferrets immunized with canine virus / FL / 04.

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Materials and methods for examples 12 to 15

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Canine influenza virus inoculum

The virus inoculum was prepared by inoculating Madin-Darby canine kidney epithelial cells (MDCK) with an A / canine / FL / 43/04 (H3N8) pool representing passage 3 of the original isolate previously described (Crawford et al. al., 2005). MDCK cells inoculated in Dulbecco's minimal essential medium (DMEM) supplemented with 1 pg / mL trypsin treated with TPCK (Sigma-Aldrich Corp., St. Louis, MO) and antibiotics (gentamicin and ciprofloxacin), were developed in 250 cm flasks² at 37 ° C in a humidified atmosphere containing CO₂ to 5%. Cultures were observed daily for morphological changes, and were harvested 5 days post-inoculation. The harvested cultures were clarified by centrifugation, and the supernatants were stored at -80 ° C, pending inoculation of the dogs. An aliquot of supernatant was used for virus titer determination by the Reed and Muench method. The title was 10⁷ infectious doses of medium tissue culture (TCID50) of A / canine / Florida / 43/04 (canine / FL / 04) per mL.

Experimental inoculation

Eight crossbred dogs were used for 4 month colony procreation (Marshall BioResources, North Rose, NY) (4 males and 4 females) for the experimental inoculation study approved by the

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University of Florida Institutional Animal Care and Use Committee. The body weights of the dogs ranged from 13 to 17 kg. The dogs were healthy based on physical examinations, baseline blood tests, and recording of body temperatures for 2 weeks prior to inoculation. All dogs were free from prior exposure to canine influenza virus based on serological tests performed on paired serum samples collected upon arrival at the facility and 2 weeks thereafter. Dogs were anesthetized by intravenous injection of propofol (Diprivan®, Zeneca Pharmaceuticals, 0.4 mg / kg body weight for effect) by intubation with endotracheal tubes. Each of six dogs (3 males and 4 females) was inoculated with 1O⁷TCID from the canine virus / FL / 04 in 5 mL of sterile saline administered into the distal trachea through a small diameter rubber catheter inserted into the endotracheal tube. Two dogs (1 male and 1 female) were pseudoinoculated with an equal volume of sterile saline. The pseudoinoculated control dogs were housed in a different location than the virus inoculated dogs, and cared for by different personnel. Physical examinations and rectal temperature records were performed twice daily for 6 days post-inoculation (pi).

Rectal and pharyngeal swab collection

To monitor the spread of the virus, oropharyngeal samples were collected twice daily from each dog on days 0 to 6.

pi using polyester swabs (Fisher Scientific International Inc., Pittsburgh,

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PA). The swabs were placed in 1 mL of sterile pH-regulated phosphate (PBS) saline containing 0.5% bovine serum albumin (BSA). Rectal swabs were collected from each dog daily from days 0 to 6. Extracts of the swabs were prepared by clarifying the swab transport medium by centrifugation. An aliquot of the swab extract was immediately tested for influenza virus nucleoprotein using the Directigen ™ commercial immunoassay kit (BD, Franklin Lakes, NJ), according to the manufacturer's instructions. The remaining extract was stored at -80 ° C with other virological tests pending.

Post mortem exams

On day 1 pi, a pseudoinoculated dog and a virus-inoculated dog were euthanized by intravenous inoculation of Beuthanasia-D® solution (1 mL / 5 kg of body weight; Schering-Plow Animal Health Corp). A virus inoculated dog was also euthanized every day from days 2 to 5 pi. On day 6 pi, the pseudoinoculated dog and the remaining virus inoculated dog were euthanized. Full post mortem examinations were performed by one of the investigators (WLC). Tissues were fixed in 10% neutral pH-regulated formalin, embedded in paraffin, and 5 µπι sections were stained with hematoxylin and eosin for histopathological diagnosis, or processed for immunohistochemistry as described below. Unattached lung tissues were referred to the Diagnostic Clinical

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Microbiology / Parasitology / Serology Service at the University of Florida College of Veterinary Medicine for the isolation and identification of bacteria. Samples were grown in non-selective media, as well as selective media for Bordetella species (Regan-Lowe; Remel, Lenexa, KS) and Mycoplasma species (Remel). All cultures were maintained for 21 days before any growth was reported. Non-fixed tissues were also stored at -80 ° C, pending virological analysis.

Immunohistochemistry

Deparaffinized and rehydrated 5 gm lung and tracheal tissue sections were mounted on Bond-Rite ™ slides (Richard-Allan Scientific, Kalamazoo, MI), and subsequently treated with proteinase K (DAKOCytomation Inc., Carpenteria, CA), followed by peroxidase blocking reagent (DAKO® EnVision peroxidase kit, DAKO Corp.,

Carpenteria, CA). Sections were incubated with a 1: 500 dilution of influenza A H3 monoclonal antibody (Chemicon International, Inc., Ternecula, CA) for 2 hours at room temperature. Controls included incubation of the same sections with mouse IgG (1 mg / mL, Serotec, Inc. Raleigh, NC), and incubation of the monoclonal antibody with normal canine lung sections. After treatment with the primary antibody, the sections were incubated with secondary immunoperoxidase and peroxidase substrate reagents (Dako® EnVision ™ peroxidase kit,

Dako Corp.), according to the manufacturer's instructions. The sections are

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INXlSTUtAL were counterstained with hematoxylin, treated with clarifier # 2 and indigo reagent (Richard-Allan Scientific, Kalamazoo, MI), dehydrated, and coverslips were applied with Permount (ProSciTech, Queensland, Australia).

