Newcastle disease virus infectious clones, vaccines and diagnostic assays

Description of the invention

The invention relates to an avian paramyxovirus cDNA, RNA, a method of producing an infectious copy of an avian paramyxovirus, an infectious copy of an avian paramyxovirus, a vaccine, a method of distinguishing animals, and a diagnostic kit.

In general, the invention relates to the infection of poultry with the Newcastle disease virus. Newcastle disease virus (NDV) is one of the most volatile and deadly avian pathogens. The almost simultaneous appearance of Newcastle disease in many different geographic locations as an obvious new disease and the high variability in the type and severity of the disease have caused some nomenclature problems.

The disease was called pseudo fowl pest (Polish name Newcastle disease), pseudo poultry plague, avian pest, avian distemper and avian pneumoencephalitis. The development of this disease was mainly due to the development of the poultry industry in the twentieth century into a highly efficient, international industry dependent on intensive trade between countries.

It is generally assumed that the first outbreaks of Newcastle disease occurred in 1926 in Java, Indonesia and Newcastle-upon-Tyne, England (Kraneveld, 1926; Doyle, 1927). The name Newcastle disease was coined by Doyle as a temporary one, avoiding a descriptive name that could be confused with other diseases. Later it became clear that other less severe diseases were caused by viruses indistinguishable from NDV. In the US, a relatively mild respiratory disease has been named avian pneumoencephalitis and has been shown to be caused by NVD (Beach, 1944). Over the course of several years, a number of NVD isolates have been collected from chickens around the world that cause extremely mild disease or no disease at all.

The following factors contributed to the spread of the disease: 1) the movement of live birds, wild birds, herd birds, racing pigeons and commercial poultry; 2) movement of people and equipment; 3) the movement of poultry products; 4) air propagation; 5) contaminated poultry feed; 6) contaminated water; 7) incompletely inactivated or heterogeneous vaccines. According to the OIE, Newcastle disease is a disease of poultry caused by avian paramyxovirus serotype 1 (APMV-1) virus having an intracerebral pathogenicity index (ICPI) in day-old chickens of 0.7 or higher. The presence of a virulent virus can also be confirmed by the presence of multiple basic amino acids at the C-terminus of the F2 protein and the F (phenylalanine) at residue 117 of the N-terminus of the F1 protein. If this amino acid sequence cannot be demonstrated, characterization by ICPI tests is required. The word poultry refers to domestic fowl, turkeys, guinea fowl, ducks, geese, quails, pigeons, pheasants, partridges and ratites which are bred or kept in captivity for breeding, the production of meat or eggs for consumption, or for the restocking of flocks.

According to Alexander (1988), there have been three panzo-feelings of Newcastle disease since the first diagnosis of the disease. The first is the primary outbreaks of the disease, and appears to have originated in Southeast Asia. Isolated outbreaks, such as the one in England in 1926, happened by chance before the mainstream epidemic was slowly moving across Asia to Europe.

The second panzootion appears to have originated in the Middle East in the late 1960s and reached most countries by 1973. The faster spread of the second panzo was likely due to a revolution in the poultry industry in which international trade played an important role.

The third panzopia originally attacked domestic birds such as various species of pigeons (Vindevogel and Duchatel, 1988). The disease apparently arose in the Middle East in the late 1970s. By 1981 it reached Europe and then spread rapidly to all parts of the world, most of it as a result of contact between birds during racing and at shows and international trade in these birds.

Today, Newcastle disease is still widespread in many countries in Asia, Africa, the Americas and Europe. Only the Oceania countries appear to be relatively free from this disease (Spradbrow, 1988).

NDV belongs to the order Mononegavirales, family Paramyxoviridae, subfamily Paramyxovirinae, genus Rubulavirus. In addition to NDV, usually called avian paramyxovirus, type-1, eight other serotypes can be distinguished, designated avian paramyxovirus types-2 through -9 by antigenic relatedness in hemagglutination inhibition tests and in serum neutralization tests (Alexander, 1993).

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Despite the uniqueness of their serological grouping, there are some cross-relationships between viruses of different serotypes.

The NDV genome is a negative-stranded RNA molecule complementary to messenger RNAs encoding viral proteins. The RNA genome is approximately 15,200 nucleotides (nt) in size and encodes the following gene products (listed from the 3 'end to the 5' end of the genomic RNA): nucleocapsid protein (NP), phosphoroprotein (P), matrix protein (M), fusion protein (F ), haemagglutinin-neuraminidase (HN), and large polymerase protein (L) (Chambers et al., 1986).

RNA complexes with the NP, P and L proteins and forms a ribonucleocapsid (RNP) molecule which is surrounded by an envelope lined internally by the M protein. The shell contains the F and HN proteins which are required for attachment and entry into the host cell.

The NDV replication strategy is similar to that used by other paramyxovirinae. The first step is the attachment of the virus to host cell receptors via HN white. The fusion of the viral envelope with the host cell membrane depends on the action of both HN and F proteins and as a result RNP is released into the cytoplasm where viral replication takes place.

Viral RNA-dependent RNA polymerase (which is part of RNP) produces complementary transcripts that act as mRNA molecules and are used by the cell's translation machinery to synthesize viral proteins. As the NP protein accumulates, the RNA polymerase complex switches from transcription to replication to synthesize full-length genome and antigenomic RNA molecules.

The newly produced RNPs are packaged in a capsid on the cell membrane due to the action of the M protein and the F and HN proteins, which are accumulated in the plasma of the cell membrane. Newly formed virus particles are released from infected cells by budding. More detailed information on NDV replication is provided in Peeples (1988). The most recent review of the molecular biology of paramyxoviridae is given in Lamb and Kolakofsky (1996).

In addition to commercial domesticated poultry (e.g. chickens, turkeys, pheasants, guinea fowl, ducks, geese, pigeons), a wide range of captive, semi-domestic and free-living birds, including migratory waterfowl, are sensitive to NDV and can be primary sources of infection (Kaleta and Baldauf, 1988).

The pathogenicity of NDV strains varies significantly from host to host. Waterfowl seem to be the most resistant hosts, while flock birds forming temporary or permanent groups are the most susceptible. Chickens are very sensitive, but ducks and geese can also be infected, although they show little or no clinical signs, even if they are infected with strains that are lethal to chickens.

Newcastle disease is complicated by the fact that different virus isolates and strains can induce huge variations in the severity of the disease. Beard and Hanson (1984) grouped NDV strains and isolates into different phenotypes with respect to disease symptoms that are observed in fully susceptible chickens: 1) Visceral-affinity velogenic NDV, producing acute lethal infections in which haemorrhagic lesions are emphasized in the intestine; and velogenic NDV with affinity for the nervous system, which gives high mortality preceded by respiratory and neurological symptoms in the absence of intestinal damage; mesogenic NDV, giving low mortality, acute respiratory disease and nervous system symptoms in some birds; 3) lentogenic NDV, which produces mild or invisible respiratory infections or even asymptomatic intestinal NDV, avirulent viruses which are found to replicate primarily in the gastrointestinal tract. There have been reports of overlapping of some symptoms related to different groups.

Viruses enter the body through the respiratory system and digestive tract, and through the eyes. In the trachea, the virus spreads through the movement of the cilia and from cell to cell. After initial multiplication at the injection site, the virus is transmitted during viremia to the spleen, liver, kidney and lungs. Viruses of some strains reach the desired organs such as the liver and kidney very quickly, so that birds may die before symptoms of the disease develop.

Most viruses reach the nervous system through the blood before any significant amount of antibodies can appear. The long, asymptomatic carrier state that is supposed to occur in parrots poses a potential threat to the poultry industry. Long-term carrier status of both lentogenic and velogenic viruses may also exist in chickens (Heuschele and Easterday, 1970).

During NDV replication, it is necessary to cleave the precursor Fo glycoprotein to the daughter virus F1 and F2 to achieve infectivity (Rott and Klenk, 1988). This is a post-translational cut of the West

This is mediated by host proteases. If cleavage is not performed, non-infectious virus particles are produced and virus replication cannot take place. The Fo protein of a virulent virus can be cleaved by a wide variety of proteases, but the Fo proteins of low virulence viruses have limited sensitivity and these viruses can be grown in vivo in certain cell types and generally cannot be grown in vitro.

Lentogenic viruses replicate only in regions with trypsin-like enzymes such as the respiratory system and gastrointestinal tract, while virulent viruses can replicate in a variety of tissues and organs resulting in a lethal systemic infection.

The Fo precursor amino acid sequencing showed that low virulence viruses have a single arginine (R) linking the F2 and F1 chains, while virulent strains have additional basic amino acids forming two pairs such as K / RXK / RRF at the cleavage site. Moreover, the F2 chain of virulent strains generally begins with a phenylalanine residue, while for non-virulent strains it begins with leucine.

In several NDV strains also the HN protein is produced as a precursor that requires cleavage to become biologically active (Garten et al., 1980; Millar et al., 1988).

In addition to cleavage of the F and HN proteins, other viral factors may be involved in the pathogenicity. Madansky and Bratt (1978, 1981a, 1981b) have shown that altering transcription or translation can modulate viral growth and spread from cell to cell and / or cytopathogenicity.

The primary immune response to NDV infection is cell mediated and can be detected at the earliest 2-3 days after infection with a live strain vaccine. This probably explains the early defense against infection that was recorded in vaccinated birds before measurable levels of antibodies were observed (Gough and Alexander, 1973).

At about 1 week after infection, the circulating antibodies can protect the host from reinfection. IgM is required in the early phase, followed by IgG. Titer and defense peak after about 3 weeks and gradually decline if not reinforced. This means that older birds will need to be revaccinated.

Only live vaccines, administered by the respiratory tract, stimulate antibodies on all mucosal surfaces as well as in the serum. Inactivated vaccines, even when administered via the mucosal route, do not elicit a local respiratory response, despite the high concentration of antibodies in the serum.

This points to the important role of live vaccines that are able to present viral antigens in the upper respiratory tract to induce local and systemic immunity. Small droplets penetrate the lower respiratory tract, thereby stimulating a major humoral immune response, while large droplets stimulate a local response in the upper respiratory system.

Thus, aerosols with a wide range of droplet sizes produce the best local and humoral response.

However, it should be noted that despite extensive vaccination with current vaccines that produce high levels of antibody titers, the virus can still be shed from the mucosa surfaces.

The identification of Newcastle disease in the US has led to the use of inactivated vaccines (Hofstad, 1953). The observation that certain enzootic viruses only cause mild disease led to the first mesogenic, live Roakin vaccine (Beaudette et al., 1949), followed by the development of milder strains of Hitchner B1 (Hitchner and Johnson, 1948) and LaSota (Goldhaft, 1980), which are the most widely used live vaccines today.