Extraction of RNA from swabs and tissues

Lung and trachea tissues of each dog were thawed and homogenized in minimal essential medium (MEM) supplemented with 0.5% bovine serum albumin (BSA) and antibiotics (gentamicin and ciprofloxacin), using a disposable tissue mill (Kendall , Lifeline

Medical Inc., Danbury, CT). Total RNA was extracted from tissue homogenates, as well as extracts from rectal and oropharyngeal swabs using a commercial kit (RNeasy® mini-kit, QIAGEN Inc., Valencia, CA), according to the manufacturer's instructions, and eluted in one volume. 60 μΙ_ end of pH regulator.

RT-PCR in real time

One-step quantitative real-time RT-PCR was performed on total RNA using the QuantiTect® probe RT-PCR kit containing ROX as the passive reference dye (QIAGEN Inc., Valencia,

CA), and a primer-probe series that targeted a highly conserved region of the influenza virus type A matrix protein (M) gene (Payungporn S. et al., 2006a; Payungporn S. et al. , 2006b).

For each real-time RT-PCR reaction, 5 μΙ_ of the RNA was added ¹¹⁰ IMPI

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Total INDUTTWAL extracted to an action mix containing 12.5 µl of 2X RT-PCR master mix with QuantITech® probe, 0.25 µί RT mix

QuantiTech®, Front and Reverse Initiators (final concentration of 0.4 μΜ for each), probe (final concentration of 0.1 μΜ) and ribonuclease-free water in a final volume of 25 μΐ_. TaqMan® GAPDH Control Reagents (Applied Blosystems, Foster City, CA) were used according to the manufacturer's instructions for the detection of GAPDH as an endogenous Internal Control for the presence of RNA extracted from swab and tissue samples and as a standardization control.

Quantitative one-step real-time RT-PCR was performed on the reaction mixtures in a QPCR Mx3000P® system (Stratagene, La Jolla, CA). Cyclic conditions included a Reverse transcription step at 50 ° C for 30 minutes, an Initial denaturation step at 95 ° C for 15 minutes to activate Thermus aquaticus HotStar® DNA polymerase, and amplification for 40 cycles. Each cycle of amplification included denaturation at 94 ° C for 15 seconds, followed by binding / extension at 60 ° C for 1 minute. Fluorescent FAM (518 nm emission wavelength) and VIC (554 nm emission wavelength) signals were recorded at the end of each cycle. The threshold cycle (Ct) was determined by adjusting the threshold fluorescence (dR) to 1000 in each individual experiment. The software program was used

Mx3000P® version 2.0 (Stratagene, La Jolla, CA) for data acquisition and analysis. The positive control consisted of amplification of RNA extracted from virus A / canlno / FL / 242/03 (H3N8). The results were

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Normalized IMPI dividing the Ct value of M by the corresponding GAPDH Ct value for each sample.

Tissue virus re-isolation

Frozen trachea and lung tissues from virus inoculated dogs were thawed and homogenized in 10 volumes of DMEM supplemented with 0.5% BSA and antibiotics. Solid debris was removed by centrifugation, and supernatants were inoculated into DMEM-grown cells in DMEM supplemented with 1 gg / mL trypsin treated with TPCK (Sigma-Aldrich Corp., St. Louis, MO) and antibiotics, as described above. . Cells were grown in 25 cm flasks² at 37 ° C in a humidified atmosphere containing 5% CO2. Cultures were observed daily for morphological changes, and were harvested 5 days post-inoculation. The harvested cultures were clarified by centrifugation, and the supernatants were inoculated into fresh MDCK cells as described for the initial inoculation; Two additional passages were performed for samples that showed no evidence of influenza virus by hemagglutination or RT-PCR. Hemagglutination activity in clarified supernatants was determined using 0.5% turkey erythrocytes as previously described (Crawford et al., 2005). RT-PCR was performed as described below.

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RT-PCR, nucleotide sequencing and phylogenetic analysis

Viral RNA was extracted from the MDCK cell supernatant using the QlAamp® viral RNA mini-kit (QIAGEN Inc., Valencia, CA), according to the manufacturer's instructions. Viral RNA was reverse transcribed to cDNA using the QIAGEN® One-Step RT-PCR Kit (QIAGEN Inc., Valencia, CA), according to the manufacturer's instructions. PCR amplification of the coding region of the 8 viral influenza genes in the cDNA was performed as previously described (Crawford et al., 2005), using primer sets specific for universal genes (primer sequences available upon request). The resulting DNA amplicons were used as templates for automated sequencing on the ABI PRISM® 3100 automated DNA sequencer using cyclic sequencing dye terminator chemistry (Applied Biosystems, Foster City, CA). Nucleotide sequences were analyzed using the Lasergene 6® package (DNASTAR, Inc., Madison, Wl). Nucleotide sequences for viruses recovered from infected dogs were compared with virus sequences in the inoculum to determine if any changes had occurred during replication in the respiratory tract.

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EXAMPLE 12

Clinical disease

All 6 virus-inoculated dogs developed transient fever (rectal temperature&gt; 39 ° C) for the first 2 days pi, but none exhibited respiratory signs such as cough or runny nose during the 6-day observation period. Pseudoinoculated dogs continued to be clinically healthy.

EXAMPL013

Spread of the virus

Influenza A nucleoprotein was detected in the oropharyngeal swab collected from one of the dogs inoculated with virus at 24 hours.

p. The oropharyngeal swabs collected from one dog at 72, 84, and 120 hp, and another dog at 108, 120, and 132 hr pi, were positive for virus using quantitative real-time RT-PCR (Table 11). The absolute copy number of the influenza M gene per pL of hyssop extract increased with time from 3 to 6 days pi No virus was detected in the rectal swabs.

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EXAMPLE014 _

Post mortem exams

In contrast to previous experimental infection using specific pathogen-free hounds 5 (Crawford et al., 2005), crossbred dogs inoculated with virus had pneumonia, as demonstrated by general and histopathological analysis of lungs from days 1 to 6 pi In addition to pneumonia, the dogs had rhinitis, tracheitis, bronchitis, and bronchiolitis similar to those described in naturally infected dogs (Crawford et al.,

2005). There was epithelial necrosis and erosion of the lining of the airways and bronchial glands with infiltration of neutrophils and macrophages from submucosal tissues (Figure 5, upper panels). Immunohistochemistry detected viral H3 antigen in epithelial cells of bronchi, bronchioles, and bronchial glands (Figure 5, lower panels).

Bacterial superinfection was not present. The respiratory tissues of the 2 pseudoinoculated dogs were normal.

EXAMPLE 15

Replication of the virus in the trachea and lungs

The trachea and lungs were positive for viruses by

Quantitative real-time RT-PCR in all dogs from 1 to 6 days pi

(table 12). The absolute number of copies of the influenza M gene per pL

115 of trachea homogenate, increased from 1 to 5 days pi. v decreased

IMPI

INSTITUTO MIKICANO DI LA MtONSDO INDUSTRIAL then on day 6. The absolute number of copies of the M gene per pL of lung homogenate decreased from 1 to 6 days pi In general, the trachea contained&gt; one log more virus than the lung in each one of the 6 days pi

TABLE 11

Detection of virus spread in the oropharynx of crossbred dogs inoculated with canine influenza virus by quantitative real-time RTPCR

<td>Dog identification</td><td>Pi time (hours)³</td><td>Relationship M / GAPDH^b</td><td>Matrix gene (copies / pL)^c</td>

<td rowspan="3"> 860</td><td> 72</td><td> 1.20</td><td>1.57E + 02</td>

<td> 84</td><td> 1.30</td><td>8.25E + 02</td>

<td> 120</td><td> 1.23</td><td>1.47E + 03</td>

<td rowspan="3"> 894</td><td> 108</td><td> 1.17</td><td>1.17E + 02</td>

<td> 120</td><td> 1.41</td><td>1.37E + 02</td>

<td> 132</td><td> 1.27</td><td>3.74E + 02</td>

^to Time in which oropharyngeal swabs were collected from dogs after inoculation with virus A / canino / FL / 43/04 (H3N8).

^b Normalization ratios were calculated by dividing M (Ct) by GAPDH (Ct) for each extract of the swab.

^c Absolute number of couplets of the matrix protein gene per uL of 20 swab extract.

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TABLE012 _

Detection of virus replication in the trachea and crossed dog lung inoculated with Canine Influenza virus using quantitative real-time RTPCR

<td rowspan="2">Dog identification</td><td rowspan="2">Weather pi (hours)³</td><td colspan="2">Relationship M / GAPDH^b</td><td colspan="2">Matrix gene (copies / pL)^c</td>

<td>Lung</td><td>T rachea</td><td>Lung</td><td>Windpipe</td>

<td> 797</td><td> 24</td><td> 1.20</td><td> 1.43</td><td>8.22E + 05</td><td>3.11E + 04</td>

<td> 801</td><td> 48</td><td> 1.33</td><td> 0.99</td><td>1.15E + 05</td><td>6.52E + 06</td>

<td> 789</td><td> 72</td><td> 1.44</td><td> 1.12</td><td>2.39E + 04</td><td>1.56E + 05</td>

<td> 819</td><td> 96</td><td> 1.40</td><td> 1.27</td><td>3.19E + 04</td><td>1.43E + 05</td>

<td> 860</td><td> 120</td><td> 1.59</td><td> 1.04</td><td>3.48E + 03</td><td>1.17E + 06</td>

<td> 894</td><td> 144</td><td> 1.70</td><td> 1.15</td><td>4.78E + 02</td><td>1.50E + 03</td>

³ Time in which tissues were collected from dogs after inoculation with A / canine / FL / 43/04 virus (H3N8).