Live NDV vaccines can be divided into two groups, lentogenic and mesogenic. Mesogenic strains are only suitable for the revaccination of birds due to their greater virulence. The immune response increases as the pathogenicity of the live vaccine increases. Thus, in order to obtain the desired level of defense without serious reactions, vaccination programs are currently employed that require the sequential use of progressively more virulent vaccines or live vaccines after inactivated vaccines.

One of the main advantages of live vaccines is that they can be administered using inexpensive mass administration techniques. A common method of administration is by drinking water. However, the administration of drinking water must be carefully monitored as viruses can be inactivated by excessive heat or light and with virucidal contaminants in the water.

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Mass administration of live vaccines via sprays and aerosols is also very popular due to the ease with which a large number of birds can be vaccinated in a short time. It is important to achieve the correct particle size by controlling the conditions under which the particles are produced.

Recently used live vaccines have several disadvantageous properties. The vaccine may still cause symptoms of the disease depending on environmental conditions and the presence of complicated infections. Thus, it is important to use extremely mild viruses for primary vaccination and, as a result, multiple vaccinations are usually required. Moreover, maternal antibodies can successfully prevent the effects of primary vaccination with live lentogenic vaccines.

Inactivated vaccines are usually made from an infectious allantoic fluid that is treated with formalin or betapropiolactone to kill the virus and mixed with an appropriate adjuvant. Inactivated vaccines are administered by injection into the muscle or under the skin. Inactivated vaccines are expensive to produce and administer.

However, the effect of inactivated vaccines in oily emulsions is not so impaired by the maternal immune response as with live vaccines and they can be used in day-old chickens. The advantages of inactivated vaccines are the low level of adverse reactions in vaccinated birds, the high level of defensive antibodies and the long duration of defense. Neither of the above vaccines can be serologically differentiated from wild-type NDV.

For many years there has been an interest in the development of recombinant viral vaccines from the poultry industry. The concept is to introduce genes with critical epitopes immunizing the pathogen of interest into an irrelevant gene of the vector virus. Vaccination with the recombinant virus results in immunization against both the vector virus and the pathogen of interest.

Many types of viruses have been evaluated as potential live viral vaccines for poultry. The two bird viruses that have received more attention are the avian pox virus (FPV) and the turkey herpes virus (HVT). The avipox virus is a DNA virus which has a large genome and is therefore believed to have plenty of room to carry foreign DNA.

When attenuated, FPV does not cause clinical disease and is commonly used as a vaccine for chickens. HVT is also a DNA virus and is classified as serotype III of the Marek's disease virus (MDV) family. HVT is non-pathogenic for chickens although it induces cross-defense against MDV and is commonly used to vaccinate chickens against Marek's disease.

It has been shown that defense against Newcastle disease can be induced using recombinant HVT or FPV vaccines (Morgan et al., 1992, 1993; Heckert et al., 1996; Boursnell et al., 1990; Taylor et al., 1990).

However, the onset of defense against Newcastle disease after vaccination with such a recombinant vaccine that produces the F protein from NDV or both F and HN proteins was severely delayed compared to that following vaccination with traditional live or inactivated NDV vaccine, which is possible due to that recombinant vaccines do not provide a sufficiently broad immunological spectrum of NDV epitopes with antigenic significance different from those found in the NDV protein, which is produced by a recombinant vaccine or is not properly presented to the immune system.

Moreover, local defense (mucosal, respiratory or intestinal) was not efficiently induced in birds vaccinated with recombinant vaccines. This is a serious drawback since vaccines used for primary vaccination against respiratory diseases must induce a local response to prevent infection and spread of virulent viruses that infect field chickens.

Antibodies to NDV that are capable of defending the host can be tested by virus neutralization assays. However, as long as a neutralizing response occurs with a concurrent haemagglutination inhibition (HI) response, the latter test is often used to assess a defense response, particularly after vaccination.

Antibodies to both the F and HN proteins can neutralize NDV. However, antibodies against F protein appear to induce greater neutralization than those directed against HN in in vivo and in vitro tests (Meulemans et al., 1986).

The presence and specificity of anti-NDV antibodies in avian serum gives little information about the infecting NDV strain and therefore has limited diagnostic value.

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The ubiquitous lentogenic NDV strains in birds in most countries and the near universal use of live vaccines that cannot be distinguished, at least not serologically from wild-type NDV, means that demonstration of infection alone is an extremely appropriate basis for ordering control testing. Since field disease can be an uncertain measure of the true virulence of the virus, it is necessary to further characterize the virus found.

Currently, the only method of diagnosing Newcastle disease that characterizes the infectious strain is virus isolation followed by pathogenicity testing. For this purpose, three in vivo tests are currently used: 1) egg mean time of death (MDT); 2) intracerebral pathogenicity index (ICPI) in day-old chickens; 3) intravenous pathogenicity index (IVPI) in six-day-old birds.

These tests suffer from a number of disadvantages, such as the availability of the animals, poor stability, and the relatively long duration of the tests. Finally, although not the last drawback, these tests do not allow straightforward serological identification of poultry vaccinated with the vaccine or infected with a wild-type strain.

As an alternative in vivo test, the polymerase chain reaction (PCR) has been successfully used to distinguish between virulent and non-virulent isolates (Stauber et al., 1995; Kant et al., 1997), although again serological differentiation is not possible here.

The development of poultry farming and trade in their products is currently organized on an international basis, often under the management of multinational companies. The threat of Newcastle disease has proved to be a major constraint on this trade.

Successful control of Newcastle disease will only be achieved if all countries report on outbreaks. However, international agreements are not straightforward due to the huge variation in the size of disease surveillance in different countries. Some countries do not vaccinate and would not want any form of NDV introduction into domestic poultry because vaccinated poultry cannot be distinguished from wild-type NDV infected poultry.

Others only allow the use of specific live vaccines and consider other vaccines to be unacceptably virulent. Still other countries have a still circulating highly virulent virus that is not recognized as such because overt disease is masked by vaccination.

Many countries have legislation in place controlling outbreaks that may arise. National control research is aimed at preventing the introduction and spread of diseases. Most countries have trade restrictions on poultry products, eggs and live poultry. Most countries have established import quarantine procedures, especially for parrot birds.

Some countries have adopted an extermination policy with the compulsory disposal of infected birds, their contact items and products. Others require prophylactic vaccination of birds, even in the absence of outbreaks, while some apply a vaccination policy in a circle around the epidemic area to create a buffer zone.

Clearly there is a need for better vaccines and better diagnostic methods that can be used to control Newcastle disease. Due to both the large variation in the dose received by individual birds during the mass administration of live vaccines, and the variation in the levels of immunity in young chickens, reactions following vaccination with live vaccines are inevitable. This is one of the main problems faced by farmers in countries where vaccination is compulsory.

Moreover, many vaccines are mixtures of a sub-population. When cloned, these subpopulations can differ significantly from one another in their immunogenicity and pathogenicity (Hanson, 1988).

However, the greatest disadvantage of the live and inactivated vaccines used today is that vaccinated animals cannot be distinguished from infected animals using current screening techniques such as hemagglutination inhibition or virus neutralization tests.

Virulent field viruses may still spread in vaccinated herds as signs of disease are masked by vaccination. Since it is not possible to carry out large-scale virus isolation and characterization of virulence by in vivo techniques, there is a great need for new and effective attenuated live vaccines that can be serologically distinguished from wild-type viruses.

Such vaccines, called NDV marker vaccines (as well as accompanying diagnostic methods and kits), which should provide the fullest possible immunological spectrum of antigenically relevant NDV epitopes and, in addition, be capable of serologically differentiating from wild-type NDV are not yet available. available.

The present invention provides a method of modifying an avian paramyxovirus genome by genetic modification, provides a genetically modified avian paramyxovirus, and a paramyxovirus marker vaccine.

The advent of modern molecular biology techniques has allowed for the genetic modification of many RNA viruses, including negative strand RNA viruses. This technique is often called reverse genetics. First, a (full-length) cDNA copy of the viral RNA is provided, and then transcribed from the DNA in permitting cells to generate infectious RNAs which replicate again to form infectious viral particles.

Basically, thanks to the earlier modification of the cDNA by standard techniques of molecular biology, it is possible to obtain a genetically modified RNA virus. However, this has never been achieved with NDV or other avian paramyxoviruses, and it has not even been possible to generate minigenomic fragments or plasmids from genomic fragments of avian paramyxoviruses to study the replication behavior of avian paramyxoviruses, which would allow understanding how to construct an infectious copy of a virus.

Surprisingly, although this description fully demonstrates that the avian paramyxovirus genome is the smallest of all paramyxovirus genomes sequenced to date, especially the 5 'end sequence from the NDV genome is much longer than previously determined and expected from the comparison with other Paramyxoviridae. The invention provides for the first time the full-length genome sequence of an avian paramyxovirus and provides a full-length or minigenomic cDNA for this virus.

The invention relates to an avian paramyxovirus cDNA, the cDNA comprising at least a nucleic acid sequence corresponding to the 5 'end of the avian paramyxovirus genome capable of producing an infectious copy of an avian paramyxovirus.

The invention also relates to an avian paramyxovirus cDNA, said cDNA comprising at least a nucleic acid sequence corresponding to the 5 'end of the avian paramyxovirus genome permitting replication of the avian paramyxovirus minigenome.

Preferred embodiments for both objects of the invention are such a cDNA which may be full length cDNA and may preferably be at least partially derived from Newcastle disease virus. Even more preferably, the cDNA may be derived from Newcastle disease virus, which is a lentogenic virus, possibly derived from a vaccine strain. In the best embodiment, the cDNA is derived from the vaccine strain which is the LaSota ATCC VR-699 strain.

Also preferably, the cDNA is provided with a modification in the nucleic acid, the modification comprising a nucleic acid encoding a modified protease cleavage site, and more preferably, the protease cleavage site of the fusion protein (F). According to a preferred embodiment of the invention, the modification also comprises a nucleic acid encoding a hybrid viral protein. The protein is preferably haemagglutinin-neuramidase (HN).

The modification may also advantageously relate to a deletion in the nucleic acid encoding the viral protein, with the viral protein also preferably being a matrix protein (M).

The cDNA according to the invention is preferably further provided with a nucleic acid encoding a heterologous antigen. The antigen preferably may be derived from a poultry pathogen, cDNA according to the invention, may furthermore be provided with a nucleic acid encoding an immune stimulating protein or part thereof.

The invention also relates to RNA produced on the basis of the cDNA according to the invention as defined above in relation to both of the presented embodiments of the invention.

The invention also relates to a method of producing an infectious copy of an avian paramyxovirus which comprises the transfection of at least one cell with an inventive cDNA as defined above in relation to both of the disclosed embodiments of the invention.