^b Normalization ratios were calculated by dividing M (Ct) by 15 GAPDH (Ct) for each tissue homogenate.

^c Absolute number of copies of the matrix protein gene per uL of tissue homogenate.

Materials and methods for Example 16

Virus strains

Canine influenza virus strains, as well as strains originating from birds, horses and humans (listed in Table 15), were spread in

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DC PROPERTY k irOUSTMAL embryonated eggs or MDCK cells, and their infectivity was titrated by extreme dilution in chicken embryos or plaque test. Rapid virus quantification was performed by hemagglutination test using turkey erythrocytes.

Diagnostic samples

A total of 60 canine lung tissues collected from probable cases of viral respiratory disease during 2005 were tested for the presence of canine influenza virus.

Extraction of RNA from canine tissue samples

Lung tissue blocks weighing between 20 and 30 mg were homogenized in a disposable tissue mill (Kendal). Total RNA was extracted using a commercial kit (RNeasy mini-kit, Qiagen, Valencia,

CA), and eluted in a final volume of 60 μΙ_, following the manufacturer's recommendations.

Initiator and probe design

Multiple sequence alignments of the H3 and M genes of various subtypes and various species were performed using the CLUSTAL X program (version 1.8). Matrix protein (M) probes and primers were selected from conserved regions of known sequences corresponding to different influenza A virus subtypes, whereas the¹¹⁸ IMPI

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INDUSTRIAL series of primers and probes specific for the hemagglutinin H3 gene were selected to specifically map equine and canine influenza A virus genes and not mating of homologous bird and human genes (Table 13). Initiator design software (OLIGOS version 9.1) and the web-based analysis tools provided by EXIQON (http://lnatools.com) were used for the calculation of Tm and the prediction of secondary structure, as well as self-hybridization. A conserved region of an 18S rRNA gene was used as an endogenous internal control for the presence of RNA extracted from canine tissue samples. Test reagents

TaqMan® pre-developed for eukaryotic 18S rRNA (VIC / TAMRA) (Applied Biosystems), were used for real-time detection of 18S rRNA in tissue samples.

Real-time RT-PCR condition

One-step real-time RT-PCR was performed using the Quantitect probe RT-PCR kit containing ROX as a passive reference dye (Qiagen, Valencia, CA). In each real-time RT-PCR reaction, 5 pL of RNA sample were used as template for combination with a reaction mixture containing 10 pL of 2X RT-PCR master mix with QuantiTech probe, 0.2 pL of RT mix

QuantiTech, primers (final concentration of 0.4 pM for the H3 gene, or final concentration of 0.6 pM for the M gene), probe (final concentration of

0.1 pM for the H3 gene, or final concentration of 0.2 pM for the M gene) and

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INSTITUTO MMICANO M LA PROFKOAD INDUSTRIAL ribonuciease-free water in a final volume of 20 μί. One-step real-time RTPCR was performed on the Mx3005P real-time QPCR system (Stratagene). Cyclic conditions included a reverse transcription step at 50 ° C for 30 minutes. After an initial denaturation step at 95 ° C for 15 minutes to activate Thermus aquaticus HotStar® DNA polymerase, amplification was performed for 40 cycles including denaturation (94 ° C for 15 seconds) and binding / extension (60 ° C for 30 seconds). The fluorescence signals FAM (emission wavelength of 516 nm for detection of H3 and M) and VIC (emission wavelength of 555 nm for detection of rRNA 18), were obtained once per cycle at the end of the extension. Data acquisition and analysis of the real-time PCR test were performed using the Mx3005P version 2.02 software (Stratagene).

Specificity of the H3 initiator / probe series for canine influenza virus (H3N8) and universality of the M initiator / probe series for influenza virus type A

To test the specificity of each series of initiators / probes, RNA extracted from several known subtypes of influenza A viruses was used as a template in the real-time RT-PCR test (Table 15).

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RNA standard for determining the performance of the

RT-PCR in real time

Canine influenza A virus genes (A / canine / Florida / 242/2003 (H3N8)) were used to generate the amplicons of

PCR for H3 (nt 1-487) and M (nt 1-276), using primers linked with the T7 promoter (Table 13). The purified H3 and M gene PCR amplicons were then used as templates for in vitro transcription using the Riboprobe T7 in vitro transcription system (Promega), following the manufacturer's recommendations. The concentration of the transcribed RNA molecules was calculated by measuring the absorbance at 260 nm. The RNA molecules were then serially diluted 10 times, ranging from 10® to 10 copies / pL to perform sensitivity tests. In addition, a standard curve was generated by plotting the logarithm of the initial concentrations of the

RNA (copies / pL) against threshold cycle (Ct) obtained from each dilution to determine the general performance of real-time RT-PCR.