Preferably, in the method of the invention, the cell is capable of producing a viral neurocapsid (NP), phosphorus (P) or large polymerase (L) protein. Moreover, in the method according to the invention, it is possible to cut the fusion protein of the virus in question. It is also possible to advantageously incubate the cell in a culture medium containing proteolytic activity, the culture medium comprising an allantoic fluid containing proteolytic activity, and the cell in question may be derived from a chicken cell.

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The invention also relates to an infectious copy of avian paramyxovirus which is obtained by the method according to the invention.

Further, the invention relates to a vaccine that contains an infectious copy of the avian paramicovirus according to the invention. Preferably the vaccine is a live vaccine, and even more preferably an infectious copy of an avian paramyxovirus, at least partially derived from Newcastle disease virus (NDV).

The invention also relates to a method of distinguishing unvaccinated animals or animals vaccinated with the NDV vaccine according to the invention from wild-type NDV infected animals or those vaccinated with an unmodified mesogenic or lentogenic NDV strain, consisting in that at least one sample is taken from the animal and the presence of antibodies against an immunodominant epitope or marker produced by wild-type NDV or NDV unmodified but not produced by the vaccine in question is determined. Preferably in this method, the antibodies can be directed against the HN or F protein of NDV, more preferably the animal is selected from the group consisting of poultry, preferably chickens.

The invention also relates to a diagnostic kit for use in the method of the invention.

In recent years, a number of unsegmented negative-strand RNA viruses have been fully characterized and fundamental work on the replication and expression of their genomes has resulted in the possibility of generating whole infectious viruses by transfecting the cloned cDNA of the viruses in question (Colnzelmann review, 1996).

To date, infectious viruses from unsegmented negative strand RNA viruses have been produced from cloned cDNAs from for example rabies virus (Schnell et al., 1994, Conzelmann; EP070285A1), (Schnell et al., 1994; EP070285A1), vesicular stomatitis virus ( Lawson et al., 1995; Whelan et al., 1995) Sendai virus (Garcin et al., 1995), measles virus (Radecke et al., 1995; Schneider et al., 1997; EP0780475A1), human bronchial virus (Collins et al., 1995), bovine blight virus (Baron and Barrett, 1997) and human parainfluenza type 3 virus (Hoffman and Banerjee, 1997, Conzelmann; P0702085A1), (Schnell et al., 1994 ; EP070285A1.

However, all of the above infectious virus copies are capable of growing in vivo as in vitro in hosts, tissues or cells of various origins, allowing for easy cDNA transfection and replication and production of infectious viral particles in a suitable cell line.

This possibility does not exist for NDV, especially for the lentogenic NDV strains that provide the vaccine. The virulence of such an NDV strain is related to its ability to replicate in a wide range of cells, which is reflected by the fact that virulent strains can easily replicate in vitro and in vivo, whereas vaccine strains can only replicate in vivo.

So, with regard to NDV, it is obvious that there is no way out. Since attempts to generate an infectious copy of a virus from, for example, an infectious cDNA, may result in infectious viruses, such a virus is generally unsuitable for use as a vaccine because the infectious virus so produced is too virulent to be a vaccine due to its shortcomings; the fact that it can be produced and replicated after transfection of cDNA into cells reflects the ease with which the Fo protein is cleaved into F1 and F2, which as discussed above is a sign of NDV virulence.

Using a vaccine strain as parental material for cDNA would not solve the problem; the vaccine strain, especially of the lanthogenic type, does not contain the easily cleaved Fo protein, making it impossible to form a first generation replication-continuing virus. The cell used for transfection simply will not allow one or more rounds of vaccine-type virus replication with the Fo protein not cleaved.

The present invention, however, provides a solution to this problem and with it provides an infectious copy of NDV, for example for use in a vaccine.

The invention provides a method for producing an infectious copy of Newcastle Disease virus comprising, transfected cells capable of producing viral NP, P and L proteins complexing with viral RNA cloned full-length or cDNA-length cDNA of the virus in question, and further comprising incubating said cells. in a culture medium containing proteolytic activity allowing for cleavage of the Fo protein from the virus in question.

In this system, cotransfection with an NP-producing plasmid can be omitted. NP is likely to be produced from full-length cDNA since the NP gene is the first gene after the 5 'end of the antigenomic RNA. Since eukaryotic mRNAs are typically monocistronic, expression of distal genes is not expected. However, it is possible to generate full-length cDNAs in which the corresponding positions of the NDV genes are altered. If the first gene of such cDNA is the P or L gene, it is not necessary to generate the corresponding gene product from the cotransected plasmid.

Instead of using full-length cDNAs, it is possible to use two or more subgenomic cDNAs which produce replication-competent subgenomic RNAs and which together make it possible to produce the complete set of avian paramyxovirus proteins. Even when the RNAs are packaged separately, the produced virus-type particles can be used for successful rounds of replication due to co-infection and complementation of gene functions.

In a preferred embodiment, the invention provides a method wherein the proteolytic activity is derived from an enzyme such as a trypsin type enzyme or is derived from a composition that comprises such proteolytic activity. In a more preferred embodiment, said culture medium comprises allantoic fluid containing proteolytic activity. Cleavage of the Fo protein is required to generate an infectious virus. It is possible to generate an infectious virus from a lentogenic strain without adding exogenous proteolytic activity. Due to the inoculation of the supernatant from the transfected cells into the allantoic cavity of embryonic eggs, the proteolytic activity present in the allantoic fluid is able to cleave the Fo protein, producing the F1-F2 complex sufficient for fusion. Virions with the F protein thus activated are capable of infecting susceptible cells and replicating in cells producing the desired proteolytic activity, resulting in infectious progeny. An alternative to delivering the desired proteolytic activity to the supernatant of the transfected cells is, for example, the possibility of using a cell which is NDV permissive and which already produces proteolytic activity. Such a cell line is used to produce infectious lentogenic NDV without adding any exogenous proteolytic activity. Such a cell line can also be generated by stably transfecting a cell line with a gene conferring such activity. Moreover, it is possible to generate a stably transfected cell line producing wild-type F protein in the viral envelope, delivering infectious particles (not themselves provided by the genomic information encoding the wild-type F protein) entering the cell. The recovery of the infectious lentogenic virus is also possible by infection of the transfected cells with the NDV helper virus. An essential requirement for such a helper virus is that it be selected by, for example, neutralizing antibodies that eliminate the helper virus and which do not react with the lentogenic virus.

Finally, a stably transformed cell line can be constructed to produce one, two, or all of the three necessary proteins NDV, NP, P, and L. Such cell lines require the co-production of a subset of the three necessary proteins or do not require co-production at all to maintain production of an infectious copy of the virus. .

In a preferred embodiment, the invention provides a method wherein the cells used for transfection are derived from primary or secondary chicken cells or cell lines. The description provides, for example, CER or CEF cells which, like most in vitro cultured cells, essentially lack the appropriate proteases required for cleavage of the Fo protein from NDV, for example from a LaSota strain. However, cells derived for example from other birds may also be used.

The invention further provides a method for producing an infectious copy of Newcastle Disease virus, which comprises providing a cell transfected with cloned cDNA of the full-length or genome length of the virus in question, for example as identified in Fig. 3 and further, and optionally, incubating said cells in a culture medium containing a proteolytic activity allowing cleavage of the Fo protein of said virus, and then recovering the infectious virus by culturing said cells and inoculating material derived from said cultured cells into the allantoic cavity of embryonic eggs. The material in question includes, for example, (freshly harvested or frozen-thawed) cells or cell debris, or supernatant derived from the cell culture in question.

For example, the description discusses a method of recovering infectious virus wherein the supernatant of the transfected monolayer CEF is inoculated into the allantoic cavity of embryonic eggs. After four days, the allantoic fluid was collected and analyzed by hemagglutination test and passaged in eggs.

Furthermore, the invention provides a method further comprising passaging said infectious Newcastle disease virus copy by collecting the allantoic fluid and re-inoculating the embryonic eggs.

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In a preferred embodiment of the method according to the invention, said virus is a lentogenic virus, for example derived from an avirulent field NDV or from an NDV vaccine strain such as the LaSota NDV strain.

In addition, provided is a method of modifying an avian paramyxovirus genome by genetic modification that allows one or more mutations, deletions and / or insertions or other modifications to be introduced. For example, a method is provided for attenuating or modifying the virulence of an avian paramyxovirus via a modified cDNA, for example encoding a viral protein such as protein V and cloning such a modified cDNA into full-length cDNA, and producing an infectious copy of the virus from the full-length cDNA in question, thereby generating new NDV strains or new attenuated live vaccines with improved properties.

In addition to attenuation by modifying the gene products, it is also possible to attenuate the avian paramyxovirus by modifying the nucleotide sequence as required for transcription and / or replication. Such modifications result in attenuated strains that produce wild-type F proteins that are cleaved both in vitro and in vivo in a wide range of cells and as a result are more immunogenic than classical vaccine strains.

In a preferred embodiment, the invention provides a method of attenuating or modifying the virulence of an avian paramyxovirus such as Newcastle disease virus comprising modifying a viral protein protease cleavage site by modifying a cDNA encoding said cleavage site, and cloning said cDNA into a genomic length cDNA, e.g. Newcastle disease virus and production of infectious copies of Newcastele disease virus. This cleavage site is, for example, a protease cleavage site in the F or HN protein of the Newcastle disease virus. Attenuation is essentially limited to the reduction of virulence, although it is now also possible to use a relatively avirulent NDV strain and to provide progeny of such a strain with increased virulence, for example by providing a strain that has an elevated tendency to replicate in specialized cell types. It is therefore now possible to impart different virulence characteristics to the NDV virus.

The invention provides an antigenic method of a modified avian paramyxovirus such as Newcastle disease virus, characterized by providing a modified cDNA encoding at least a portion of a viral protein bearing at least one immunodominant epitope, further comprising cloning said cDNA in a genomic length Newcastle disease virus cDNA and producing an infectious copy of Newcastle Disease Virus.

For example, the invention also provides a method for (additional) modification of NDV, using the provided method for producing an infectious copy of NDV (vaccine), providing a method for producing a recombinant NDV marker vaccine, a marker vaccine containing the fullest possible or necessary immunological spectrum of NDV epitopes of antigenic significance, and is provided. serologically distinguishable from wild-type NDV because different, having serological significance, epitopes or markers have been removed by recombinant techniques. The invention provides a method of modifying the antigenic composition of an avian paramyxovirus such as NDV, which allows for the production of e.g.

In one embodiment, the invention provides an infectious copy of NDV, characterized in that the HN protein from NDV has been modified by recombining a cDNA encoding part of the protein in question with a cDNA encoding part of an HN protein derived from an avian paramyxovirus, for example type 2 or type 4. The said hybrid HN protein serves as a serological marker for the strain of the infectious NDV copy so produced or may serve to alter the tropism of the avian paramyxovirus to other cells and / or tissues. These so-called marker strains as introduced according to the present invention allow the production of vaccines which are an invaluable tool in estimating the prevalence of NDV in commercial herds worldwide. Moreover, the large-scale use of such marker vaccines will lead to the complete eradication of NDV through a process of intensive screening and eradication of infected herds.