Comparative sensitivity tests between real-time RT-PCR and the Directigen Flu A test set

Reserve virus of two viral strains that included

A / Wyoming / 3/2003 (H3N2) a 10® ⁶⁷ EID₅₀/ mL (HA = 64) and

A / canine / Florida / 242/2003 (H3N8) at 10⁷¹⁷ EID₅₀/ mL (HA = 16), were used for the detection threshold test. The logarithmic dilution of samples in phosphate-buffered saline with phosphate (PBS) (125 pL) was used in a kit

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IWnWTO MMICANO OE INDUSTRIAL PROPERTY rapid detection of influenza A antigen, Directigen Flu A (Becton, Dickinson and Company), following the manufacturer's instructions. Each Directigen Flu A test device has a dot of H1N1 influenza antigen in the center of the membrane, which develops as a purple dot and indicates the integrity of the test, which is based on a monoclonal antibody to nucleoprotein. (NP). The development of a purple triangle around the point is indicative of the presence of influenza NP in the sample tested. The intensity of the purple signal of the triangle was rated as + (outline of triangle), ++ (slightly colored triangle), +++ (dark purple triangle) and ++++ (very dark purple triangle). Viral RNA was extracted in 125 pL aliquots from each virus dilution using the QlAamp viral RNA mini-kit (Qiagen, Valencia, CA), and eluting in a final volume of 50 pL. A volume of 5 pL of the extracted viral RNA molecules was tested by real-time RT-PCR for comparative sensitivity testing with the Directigen Flu A kit.

EXAMPLE 16

The real-time RT-PCR test for canine influenza virus depends on information from three molecular probes that target the host cell 18S rRNA, as well as M and H3 from the influenza A virus genome ( table 14). Amplification of the host gene¹²² IMPI £

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INDUrnUAL is a reporter of the quality and integrity of the sample. Clinical, necropsy, or laboratory samples containing canine influenza (H3N8) viruses are expected to give an amplification signal with all three probes.

Samples that give an amplification signal with the 18S rRNA and M probes, but negative for H3, would be indicative of an H3 subtype of the influenza virus originated from human, pig or bird, or of non-H3 subtypes. These rare cases could be resolved by RT-PCR using universal HA primers to generate amplicon cDNAs that can be analyzed by sequencing. Properly collected and handled samples lacking the influenza A virus give only an 18S rRNA amplification signal. Situations in which only the 18S rRNA probe and the H3 probes give an amplification signal are indicative of a failed technique, unless otherwise demonstrated; A false negative with M probes or a false positive for H3 needs to be demonstrated. Finally, samples that cannot give amplification signals with all three probes are indicative of defective sample collection, degradation, failed RNA extraction, or the presence of polymerase inhibitors used in the

PCR.

To test the specificity of the H3 primer / probe set for canine influenza A (H3N8) virus and the universality of the M primer / probe set for type A influenza, various virus subtypes of Influenza A were tested by real-time RTPCR. The results show that the series of¹²³ IMPI

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INDUSTRIAL H3 primers / probes gave a positive amplification signal only with canine influenza virus (H3N8). No significant nonspecific or false positive amplification signals were observed in other human H3 subtypes or strains. The series of M primers / probes gave a positive amplification signal with all the strains tested (Table 15). These results indicated that the H3 primer / probe series specifically detects canine influenza A virus (H3N8), while the M primer / probe series detects multiple subtypes of type A influenza virus.

The performance of real-time RT-PCR tests was evaluated by extreme dilution of in vitro transcribed M and H3 RNA molecules. As expected, the threshold cycle (Ct) was increased in direct correlation with dilution of the RNA standards. Fluorescent signals can be detected at standard dilutions of M and H3 RNA as low as 10³ and 10² copies / uL, respectively (figure 6A and 6B). The standard M and H3 gene curves were constructed by plotting the logarithm of starting RNA concentrations against the threshold cycle (Ct) obtained from each dilution (Figure 6C and 6D). The slope of the standard curve is used to determine the efficiency of the PCR reaction, which is theoretically exponential; 100% amplification efficiency would involve doubling the concentration of amplicons each cycle. Standard curves with a slope between approximately -3.1 and -3.6 are typically acceptable for most applications that require precise quantification (efficiency

124

IMPI tWTnVTO MIKICANO DI LA WOMOAD INDUSTRIAL (90 to 110% reaction). A value of Rsq is the fit of all the data to the graph of the standard curve. If all the data is perfectly on the line, the value of Rsq will be 1.00. Since the data is beyond the line, the value of Rsq decreases. A value of Rsq&gt; 0.985 is acceptable for most tests. The standard curve of M revealed a slope of 3,576 (efficiency = 90.4%) and Rsq = 1.00, while the standard curve of H3 gave a slope of -3,423 (efficiency = 95.9%) and Rsq = 0.999. These values ​​indicate satisfactory amplification efficiency and overall performance of real-time RT-PCR tests. The present inventors attribute the lower efficiency and sensitivity of the M primer / probe series compared to the H3 primer / probe series to the N-fold degeneration of the M primer sequences required to ensure wide coverage of variability of M gene sequences across viruses of multiple subtypes, hosts, and lineages.