Further provided is a method of producing an infectious copy of NDV strain in which one or more antigens are produced from other pathogens and which can be used for vaccination against a variety of diseases. Such an infectious copy of the NDV virus, for example, includes a heterologous cDNA encoding a heterologous protein derived for example from avian influenza virus (AI) (hemagglutinin (H5 and H7) and neuraminidase), avian leukemia virus (ALV) (env protein (gp85)), chicken anemia virus ( CAV) (VP1 + VP2), Marek's disease virus (MDV) (glycoprotein B (gpB), gH), inPL 197 722 B1 of fecal laryngotracheitis virus (ILT) (gB, gH, gD), infectious bursitis virus ( IBDV) (VP2 and VP3), rhinotracheitis virus (TRT) (fusion protein (F)), avian paramyxovirus-2, -3, -6 (PMV) (F protein, haemagglutinin-neuraminidase (HN) and others), infectious bronchitis virus (IBV) (peplomer protein, neuroprotein), reoviruses (sigma protein), adenoviruses, pneumoviruses, Salmonella enteritidis, Campylobacter jejuni, Escherichia coli, Bordetella avium (formerly Alcaligenes faecalis), Haemophilus paragallinarum, Pasteurellaithracheale, rhinotracheobacterium Riemerella (formerly Pasteurella) anatipestifer, Mycoplasmata (M. gallisepticum, M. synoviae, M. mereagridis, M. iowae) or Aspergilli (A. flavus, A. fumigatus).

The invention thus provides an avian paramyxovirus or strains derived therefrom that can be used as a vaccine vector to generate antigens from other poultry pathogens. Several features make NDV an ideal vaccine vector for vaccination against respiratory or intestinal diseases. 1) NDV can easily be multiplied to very high titers in embryonic eggs. 2) mass breeding of NDV in embryonic eggs is relatively cheap. 3) NDV vaccines are relatively stable and can be easily administered by mass administration such as drinking water or spraying, or in the form of an aerosol. 4) Natural infection with NDV occurs through the respiratory tract and / or the gastrointestinal tract, which are also natural routes of infection for many other poultry pathogens. 5) NDV can induce a local immune response independent of the presence of circulating maternal antibodies.

NDV has been shown to have potent anti-neoplastic as well as immune stimulating properties (for a review see Schirrmacher et al., 1998) [Schirrmacher V., Ahlert T., Steiner H.-H., Herold-Mende C., Gerhards R. and Hagmuller E. (1998) Immunization with virus-modified tumor cells. Seminars in Oncology 25: 677-696]. While NDV does not appear to be able to replicate efficiently in normal human cells, selected, NDV-mediated killing human tumor cells have been described. In nude mice, viral oncolysis and complete remission of human tumor transplants were observed following topical NDV therapy. This enabled the use of the NDV virus in cancer therapy. However, a problem is that administration is limited to topical application.

NDV infection induces interferons, chemokines and other potentially important gene products and introduces pleiotropic immune-stimulating properties into cancer cells. This was used to produce autologous tumor cell vaccines consisting of fresh effective samples that were infected with NDV. This type of vaccine was called an autologous cancer vaccine with NDV or ATV-NDV ( autologous tumor vaccine-NDV, Schirrmacher et al., 1998). NDV infected cells are inactivated by gamma radiation, which prevents cell division but allows NDV to replicate in the cytoplasm of infected cells. After a patient is vaccinated with ATV-NDV, T cells are recruited via NDV-induced chemokines. Some of these T cells can produce a T cell receptor that interacts with tumor associated antigen peptides in complex with class I molecules of the major histocompatibility complex on the cell surface. As a result of these interactions, cytotoxic T cell responses are induced and autologous tumor cells are killed as a result.

The invention provides the ability to modulate the repertoire and amount of chemokines and immune stimulating proteins induced by NDV infection. The present invention provides a method of producing recombinant NDV that has been modified to introduce and express (a) heterologous gene (s). Such recombinant NDV can be used to modify the repertoire and amount of chemokines and proteins that stimulate the immune response in infected cells. In one embodiment, the invention provides recombinant NDV that introduces and enables expression of genes encoding human interferons, chemokines, or other proteins that stimulate the immune response. This recombinant NDV is used to produce ATVNDV which is more potent than conventional ATV-NDV. (For example: IFN-α, β, TNF-α, IL-1, IL-6 digitokines; RANTES, IP-10 chemokines; other genes such as HSP, ACTH, endorphin, iNOS, EPA / TIMP, NFkB). The pleiotropic properties of NDV to stimulate the immune system can also be used as adjuvants to vaccinate animals and humans against infectious diseases. In one embodiment of the invention, the foreign genes encoding the essential antigen (s) of the infectious agent (s) are inserted into the genome of the NDV and the simultaneous production of the antigen (s) and immune stimulating proteins by the infected cells can induce a strong immune response against the infectious agent. In another embodiment

According to the invention, the immune stimulating properties of NDV can be further enhanced by the use of NDV recombinants which simultaneously produce antigens and specific immune stimulating proteins. In a preferred embodiment, the invention is used to produce an AIDS (acquired immune deficiency syndrome) vaccine using recombinants of NDV producing essential human immunodeficiency virus (HIV) antigens, alone or in combination with immune stimulating proteins.

NDV is also used as an adjuvant to vaccinate animals and humans against infectious diseases. In one embodiment of the invention, heterologous or foreign genes encoding the relevant antigen (s) of the infectious agent (s) are inserted into the genome of the NDV and the simultaneous production of the antigen (s) and immune stimulating proteins by the infected cells can induce a strong immune response against the infectious agent. . In another embodiment of the invention, the immune stimulating properties of NDV are further enhanced by the use of NDV recombinants that simultaneously produce immune stimulating antigens and specific proteins. In a preferred embodiment, the invention is used to produce an AIDS (acquired immune deficiency syndrome) vaccine using recombinants of NDV producing essential human immunodeficiency virus (HIV) antigens, alone or in combination with immune stimulating proteins.

Also provided is a method for producing NDV conditionally lethal deletion mutants that can be used as self-limiting, non-transfer (carrier) vaccines. An NDV deletion mutant was generated which is unable to produce the (M) matrix protein required for NDV budding on the inner membrane of the cell. For example, the invention provides a phenotypically complemented NDV strain that is capable of producing the M protein, and is capable of infecting cells and spreading from cell to cell. However, the mutant virus is unable to produce infectious progeny in non-complementing cells. This shows that phenotypically complementing NDV deletion mutants can be used as safe self-limiting vaccines that are incapable of spreading into the environment. This non-passed vaccine combines the most important advantage of live vaccines, i.e. efficacy, with the most important advantage of killed vaccines, i.e. safety.

The invention provides Newcastle disease virus or a strain derivative thereof, for example by passaging or further culturing in embryonic eggs or suitable cells, which is derived from an infectious copy of the virus produced by the method provided by the invention.

For example, provided is NDV that has been modified by at least one way of producing an infectious copy of Newcastle Disease virus, which is attenuated, virulence-modified, antigen-modified, produces a heterologous antigen, or is not transmitted, or has combinations of the above characteristics.

The present invention provides NDV vaccines characterized, for example, in that they carry different virulence properties or different antigenic properties for the purposes of a marker vaccine and / or for the production of heterologous antigens derived from other pathogens in a transferable and / or non-transferable form.

Such vaccine may be a killed or live vaccine. Preferably the vaccine is a live vaccine, however, killed vaccines provided according to the invention are preferred in circumstances where a live vaccine is not suitable or is only suitable in small amounts, for example due to commercial limitations or other conditions set by disease control authorities.

The invention also provides a method of diagnosis and suitable test kits for detecting antibodies against a serologically relevant immunodominant epitope or marker, providing methods and methods for carrying out a method for controlling and / or eradicating NDV and / or other poultry diseases. The invention provides new and effective vaccines that can be distinguished serologically from field viruses and old-type vaccines. Such new vaccines, called NDV marker vaccines, provide the fullest possible immunological spectrum of antigenically relevant NDV epitopes, and are serologically distinguished from wild-type NDV by the use of the included methods and diagnostic kits.

The invention provides a method of distinguishing unvaccinated animals or animals vaccinated with the NDV vaccine of the invention from wild-type NDV infected or vaccinated animals vaccinated with an unmodified mesogenic or lentogenic NDV strain by collecting at least one sample (such as plasma, blood, eggs or liquid

From the eye) from the animal in question and to determine in said sample for the presence of antibodies directed against an immunodominant epitope or marker produced by said wild-type virus or unmodified NDV but not produced by a vaccine according to the invention.

The invention provides a method wherein said antibodies are directed against an HN or F protein from NDV, for example a hybrid protein as described in the experimental section of this description. The invention provides, for example, a diagnostic method which consists in that the animal in question is selected from the group consisting of poultry, preferably chickens.

The invention also provides a diagnostic kit for use in a method of serologically differentiating animals. In one embodiment of the invention, a simple and rapid haemagglutination inhibition (HI) test is used to differentiate between vaccinated animals and infected animals. Animals vaccinated with a marker vaccine in which the entire globular NDV HN head has been replaced with the corresponding portion with the HN of a different serotype will not induce NDV HN antibodies and therefore will not inhibit erythrocyte haemagglutination by NDV virions.

By using marker vaccine virions in the HI test, antibodies against the HN hybrid protein are detected and can be used to measure vaccination efficiency. Alternatively, an ELISA test which detects antibodies against the NDV F protein is used to measure the vaccination efficiency.

In addition to the HI test, an ELISA test can be used to determine the presence of anti-HN antibodies from NDV. The antigen to be used in such an assay is, for example, HN from NDV, which is produced by recombinant DNA techniques, or a conserved peptide from HN from NDV.

A blocked ELISA can also be used. In this case, one or more monoclonal antibodies against conserved HN epitopes of NDV were used to determine whether competitive antibodies are present in the samples from the vaccinated animals. ELISAs can advantageously be used when the marker vaccine contains only chimeric HN proteins or when several HN epitopes from NDV are swapped.

The invention is further elucidated in the experimental part of this description, which does not limit the present invention.

Experimental part

Materials and methods

Standard cloning procedures were performed according to Sambrook et al. (1989) unless otherwise stated. All constructs requiring DNA fragments that were produced using the polymerase chain reaction (PCR) were confirmed by sequence analysis. In the primer sequences given below, the nucleotides corresponding to the NDV sequences are underlined and the positions in the NDV genome are marked. The nucleotide sequence of the restriction sites used for cloning is shown in bold.