The sensitivity of the real-time RT-PCR test was also compared to the commercial rapid antigen detection test (Directigen Flu A). Logarithmic dilutions of AM / yoming / 3/2003 (H3N2) and A / canine / Florida / 242/2003 (H3N8) were analyzed with Directigen Flu A and by real-time RT-PCR. The Directigen Flu A results showed that the sensitivities against both viral strains are an approximately 100-fold dilution of the stock viruses used in these experiments (Figure 7). The signals (purple color) generated by the canine virus samples (A / canine / Florida / 242/2003: 10^{6 x} PFU / ml), were

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IMPI Mexican institute OF INDUSTRIAL FROFIDITY much weaker than those present in the human virus (A / Wyoming / 3/2003: 10^7x PFU / ml), according to the lowest concentration of virus in these samples. Alternatively, the lower signal for canine influenza could be attributed to the molecular specificity of monoclonal antibodies against NP, that is, poor conservation of amino acids within the NP epitope of canine influenza A viruses.

Real-time RT-PCR of the M gene gave Ct values ​​above the threshold with 10 and 30 equivalents of virus PFU by reaction of A / canine / Florida / 242/2003 and A / Wyoming / 3/2003, respectively ( table 16). The differences were between the sensitivity value of 2 viral strains due to the differences in the original viral titers. Comparison of H3 gene detection between canine and human influenza viruses was not performed because the H3 primer / probe signal in real-time RT-PCR tests exclusively amplifies influenza virus. A canine. RT-PCR was 10⁵ times more sensitive than rapid antigen detection equipment.

To evaluate the performance of the RT-PCR test in necropsy samples from dogs with acute respiratory disease, 60 canine lung tissue samples referred during the year of 2005 were tested for the presence of influenza A virus. canine by real-time RT-PCR. A total of 12 out of 60 samples (20%) were positive for the M and H3 genes, while the remaining 48 samples were negative for the M and H3 gene. Attempts were made to

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isolate the virus by inoculating MDCK cells and eggs to assess the specificity of the test in real time; 2 of 12 samples that were positive for canine influenza by RT-PCR yielded canine influenza virus (data not shown, manuscript in preparation). Although all tissues were collected from dogs with a history of severe respiratory disease, the majority of samples did not yield canine influenza virus by real-time PCR or conventional isolation, suggesting a high incidence of other respiratory pathogens such as Bordetella bronchiseptica, canine parainfluenza virus or canine distemper virus. The real-time, one-step RT-PCR test herein provides a fast, sensitive, and cost-effective procedure for the detection of canine influenza A (H3N8) virus. Rapid laboratory diagnosis of infections by the canine influenza A virus (H3N8) in the early stage of the disease can provide relevant information for the clinical management of the facility and the patient.

Initiators and probes used for real-time RT-PCR detection and in vitro transcription

<img file="MX339202B_D0055.tif" />

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TABLE 14

Interpretation of the RT-PCR test in real time

<td rowspan="2">Interpretation</td><td colspan="3">Results</td>

<td>M</td><td>H3</td><td>RRNA 18S</td>

<td>Positive for canine influenza A virus (H3N8)</td><td> +</td><td> +</td><td> +</td>

<td>Positive for influenza A virus (unknown subtype)</td><td> +</td><td> -</td><td> +</td>

<td>Negative for influenza A virus</td><td> -</td><td> -</td><td> +</td>

<td>RNA extraction error or presence of PCR inhibitor</td><td> -</td><td> -</td><td> -</td>

TABLE015

Evidence of the specificity of the canine H3 primer / probe series, and universality test of the M primer / probe series with various influenza A virus subtypes

<td rowspan="2">Subtypes</td><td rowspan="2">Strain name</td><td rowspan="2">Host</td><td colspan="2">Real-time RT-PCR detection</td>

<td>H3 gene (Ct)</td><td>M gene (Ct)</td>

<td rowspan="2">H1</td><td>A / Ohio / 1983</td><td>Human</td><td>Without Ct</td><td> 15.40</td>

<td>A / WSN / 1933</td><td>Human</td><td>Without Ct</td><td> 20.09</td>

<td rowspan="3">H3</td><td>A / Wyom ¡ng / 3/2003</td><td>Human</td><td>Without Ct</td><td> 28.85</td>

<td>A / Victoria / 3/1975</td><td>Human</td><td>Without Ct</td><td> 16.62</td>

<td>A / canine / FL / 242/2003</td><td>Canine</td><td> 28.43</td><td> 29.25</td>

<td rowspan="2">H4</td><td>Turkey / MN / 1066/1980</td><td>Bird</td><td>Without Ct</td><td> 17.49</td>