Cells and viruses

CER cells (Smith et al., 1976) were grown in GMEM / EMEM (1: 1) medium containing 5% fetal calf serum and a 2% antibiotic mixture containing 1000 U / ml Penicillin, 1000 µg / ml Streptomycin, 20 µg / ml ml Fungizon, 500 pg / ml Polymixin B and 10 mg / ml Kanamycin. QT35 cells (Moscovici et al., 1977; Cho, 1982) were grown in medium supplied by GibcoBRL / Life Technologies (Cat # 041-91536; Composition Properties from Fort Dodge) supplemented with 5% FCS and a 2% mixture of antibiotics. QM5 cells (Antin and Ordahl, 1991) were grown in M199 medium supplemented with 10% tryptose phosphate medium, 10% FCS and a 2% mixture of antibiotics.

The NDV LaSota strain was obtained from ATCC (ATCC VR-699) and was passaged twice in embryonated eggs. Before starting constructing and cDNA cloning, viruses were plaque purified using three rounds of plaque purification on primary chicken embryonic fibroblasts (CEF). Finally, the virus titer was determined on CEF cells grown in GMAM / EMAM (1: 1) medium containing 5% fetal calf serum and 2% antibiotic mixture, 5% allantoic fluid, 30 mM MgCl.₂, 200 pg / ml DEAE dextran (Sigma) and 0.8% Nobel agar (Difco). The virus from the third round of plaque purification (designated clone E13-1) was grown in embryonic eggs and the allantoic fluid was harvested four days after inoculation and stored in aliquots at -70 ° C. Recombinant avipox virus fpEFLT7pol (Britton et al., 1996; hereinafter referred to as FPV-T7), producing T7 RNA polymerase, was obtained from Dr. Michael Skinner and grown on QT35 cells.

Viral RNA isolation

All operations were carried out in RNase-free glass or plastic vessels, and all solutions were made up in RNase-free water that had been treated with 1% diethyl ether.

Hydrocarbonic acid (DEPC) and sterilized by autoclaving. Viruses were pelleted from the allantoic fluid by centrifugation at 21,000 rpm for 70 min. in a Beckman SW40 rotor at 4 ° C. The pellet was resuspended in homogenization buffer (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 5 mM EDTA, 0.5% SDS) and treated with Proteinase K (200 pg / ml) for 90 min. at 37 ° C with constant shaking. Lysates were extracted twice in the presence of an equal volume of phenol / chloroform (1: 1) pH 4.5 and once in the presence of an equal volume of chloroform. The viral RNA was precipitated from the aqueous phase by adding 0.1 volumes of 3M NaOAc pH 5.3 and 2.5 volumes of 100% ethanol. The precipitate was pelleted by centrifugation, washed once with 70% ethanol, resuspended in water and stored in aliquots at -70 ° C.

Reverse transcription

Viral RNA (1.5 µg) was mixed with 500 ng of primer in a volume of 12 µl and incubated for 10 min. at 70 ° C. 4 pl of 5x RT buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCL2, GibcoBRL / Life Technologies) were added 2 pl 0.1 M DTT and 2 pl 10 mM dNTP (2.5 mM each each) and the mixture was incubated for 2 min. at 42 ° C. Reverse transcription was performed in a final volume of 20 µl by adding 200 units. reverse transcriptase (Superscript II; GibcoBRL / Life Technologies) and incubation for 60 min. at 42 ° C.

Polymerase chain reaction (PCR)

All PCR reactions used to determine the 3 'and 5' ends of the NDV genome (see below) were performed using Taq DNA polymerase (Perkin Elmer). For the cloning of individual genes from NDV or large subgenomic cDNA molecules, according to the supplier's (Boehringer Mannheim) recommendations, either Pwo correction DNA polymerase or a mixture of Taq and Pwo (Expand High Fidelity Kit or Expand Long Templete Kit) were used. All samples were incubated for 2 min. at 94 ° C before starting the number of PCR cycles indicated. After the indicated number of PCR cycles were completed, the samples were incubated at the elongation temperature for at least 3 times the extension phase of the PCR cycle. The PCR fragments were purified directly using the High Pure PCR Product Purification Kit (Boehringer Mannheim) or after agarose gel electrophoretic separation using the Qiaex II extraction kit (Qiagen) essentially as described by the manufacturer.

Sequence analysis

All sequences were determined using the PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kit (Perkin Elmer). Reaction mixtures (5 µl) were subjected to 25 cycles of linear amplification (10 sec. 94 ° C, 5 sec. 50 ° C, and 4 min. 60 ° C) in a GeneAmp2400 thermocycler. The reaction mixtures were then triturated with ethanol, rinsed with 70% ethanol, resuspended in 15 µl TSR buffer (Perkin Elmer) and heated for 2 min. at 94 ° C before loading into the Applied Biosystems AB310 automated sequencer.

The nucleotide sequences of the primers used to sequence the entire genome of the LaSota strain NDV were either derived from published sequences or from sequences established during this sequencing project. The primers are shown in Table 1.

Cloning and sequencing of the 3 'and 5' ends of the NDV genome of the LaSota strain

The nucleotide sequence of the 3 'and 5' ends of the NDV genome was determined using RACE (rapid amplification of cDNA ends) procedures. The NDV RNA was used in a reverse transcription reaction carried out in a final volume of 20 µl using the p360 primer (5'-GGCGATGTAATCAGCCTAGTGCTT-3 '; nt 14756-14779) which was derived from the published NDV L gene sequence (Yusoff et al., 1987) . Single stranded cDNA (2.5 µl RT mixture) was added to 8 pmol ALG3 anchor primer (5'-CACGAATTCACTATCGATTCTGGATCCTTC-3 ') and ligated overnight at room temperature in 20 µl reaction mixture containing 50 mM Tris-HCl, pH 8.0, 10 mM MgC); 10 pg / ml BSA, 25% PEG, 1 mM HCC, 20 pM ATP and 10 U. T4 RNA ligases (New England Biolabs) as described in Tessier et al. (1986). One pI of the ligation reaction was used as a template in a PCR reaction with primers p375 (5'-CAATGAATTCAAAGGATATTACAGTAACT-3 '; nt 14964-14984) and ALG4 (5'-GAAGGATCCAGAATCGATAG-3'). The last primer is complementary to the ALG3 anchor primer. The PCR reaction conditions (40 cycles) were as follows: 1 min. 94 ° C, 1 min. 55 ° C and 2 min. 72 ° C. The PCR products were purified and cloned into the pBluescriptII-TSK T vector (Ichihara and Kurosawa, 1993). Alternatively, the purified PCR products were treated with the Klenow fragment of DNA polymerase I to blunt ends and cloned into the HincII site of the pGEM4Z plasmid (Promega). Thirteen independent clones (8x pBluescriptII-TSK and 5x pGEM4Z) were sequenced to determine the nucleotide sequence of the 5 'end of the NDV genome of the LaSota strain. The 3 'end nucleotide sequence was determined using two independent methods. In method I, the ALG3 primer was ligated to the 3 'end of the viral RNA using T4 RNA ligase as described in Schutze et al. (1995). Reaction mixture (final volume 10 μΙ contained 2.5 μg RNA from NDV, 100 pmoles ALG3, 1 μΙ 10 × buffer for T4 RNA ligase (500 mM Tris-HCl, pH 7.8, 100 mM MgCl₂, 100 mM DTT, 10 mM ATP), 1 µl RNazine (Promega) and 10 U T4 RNA ligase (New England Biolabs). The mixture was incubated overnight at room temperature and 5 µl of the ligation reaction was used as a template in a reverse transcription reaction using the ALG4 primer. One pI of the RT reaction was used in a PCR reaction using primers ALG4 and p376 (5'-GAGCCTTAAGGAGCTGCTCGTACTGATC-3 '; nt 137-164) which was derived from the published 3' NDV sequence (Ishida et al., 1986). The PCR reaction conditions were as described above for the 5'RACE reaction. In method II, the 3 'and 5' ends of the NDV RNA were ligated together using T4 RNA ligase using the same conditions as described above in method I. Five µl of the ligation mixture was used as template in a reverse transcription reaction using the p360 primer. One pl of the RT reaction was used in a PCR reaction using the primers p375 and p376 and the PCR conditions described above for the 5'RACE reaction. The PCR products were treated with the Klenow fragment of polymerase I to blunt ends and cloned into the HincII site of pGEM4Z (Promega). Ten independent clones (4 obtained by method I and 6 by method II) were sequenced to determine the nucleotide sequence of the 3 'end of the NDV genome of the LaSota strain.

Construction of a transcription vector

Low-copy transcription vectors were constructed using plasmid pOK12 (Vieira and Messing, 1991) as the primary replicon. Plasmid pOK12 was digested with PvuII and a DNA fragment containing the origin of replication and the kanamycin resistance gene was isolated. The DNA fragment was ligated to the Eco47III-AflII fragment (the AfIII site was blunted using the Klenow fragment of polymerase I) from the transcription vector 2.0 (gift from Dr. Andrew Ball; Pattnaik et al., 1992). The XbaI-NheI fragment was deleted from the generated plasmid in order to eliminate as many unique restriction sites as possible. The plasmid produced was designated pOLTV5 (Fig. 1). The pOLTV5 transcription vector contains the T7 phage DNA dependent RNA polymerase promoter followed by the unique StuI and Smal restriction sites, the autocatalytic ribozyme from the delta hepatitis virus (HDV) and the transcription termination signal from the bacteriophage T7. DNA fragments cloned between the StuI and Smal sites can be transcribed both in vitro and in vivo using T7 RNA polymerase. After transcription, the 5 'end of the generated transcripts contain the two G residues encoded by the plasmid. Due to the autocatalytic activity of the HDV ribozyme, the 3 'end of the transcripts corresponds exactly to the terminal nucleotide of the cloned DNA fragment (Pattnaik et al., 1992).

Construction of minigenomic plasmids

To test the replication and transcription requirements of the NDV virus, minigenomic plasmids were constructed that contained the 3 'and 5' end regions of NDV flanking a reporter gene that replaced all genes from NDV (Fig. 2). DNA fragments corresponding to the 3 'and 5' end regions of NDV were generated by PCR using Pwo DNA polymerase (30 cycles: 15 sec. 94 ° C, 30 sec. 50 ° C and 30 sec. 72 ° C) and using plasmids containing 3 'and 5' RACE fragments as templates (see above).

The 3 'region (nt 1-119) was generated with primers 3UIT (5'-ACCAAACAGAGAGAATCCGTGAGTTACGA-3'; nt 1-27) and SEAP3 (5'-ATCGATACTGGTCAGCATGCTGGCAGAAGGCTTTCTCG-3 '; nt 102-119) Region 5' (14973) -15186) was generated with the primers SEAP5 (5'-GCATGCTGACCAGTATCGATATTACAGTAACTGTGACT-3 '; NT 14973-14990) and 5NDV (5'-ACCAAACAAAGATTTGGTGAATGACGA-3'; nt 15158-15186). The two DNA fragments were joined by "overlapping PCR (overlapping sequence is in italics in the primer sequences shown above) using the primers 3UIT and 5NDV. The generated DNA fragment being a 3 'and 5' fusion of NDVs separated by 20 nucleotides was phosphorylated by the action of T4 polynucleotide kinase and cloned in both orientations in the pOLTV5 transcription plasmid (Fig. 1) that was cut with StuI and Smal and dephosphorylated with calf intestinal phosphatase (Boehringer Mannheim). Finally, the SEAP gene (encoding the secreted alkaline phosphatase) was recovered from the pSEAP-Basic plasmid (Clontech) by digestion with Sphl and ClaI and cloning between the Sphl and ClaI sites between the 3 'and 5' ends of NDV. The resulting plasmids were designated pOLTV535 and pOLTV553, respectively. In vivo and in vitro transcription using T7 RNA polymerase from pOLTV535 resulted in antigenomic RNA ([+] - RNA), while transcription from pOLTV553 resulted in genomic RNA ([-] - RNA).

The plasmids pOLTV535N0 through -N5 and pOLTV553N0 through -N5 were generated by inserting complementary oligonucleotides into a ClaI site located between the SEAP gene and the 5 'end of NDV in pOLTV535 and pOLTV553, respectively (see Fig. 2). The following oligo16 were used

Nucleotide N0,5'-CGCGAGCTCG-3 '; N1,5'-GCGGAGSCTCG-3 '; N2,5'-CGCGAGCGCTCG-3 '; N3,5'-CGCGAGCWGCTCG-3 '; N4,5'-CGCGAGCATGCTCG-3 '; N5,5'-CGCGAGCWGCTCG-3 '; N4,5'-CGCGAGCATGCTCG-3 '; N5,5'-CGCGAGCASTGCTCG-3 '(W = A or T; S = C or G).

Modification of the T7 promoter in plasmids pOLTV535 and pOLTV553

To generate in vivo and in vitro transcripts containing true 5 'and 3' ends from NDV, the T7 promoter in plasmids pOLTV535 and pOLTV553 was modified so that transcription could start at the first nucleotide of the 3 'and 5' ends of NDV.

Primers were designed that contained, 1) a Bgl1 restriction site, 2) a T7 promoter sequence (shown in italics) that was modified such that the G residues at the end of the T7 promoter were replaced with an A residue and the -3 'end (nt 1-21), or 5 '(nt 15164-15186) from NDV. The primers BGL3F2 (5'-GATATGGCCATTCAGGCTTAATACGACTCACTAACCAAACAGAGAATCCGTGAG-3 ') and SEAP3 (see above) were used to generate a DNA fragment containing the modified T7 promoter and the entire 3' end from NDV to the start site of the SEAP gene in pOLTV535. Similarly, a DNA fragment containing the modified T7 promoter and the entire 5 'end from NDV to the SEAP gene end site in pOLTV553 was produced using primers BGL5F2 (5'-GATATGGCCATTCAGGATTAATACGACTCACTATAACCAAACAAAGATTTGGTGAATG-3') and SEAP5. The generated fragments were digested with Bgll and SphI (3 'end) or Bgll and ClaI (5' end), respectively, and used to replace the Bgll-Sph1 fragment in pOLTV535 or the Bgll-Clal fragment in pOLTV553. The resulting plasmids were designated pOLTV735 and pOLTV753, respectively. The plasmids pOLTV735N3 and pOLTV753N3 were generated by introducing a complementary oligonucleotide (5'-CGCGAGCWGCTCG-3 '; W = A or T) in the ClaI site located between the SEAP gene and the 5' end of NDV in pOLTV735 and pOLTV753, respectively.

Construction of plasmids with a SEAP reporter

The plasmid pCIneoSEAP was constructed by cloning the XhoI-ClaI fragment (the Clal site was blown out with the Klenow fragment of polymerase I) containing the SEAP gene from the pSEAP-Basic plasmid (Clontech) between the Xhol and Smal sites of the eukaryotic expression vector pCIneo (Promega). The latter plasmid contains the human cytomegalovirus (hCMV) promoter in addition to that of the bacteriophage T7. To test and quantify SEAP expression through transcripts produced only from the T7 promoter, another plasmid was constructed, one that lacked the hCMV promoter. For this, the hCMV promoter was removed from pCIneo by partial digestion with HindII followed by complete digestion with BglII. The DNA fragment (nt 756-5469) according to Clontech numbering) from which the hCMV promoter had been removed was isolated, treated with T4 DNA polymerase to produce blunt ends, and re-circled with T4 DNA ligase. The resulting plasmid was designated pCIneoD. Finally, the SEAP gene was recovered from pSEAP-Basic as a MluI-AccI fragment and cloned into pCIneoD between the Mlul and Body sites. The resulting plasmid was designated as pCIneoD SEAP.

Transfections

Cells were placed in 24-well culture dishes, grown overnight to 60-80% confluency, and infected with FPV-T7 at moi 1 for 1 hr. at 37 ° C. Cells were transfected with 0.5 µg minigenomic plasmid DNA with 3 µL LipofectAMINE and OptiMem, essentially as described by the supplier (GibcoBRL / Life Technoligies). After incubation for 4 hours. (CER cells) or 16 h. (QM5 cells) at 37 ° C cells were either infected with NDV (Dutch virulent isolate No. 152608; 200 µl per well) for 1 h. at my 5 or left uninfected. The inoculum was aspirated and 1 ml of complete medium was replaced and the cells were further incubated at 37 ° C.

For cotransfection, cells were grown in 6-well culture dishes and infected with FPV-T7 as described above. Cells were co-transfected with 0.25 µg minigenomic plasmid DNA, 0.4 µg pCIneoNP, 0.2 µg pCIneoP and 0.2 µg pCIneoL (c) or pCIneo by adding 8 µl LipofectAMINE or 9 µl FuGene6 (Boehringer Mannheim). To generate virus, the minigene plasmid was exchanged with a transcription plasmid containing the full length NDV cDNA.

Quantification of SEAP activity

The amount of SEAP that was secreted into the medium with transfected cells was measured in 96-well disposable plates using the Phospha-Light Chemiluminescent Reporter Assay for Secreted Alkaline Phosphatase kit essentially as described by the manufacturer (Tropix). Chemiluminescence was determined using a fluid scintillation counter (Wallac 1450 microbeta PLUS).

Cloning and sequencing of cDNA molecules covering the entire genome of the NDV LaSota strain

For cloning and sequencing of the entire NDV genome of the LaSota strain, clones with large subgenomic cDNAs were generated by RT-PCR and pGEM-T cloning. First strand cDNA synthesis was performed using the 3UIT primer as described above and 1 µΙ of the RT reaction was used in the PCR reaction using the Expand Long Template PCR kit (Boehringer Mannheim). The PCR reaction consisted of 5 cycles with the profile: 10 sec. 94 ° C, 30 sec. 58 ° C and 6 min. 68 ° C followed by 10 cycles with a 10 sec profile. 94 ° C, 30 sec. 58 ° C and 6 min. 68 ° C, in which the elongation time at 68 ° C increased by 20 sec. per cycle. The PCR fragments were cloned into pGEM-T using the pGEM-T cloning kit essentially as described by the manufacturer (Promega). The ligation mixture was transformed with E.coli strain SURE II (Stratagee). Two independent RT-PCR reactions (A and B) were performed and each yielded a similar set of cDNA clones. The nucleotide sequences of the subgenomic cDNA clones were determined using NDV-specific primers (Table 1) and primers flanking the insert. After comparing the nucleotide sequence of clones from series A and B, remaining ambiguities were resolved by sequencing the corresponding regions from the third independent series of cDNA molecules (series C). The NV nucleotide sequence of the LaSota strain is shown in Figure 3. Construction of a full genome length cDNA clone from NDV

The full length NDV cDNA was integrated into the pOLTV5 transcription plasmid using pOLTV535 as the starting plasmid. The DNA fragments were overlapped using commercially available restriction enzymes as detailed in Fig. 4B. In a series of cloning steps, a plasmid (designated p535-DI) was constructed which contained nucleotides 1-3521 and 12355-15186 separated by a Clal site generated by joining the Clal sites at position 3521 and 12355. In another series of cloning steps, a plasmid (designated pGEM-B) was generated that contains a portion of the NDV genome spanning nucleotides 3521-12355 (Clal fragment). For cloning convenience, the last Clal fragment was tagged with the chloramphenicol (Cm) resistance gene from plasmid pACYC184 (Chang and Cohen, 1978). To this end, the Cm gene was recovered from pACYC184 by PCR using the primers CAT-F (5'-GCGTACGTCTAGACTGGTGTCCCTGTTGATACCGG-3 ') and CAT-R (5'-GCTCTAGACGTACGACCCTGCCCTGAACCGACG-3'). The PCR reaction was carried out with the use of Pwo DNA polymerase in 30 cycles with the profile: 30 sec. 94 ° C, 45 sec. 60 ° C and 60 sec. 72 ° C. The resulting DNA fragments were digested with BsiWI and cloned into the unique BsiWI site of pGEM-B, generating pGEM-B (CAT). The ClaI fragment from pGEM-B (CAT) was cloned into the unique Clal site of p535-DI, generating pNDFL (CAT). Finally, the CM gene was removed from the plasmid by digestion with BsiWI, religion and transformation of E.coli strain DH5a. The resulting plasmid, designated pNDFL +, contains the entire NDV cDNA sequence cloned between the T7 promoter and the HDV ribozyme in the pOLTV5 transcription plasmid.

Cloning and expression of individual genes from NDV

DNA fragments containing individual genes from the NDV LaSota strain were generated by RT-PCR and cloned into pCIneo. After cloning, all fragments were sequenced using primers flanking the inserts and gene specific primers.

NP gene: primer 386 (5'-GAGCAATCGAAGTCGTACGGGTAGAAGGTG-3 '; nt 40-69) was used for reverse transcription. Primers 365 (5'-GTGTGAATTCCGAGTGCGAGCCCGAAG-3 '; nt 77-94) and 892 (5'-TTGCATGCCTGCAGGTCAGTACCCCCAGTC-3'; nt 1577-1593) were used for the PCR reaction with Pwo DNA polymerase. The following PCR profile was used (30 cycles): 30 sec. 95 ° C, 40 sec. 65 ° C and 45 sec. 72 ° C. The resulting DNA fragment was digested with EcoRI and cloned in pCIneo between the EcoRI and Smal sites. Expression of NP was confirmed by peroxidase immunoassay (IPMA) as described by Peeters et al. (1992) using monoclonal antibody 38 (Russell et al., 1983).