<td>Clinical sample *</td><td>Bird</td><td>SinCt</td><td> 20.87</td>

<td rowspan="2">H5</td><td>A / chicken / Thailand / CUK2 / 2004</td><td>Bird</td><td>Without Ct</td><td> 20.13</td>

<td>A / fa¡sán / NJ / 1335/1998</td><td>Bird</td><td>Without Ct</td><td> 16.64</td>

<td>H6</td><td>Clinical sample *</td><td>Bird</td><td>Without Ct</td><td> 19.52</td>

<td rowspan="2">H10</td><td>Clinical sample *</td><td>Bird</td><td>Without Ct</td><td> 25.64</td>

<td>Clinical sample *</td><td>Bird</td><td>Without Ct</td><td> 19.59</td>

<td rowspan="2">H11</td><td>Clinical sample *</td><td>Bird</td><td>Without Ct</td><td> 15.72</td>

<td>Clinical sample *</td><td>Bird</td><td>Without Ct</td><td> 24.55</td>

* Note that the subtypes of the clinical samples were confirmed by nucleotide sequencing.

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TABLE 16

Comparative sensitivity tests for the detection of influenza A virus between real-time RT-PCR and Directiqen Flu A

<td rowspan="2">Virus dilutions</td><td colspan="2">Directigen Flu A</td><td colspan="2">Real-time RT-PCR of M (Ct)</td>

<td>A / canine / 242/03</td><td>A / Wyoming / 3/03</td><td>A / canine / 242/03</td><td>A / Wyoming / 3/2 003</td>

<td> 10'¹</td><td> + +</td><td> + + + +</td><td> 22.42</td><td> 19.48</td>

<td> 10'*</td><td> +</td><td> + + +</td><td> 25.85</td><td> 22.66</td>

<td> 10⁵</td><td> -</td><td> -</td><td> 29.27</td><td> 25.76</td>

<td>io³¹</td><td>Unrealized</td><td>Unrealized</td><td> 32.66</td><td> 28.66</td>

<td> 10^s</td><td>Unrealized</td><td>Unrealized</td><td> 35.48</td><td> 33.14</td>

<td>ñr⁶</td><td>Unrealized</td><td>Unrealized</td><td> 37.51</td><td> 35.06</td>

<td> 10’'</td><td>Unrealized</td><td>Unrealized</td><td> 39.09</td><td> 36.44</td>

<td> 10’°</td><td>Unrealized</td><td>Unrealized</td><td>Without Ct</td><td> 38.93</td>

TABLE 17

<td>Amino acid class</td><td>Examples of amino acids</td>

<td>Non polar Polar Uncharged Acids Basics</td><td>Ala, Val, Leu, lie, Pro, Met, Phe, Trp Gly, Ser, Thr, Cys, Tyr, Asn, Gln Asp, Glu Lys, Arg, His</td>

TABLE 18

<td>Single letter symbol</td><td>Amino acid</td><td>Single letter symbol</td><td>Amino acid</td>

<td>TO</td><td>To the girl</td><td>M</td><td>Methionine</td>

<td>B</td><td>Asparaglna or aspartic acid</td><td>N</td><td>Asparaglna</td>

<td>C</td><td>Cysteine</td><td>P</td><td>Proline</td>

<td>D</td><td>Aspartic acid</td><td>Q</td><td>Glutamine</td>

<td>AND</td><td>Glutamic acid</td><td>R</td><td>Arginine</td>

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TABLE 18 (CONTINUED)

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<td>Single letter symbol</td><td>Amino acid</td><td>Single letter symbol</td><td>Amino acid</td>

<td>F</td><td>Phenylalanine</td><td>S</td><td>Serine</td>

<td>G</td><td>Glycine</td><td>T</td><td>Threonine</td>

<td>H</td><td>Histidine</td><td>V</td><td>Valine</td>

<td>I</td><td>Isoleucine</td><td>w</td><td>Tryptophan</td>

<td>K</td><td>Power plant</td><td>AND</td><td>Tyrosine</td>

<td>L</td><td>Leucine</td><td>z</td><td>Glutamine or glutamic acid</td>

TABLE 19

Amino acid differences between the PB2 proteins of equine and canine H3N8 influenza virus

<td>Position</td><td>Equine Consensus *</td><td>Canine / FL / 03</td><td>Canine / FL / 04</td>

<td> 5</td><td>K</td><td>K</td><td>AND</td>

<td> 12</td><td>S</td><td>L</td><td>L</td>

<td> 37</td><td>G</td><td>G</td><td>AND</td>

<td> 175</td><td>R</td><td>R</td><td>I</td>

<td> 374</td><td>L</td><td>I</td><td>I</td>

<td> 375</td><td>R</td><td>R</td><td>K</td>

<td> 447</td><td>Q</td><td>Q</td><td>H</td>

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TABLE 20

Amino Acid Differences Between Equine Influenza and Canine H3N8 Influenza PB1 Proteins

<td>Position</td><td>Equine Consensus *</td><td>Canine / FL / 03</td><td>Canine / FL / 04</td>

<td> 114</td><td>V</td><td>I</td><td>I</td>

<td> 154</td><td>D</td><td>G</td><td>G</td>

<td> 221</td><td>TO</td><td>T</td><td>T</td>

<td> 317</td><td>M</td><td>I</td><td>I</td>

<td> 459</td><td>I</td><td>I</td><td>V</td>

<td> 682</td><td>I</td><td>I</td><td>V</td>

TABLE 21

Amino acid differences between the PA proteins of equine and canine influenza viruses H3N8

<td>Position</td><td>Equine Consensus *</td><td>Canine / FL / 03</td><td>Canine / FL / 04</td>

<td> 27</td><td>D</td><td>N</td><td>N</td>

<td> 62</td><td>I</td><td>V</td><td>V</td>

<td> 213</td><td>R</td><td>K</td><td>K</td>

<td> 337</td><td>TO</td><td>T</td><td>T</td>

<td> 343</td><td>TO</td><td>AND</td><td>AND</td>

<td> 345</td><td>L</td><td>I</td><td>I</td>

<td> 353</td><td>K</td><td>R</td><td>R</td>

<td> 400</td><td>T</td><td>T</td><td>TO</td>

<td> 450</td><td>V</td><td>I</td><td>I</td>

<td> 460</td><td>M</td><td>M</td><td>I</td>

<td> 673</td><td>R</td><td>R</td><td>K</td>

<td> 675</td><td>N</td><td>D</td><td>D</td>

Based on available genes from isolated viruses between 1963 and 1998.

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TABLE 22

Amino Acid Differences Between NP Proteins of Equine and Canine H3N8 Influenza Viruses

<td>Position</td><td>Equine Consensus *</td><td>Canine / FL / 03</td><td>Canine / FL / 04</td>

<td> 16</td><td>G</td><td>D</td><td>D</td>

<td> 157</td><td>TO</td><td>T</td><td>T</td>

<td> 214</td><td>R</td><td>R</td><td>K</td>

<td> 285</td><td>V</td><td>V</td><td>I</td>

<td> 286</td><td>TO</td><td>T</td><td>T</td>

<td> 359</td><td>TO</td><td>T</td><td>T</td>

<td> 375</td><td>D</td><td>D</td><td>N</td>

<td> 384</td><td>R</td><td>K</td><td>K</td>

<td> 452</td><td>R</td><td>K</td><td>K</td>

TABLE 23

Amino Acid Differences Between NA Proteins of Equine and Canine H3N8 Influenza Viruses

<td>Position</td><td>Equine Consensus *</td><td>Canine / FL / 03</td><td>Canine / FL / 04</td>

<td> 9</td><td>A / T</td><td>T</td><td>TO</td>

<td> 12</td><td>S</td><td>F</td><td>F</td>

<td> 20</td><td>L</td><td>I</td><td>i</td>

<td> 40</td><td>G</td><td>R</td><td>R</td>

<td> 42</td><td>G</td><td>D</td><td>D</td>

<td> 46</td><td>N</td><td>K</td><td>K</td>

<td> 52</td><td>AND</td><td>AND</td><td>K</td>

<td> 61</td><td>R</td><td>K</td><td>K</td>

<td> 69</td><td>N</td><td>S</td><td>S</td>

<td> 72</td><td>AND</td><td>K</td><td>K</td>

<td> 201</td><td>V</td><td>I</td><td>I</td>

<td> 261</td><td>I</td><td>V</td><td>V</td>

<td> 301</td><td>I</td><td>I</td><td>V</td>

<td> 396</td><td>N</td><td>D</td><td>D</td>

<td> 397</td><td>L</td><td>P</td><td>P</td>

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TABLE 24

Amino Acid Differences Between Equine Influenza and Canine H3N8 Virus M1 Proteins

<td>Position</td><td>Equine Consensus *</td><td>Canine / FL / 03</td><td>Canine / FL / 04</td>

<td>M1 161</td><td>S</td><td>S</td><td>TO</td>

<td>M1 208</td><td>K / Q</td><td>R</td><td>R</td>

'Based on available genes from isolated viruses between 1963 and 1998.

TABLE 25

Amino Acid Differences Between Equine and Canine H3N8 Influenza Virus NS1 Proteins

<td>Position</td><td>Equine Consensus *</td><td>Canine / FL / 03</td><td>Canine / FL / 04</td>

<td> 44</td><td>K</td><td>R</td><td>R</td>

<td> 59</td><td>R</td><td>H</td><td>H</td>

<td> 71</td><td>AND</td><td>K</td><td>K</td>

<td> 86</td><td>TO</td><td>T</td><td>T</td>

<td> 88</td><td>R</td><td>R</td><td>L</td>

<td> 140</td><td>R</td><td>G</td><td>G</td>

<td> 216</td><td>P</td><td>S</td><td>S</td>

'Based on available genes from isolated viruses between 1963 and 1998.

request.

It should be understood that the examples and modalities described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and will be included within the spirit and competence of this

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