P gene: the primer pRT1 (5'-CAAAGAATTCAGAAAAAAGTACGGGTAGAA-3 '; nt 1794-1814) was used for reverse transcription. The pRT1 and p2 primers (5'-GCAGTCTAGATTAGCCATTCACTGCAAGGCGC-3 '; nt 3053-3071) were used for the PCR reaction with Pwo DNA polymerase. The following PCR profile was used (30 cycles): 30 sec. 95 ° C, 40 sec. 65 ° C and 60 sec. 72 ° C. The resulting DNA fragment was digested with EcoRI and Xbal and cloned into pCIneo between the EcoRI and Xbal sites. Expression of P was confirmed by IPMA test using monoclonal antibody 688 (Russell et al., 1983).

M gene: for reverse transcription the primer 3UIT (5'-ACCAAACAGAGAATCCGTGAGTTACGA-3 '; nt 1-27) was used. NDV5M primers (5'GGGTGCTAGCGGAGTGCCCCAATTGTGCCAA-3 '; nt 3268-3288) and NDV3M (5'-TCTCCCCGGGGCAGCTTATTTCTTAAAAGGAT-3'; nt 4368-4389 kit) were used for the PCR reaction. The PCR reaction consisted of 10 cycles with the profile: 15 sec. 95 ° C, 30 sec. 55 ° C and 2 min. 68 ° C followed by 15 cycles where the elongation time at 68 ° C was increased by 20 sec. per cycle. The resulting DNA fragment was treated with T4 DNA polymerase to make blunt ends, digested with NheI and cloned

PL 197 722 B1 in pCIneo between the NheI and Smal sites. Expression of the M protein was confirmed by IPMA using the monoclonal antibody 424 (Russell et al., 1983).

F gene: the 3UIT primer was used for reverse transcription (see above). For the PCR reaction, the primers NDV5F (5'-ACGGGCTAGCGATTCTGGATCCCGGTTGG-3 '; nt 4508-4526) and NDV3F (5'-ACTACCCGGGAAACCTTCGTTCCTCAT-3'; nt 6212-31) were used and the High Fidelity kit described above was carried out using the kit Expand conditions described above. for the M gene. The resulting DNA fragment was treated with T4 DNA polymerase to generate blunt ends, digested with NheI and cloned in pCIneo between the NheI and Smal sites. F protein expression was confirmed by the IPMA assay using the monoclonal antibody 8E12A8C3 (ID-DLO Department of Avian Virology).

HN gene: 3UIT primer was used for reverse transcription. For the PCR reaction, primers NDV5HN (5'-GTAGGCTAGCAAGAGAGGCCGCCCCTCAAT-3 '; nt 6335-6354) and NDV3HN (5'-CGAGCCCGGGCCGGCATTCGGTTTGATTCTTG-3'; nt Fidelity 8205-8227 was performed using the kit conditions described above) were used for the PCR reaction. for the M. The resulting DNA fragment was treated with T4 DNA polymerase to generate blunt ends and after digestion with Xmal, cloned into pClneo between the blunted (Klenow polymerase I fragment) NheI site and the Xmal site. HN protein expression was confirmed by IPMA using the monoclonal antibody 86 (Russell et al., 1983).

L gene: The L gene was recovered from the pGEM-L7a cDNA clone (Fig. 4A) by digestion with SacII and SalI. Prior to SalI digestion, the SacII site was blunted by treatment with T4 DNA polymerase. The resulting fragment was cloned in pCineo between the extirpated (Klenow fragment of polymerase I) NheI site and the Sal site. The 5 'untranslated region between the T7 promoter and the ATG start codon of the L gene contained out-of-phase ATG codons, which could interfere with normal L protein production. Thus, a new plasmid was constructed in which the first ATG codon was dropped out and in which the second ATG codon was changed to AAG by PCR mutagenesis. The following primers 5LE (E) (5'-CAATGGAATTCAAGGCAAAACAGCTCAAGGTAAATAATACGGG-3 '; nt 8332-8374) and 3LE (B) (5'-GTGAATCGATCCATGTCCGA; nt 8847-8870). The PCR reaction was performed using Pwo DNA polymerase and using the following. The following PCR profile was used (30 cycles): 30 sec. 94 ° C, 45 sec. 60 ° C and 60 sec. 72 ° C. The resulting DNA fragment was digested with EcoRI and Xbal and cloned into pCIneo between the EcoRI and Xbal sites to generate the plasmid pCIneoL (N). The BsiWI-SalI fragment from pGEM-L7a containing the remainder of the L gene (nt 8852-15046) was then cloned into pCIneo between the BsiWI and SalI sites, generating the plasmid pCIneoL (c). As no antibodies to L protein were available, L protein production could not be checked by immunochemical techniques.

Introduction of a genetic marker into the F gene

To show unequivocally that an infectious virus could be generated from a full-length cDNA clone, a genetic tag was introduced into the F gene by PCT-mediated mutagenesis. For this purpose, the F gene was cloned using two overlapping PCR fragments. The first PCR fragment was generated using the NDV5F primers (see above) and the F5R primer (5'-AAAGCGCCGCTGTCTCCTCCCTCCAGATGTAGTCAC-3 '; nt 4859-4894).

The residues in bold are changes made to the primer to change the amino acid sequence of the proteolytic cleavage site between F1 and F2 from the NDV LaSota strain (GGRQGR | L) to the consensus cleavage site from the virulent form of the NDV strains (GRRQRR | F). A second PCR fragment was generated using primers F3F (5'-GGAGGAGACAGCGGCGCTTTATATAGGCGCCATTATTGG-3 '; nt 4875-4911) and IV09 (5'-CTCTGTCGACACAGACTACCAGAACTTTCAC-3'; nt 6246-6266). The PCR reaction was performed with Pwo DNA polymerase and 25 cycles with the following profile were used: 15 sec. 94 ° C, 30 sec. 55 ° C and 2 min. 72 ° C. Two overlapping PCR fragments (overlapping sequence is in italics in the primer sequences) were ligated using a second PCR reaction with primers NDV5F and IV09 under the same PCR conditions. The resulting fragment, which contains the entire ORF for the F gene encoding the virulent consensus cleavage site, was digested with NheI and Sall and cloned in pICneo between the NheI and Salh sites of the^andc ^pCIneo^Tue. ^F.ra^gmen^vol ^ u ^ oh (n^{t 4646}-⁴⁹⁵²) with ^pCIneoⁱⁿ at^ryeabout ^dthat^ymfon^y the corresponding fragment from plasmid p535-S, which was constructed by inserting the Clal-Scal fragment (nt 3521-10311) of pGEM-B in p535-DI between the ClaI and Scal sites (see Fig. 4C). Manufactured^y pdazmfo is marked as ^p535-^S. [^Fwtc]. ^F.ra^gmen^{t PCR} insist^andc^ygen o^porno^sc^and the cMoramphenicol (Cm) from pACYC184 (see above) was cloned as an XbaI fragment at a unique site ^Xband^l (^poz^ytation ⁶¹⁷² in se^kwencja^{and NDV}) with pdazmfou ^p535-^S.[^Fwtc] ^volsir^andc pdazmfo ^p535-^S. [^Fwtc] Cm.

PL 197 722 B1

Then a Cm-labeled Apal-Spel fragment (nt 2285-8094) from this plasmid was used for replacement with the corresponding fragment of the full-length cDNA clone with pNDFL +. Finally, the Cm gene was removed from this plasmid by digestion with XbaI, after which the plasmid was resealed using T4 ligase. The resulting plasmid containing the genetically tagged full-length NDV cDNA was designated as I^kabout ^pNDFL+[^Fwt].

Generation of stably transformed cell lines expressing individual NDV genes

To generate stably transformed cell lines expressing the individual ^biand^Lukeau^ryeabout ^plazm^goin pCtaeoNP, ^pCIneoP ^pClneoM^, piCIneoR ^pClneo^Fwt and ^pClneoH^N. Journal^ande^ñPrior to transfection, cells were plated in 6 cm dishes and incubated overnight until 60-80% confluence. Cells were transfected with 2 µg of plasmid DNA with 12 µL LipofectAmine and OptiMem according to the manufacturer's recommendations (GibcoBRL / Life Technologies). After 48 hours, cells were trypsinized and plated by dilution in 10 cm culture dishes in medium containing 500 pg / ml G418 (Boehringer Mannheim). Every three days, the medium was replaced with fresh medium containing increasing amounts (in steps of 100 µg / ml) of G418 until the concentration of 800 µg / ml was reached. Cells were grown in medium containing 800 µg / ml G418, and three weeks after transfection, single colonies were isolated and transferred to a 96-well culture dish. The cloned cell lines were tested for expression of the corresponding NDV gene using the IPMA assay as described above for the transient expression studies.

Cell lines constitutively producing NP, P, M or F could be identified and isolated. However, we have not been able to generate a cell line that expresses the HN protein. Perhaps constitutive expression of HN is toxic to cells.

Generation of stably transformed cell lines expressing T7 polymerase

The gene encoding T7 RNA polymerase was obtained from plasmid pRT7NT (Rene van Gennip, ID-DLO, Department of Mammalian Virology) by digestion with EcoRI and SalI. The resulting fragment contains the T7 RNA polymerase gene placed under the baculovirus p10 promoter. The fragment was cloned into plasmid pCIneo between the EcoRI and SalI cleavage sites, creating plasmid pCIneo107. Plasmid pCIneo0 lacks the T7 promoter and was obtained from pCIneo by NheI cleavage followed by partial Scal cleavage, filling the sticky ends with a Klenow fragment of DNA polymerase, and circularization using T4 DNA ligase. Baculovirus sequences were removed from pCIneo107 by digestion with EcoRI and PacI followed by blunt end formation and recirculation with T4 DNA polymerase. The resulting fragment was designated pCIneo007. Expression of T7 RNA polymerase was checked by co-transfecting cells with pCIneo007 and pRh01. The latter plasmid encodes the E2 protein of the common swine fever virus cloned downstream of the T7 promoter and having an internal ribosome binding site (Rene van Gennip, oral inf.). Expression of E2 was determined by IPMA using monoclonal antibody V4 (Wensvoort et al., 1986). Stably transformed cell lines expressing T7 RNA polymerase were generated and isolated as described above, except that 10 cm culture dishes were used and cells were transfected with 5 µg pCIneo007 DNA and 25 µl LipofectAmine. To check individual cell lines for expression of T7 RNA polymerase, they were transfected with plasmid pRh01, and E2 expression (which is independent of T7 RNA polymerase) was determined by IPMA using monoclonal antibody V4. Several lines expressing T7 RNA polymerase have been identified. One of them, designated CER-C9, was used for subsequent experiments.

Cloning and expression of HN genes and HN hybrid genes

The avian paramyxovirus serotypes 2 and 4 (APMV2 and APMV4) were used for the synthesis of single-stranded NDV cDNA as described above. All subsequent PCR reactions were performed using 25 cycles of 15s at 94 ° C, 30s at 55 ° C and 2 min at 72 ° C. The entire coding region of the APMV2 HN gene was obtained by PCR using primers IV03 (5'-GGGGGAATTCCCCATTCAATGAAGGGTCAC-3 ') and IV05 (5'-GATCCCCGGGTCTTAAACCAGGCTTCGCAATG-3') obtained from the sequence of the gene HN APMV2 1403030 . The entire coding region of the APMV4 HN gene was obtained by PCR using primers IV06 (5'-GGGGGAATTCTGGTAGGGTGGGGAAGGTAGC-3 ') and IV08 (5'-ATTGCCCGGGGGGTAACTAATCAGGATCTCAG-3') obtained from the APMV4 gene database (HNBank31 gene number) . The resulting fragments were digested (either directly or after subcloning into pGEM-T) with EcoRI and Xmal and then cloned into the pCIneo between the EcoRI and Xmal cleavage sites. The obtained plasmids were designated as pCIneoHN2 and pCIneoHN4, respectively.

Hybrids of the HN genes of the LaSota strain NDV and the HN genes of APMV2 and APMV4 were obtained by overlapping PCR reactions as follows. The N-terminal part (amino acids 1-141) of the HN gene strain 20

NDV LaSota was amplified with Pwo DNA polymerase using primers IV01B (5'-GTAGGAATTCAAGAGAGGCCGCCCCTCAAT-3 '; nucleotides 6325-6354) and IV10 (5'-AATGAGTTCTTTGCCTATCCCCCC-3'; nucleotides 6811-6834). The C-terminal part of the APMV2 HN gene (amino acids 142-580) was amplified with Pwo DNA polymerase using primers IV11B (5'-GGGGGGATAGGCAAAGAACTCATTCAAGGAGATGCATCTGCAGGC-3 ') and IV05.

The resulting fragments were linked by overlapping PCR (overlapping fragments are in italics) using primers IV01B and IV05 and the Expand High Fidelity enzyme mixture. The resulting PCR fragments were digested (either directly or after subcloning in pGEM-T) with EcoRI and Xmal and then cloned into pCIneo between the EcoRI and Xmal cleavage sites. Obtained plasmid containing the hybrid HN gene consisting of amino acids 1-141 of NDV and amino acids 142-580^APMV2 marked ^pCIneo^{HN1 / 2141}.

The C-terminal part of the APMV4 HN gene (amino acids 143-569) was amplified using primers IV14B (5'-GGGGGGATAGGCAAAGAACTCATTGTAGATGATGCATCTGCAGGCCTAAATTTCC-3 ') and IV08. This fragment was ligated to the N-terminal part of the NDV HN gene (see above) by overlay PCR using primers IV01B and IV08. The resulting PCR fragments were digested (either directly or after subcloning into pGEM-T) with EcoRI and Xmal, and then cloned into pCIneo between the EcoRI and Xmal cleavage sites. The obtained plasmid containing the hybrid HN gene consisting of NDV amino acids 1-141 and APMV4 amino acids 143-569 was designated pCIneoHN1 / 4.

Analogously to the constructs described above, HN hybrid genes consisting of amino acids 1-143 of NDV and 144-580 APMV2 or 1-143 of NDV and 145-569 APMV4 were generated. These constructs were made using the following primer pairs; NDV amino acids 1-143, primers IV01B and IV13 (5'-ATCTACAATGAGTTCTTTGCCTATC-3 ') NT 66816-6840); APMV4 amino acids 144-580, primer IV14b (5'-GGGGGGATAGGCAAAGAACTCATTGTAGATGATGCATCTGCAGGCCTAAATTTCC-3 ') and IV05; APMV4 amino acids 145-569, primer IV15b (5'-GGGGGGATAGGCAAAGAACTCATTGTAGATCAAACAGCTGACTAGAGAGCAG-3 ') and IV08. The PCR fragments were digested (either directly or after subcloning in pGEM-T) with EcoRI and Xmal, and then cloned into pCIneo

143 between EcoRI and Xmal cleavage sites. The obtained plasmids were named pCIneoHN1 / 2 and, respectively^pCIneoHN1 / 4¹⁴³. In ce^lu grain e^ks^presj^{and bi}ate^{k HN} tomórld C^{ER l}at^b Q^M5 infected ^PPV-T7 for 1 hour at mo 1, transfected with plasmids pCIneoHN, pCIneoHN2, pCIneoHN4, pCIneoHN1 / 2¹⁴¹, pCIneoHN1 / 2143, pCIneoHN1 / 4141 and pCIneoHN1 / 4143; 24 hours after transfection, monolayer cultures were covered with a 1% suspension of chicken erythrocytes in PBS and left for 45 minutes at room temperature. Subsequently, the cultures were washed three times with PBS and the erythrocyte adhesion to the transfected cells was examined microscopically. To test induction of coex cell fusion^presj^{and bi}and^Lukeand ^{HN and} F tomórld C^{ER and} Q^M5 co-infected ^pCIneo^{Fwt and pCI}neo^{HN or pCI}neo^HN2,pCIneoHN4, pCIneoHN1 / 2¹⁴¹, pCIneoHN1 / 2^M.3, pCIneoHN1 / 4141 or pCIneoHN1 / 4ⁱⁿ After incubation for 2 to 3 days, monolayer cultures were washed with PBS, stained with Giemza dye (diluted 1:30 in water) and examined microscopically.

Cloning of HN hybrid genes from full-length NDV genomic cDNA

A synthetic linker named HN12 was placed between the Not1 and Spel cleavage sites in the pGEM-T vector (Promega) using oligonucleotides HN12a (5'-GGCCGCATATTCTAGAGTTAACGACTTA-3 ') and HN12b (5'-CTAGTAAGTCGTAACTCTAGAATATGC-3'). A synthetic linker named HN14 was placed between the Not1 and Spel cleavage sites in the pGEM-T vector with oligonucleotides HN14a (5'-GGCCGCATATTCTAGAGTTAACGA-3 ') and HN14b (5'-CTAGTCGTTAACTCTAGAA-TATGC-3'). The resulting plasmids were named pGEM-HN12 and pGEM-HN14, respectively. These plasmids, after digestion with Not1 and Xbal, were used to clone the NotI-Spel fragment (nucleotides 33907488) from ^plasmid ^p535-S [Fⁱⁿc] Cm. ^P.shatter ^plazm^idy was called o ^ owterno ^pGEM-^{HN1 / 2NS}and pGEM-HN1 / 4NS. HN genes in these plasmids were replaced with hybrid genes from plasmids pCIneoHN1 / 2143 and pCIneoHN1 / 4, respectivelyⁱⁿ (see section Cloning and expression of HN genes and h^ybr^ydow^ych HN). In vol^ym purpose ^pCIneoHN1 / 2^{143 and pCI}neo^{HN1 / 4143} frawtono ^Nhe^{and S}has^Juloath^Łe ^fra^gmen^you (stop by^andce ^hybridry^dthose ^gen^{y HN1 / 2143 and HN1 / 4143}) have been made ^pohm^iaplot^y mtejsca Nhel and Hpal of the pGEM-HN1 / 2NS and pGEM-HN1 / 4NS plasmids, giving the pGEM + NH12 and pGEM + NH14 plasmids, respectively. The last plasmid was used to insert the HN hybrid genes into the full-length NDV genomic cDNA clone. For this, the plasmids pGEM + NH12 and pGEM + NH14 were digested with NotI and Spel and the segment containing the HN12 or HN14 gene was used to replace the corresponding^fra^gmen^volu pND^FL+, ^day^andc respectively ^pNDFL+ ^HN1/2¹⁴³Cm ^{and pNDFL}+^{HN1 / 4143} Cm. ^G.en Cm delete^iathis from plasmids by digestion with XbaI followed by recirculation using T4 DNA ligase. To

To fulfill the six rule, the linker was incorporated using the unique Spel site of these plasmids using self-complementary oligonucleotides. Linker H2 (5'-CTAGCGAGCGCTCG-3 ') was placed in^plasmid ^pNDFL + HN1 / 2¹⁴³ Cm and Hnter ^H3 (⁵'-^CTAGCGAGIN^GCTCG-³¹) in prtazmktóe pNDFL + HN1 / 4¹⁴³ Cm, creating plasmids pNDFL + HN1 / 21, respectively⁴³(H2) and pNDFL + HN1 / 4^M.3 (H3).

Deletion of a specific epitope in the NDV LaSota HN protein

The specific epitope, i.e. amino acids 346-354 (PDEQDYQIR) of the LaSota NDV HN protein, recognized by the monoclonal antibody 4D6 (Long et al., 1986; Meulemans et al. 1986) was removed by replacing this sequence with the appropriate sequence of the HN or APMV-2 proteins (NRTDIQQTI) or APMV-4 (PDPLQDQIL). For this purpose, plasmid pCIneoHN (see section: Cloning and expression of individual NDV genes) was used as a template to create overlapping PCR products. For APMV-2, the first fragment was generated with primers IV01 (5'-GTAGACGCGTAAGAGAGGCCGCCCCTCAAT-3 ') and 3HN2 (5'-GATAGTTTGCTGTATATCAGTCCCGATTGCATGTGTCATTGTATCGCTTGTATATATATCAC-3'). The second PCR reaction was performed with the primers 5HN2 (5'-AATCGGACTGATATACAGCAAACTATCATGGCCAAGTCTTCGTATAAGCCTGGAGCC-3 ') and NDV3-HN (5'-CGAGCCCGGGCCGGCATTCGGTTTGATTCTTG-3').

The resulting fragments were pooled and used as template for the third PCR with primers IV01B (5'-GTAGGAATTCAAGAGAGGCCGCCCCTCAAT-3 ') and NDV3-HN. For the APMY-4 sequence, the first PCR fragment was generated with primers IV01 and 3HN4 (5'-TAAGATCTGATCTTGCAGCGGGTCAGGGCATGTGTCATTGTATCGCTTGTATATCAC-3 '). A second PCR was performed with primers 5HN4 and NDV3-HN. The resulting fragments were combined and used as template for the third PCR with primers IV01B and NDV3-HN. The primer 3HN2 / 5HN2 and 3HN4 / 5HN4 are partially complementary and encode the APMY-2 (NRTDIQQTI) or APMV-4 (PDPLQDQIL) sequences, respectively. PCR reactions were performed using the Expand Long Template kit (Boehringer Mannheim) and consisted of 30 cycles of 10 sec. 94 ° C, 30 sec. 58 ° C and 2 min. 68 ° C, followed by one cycle of 68 ° C. The PCR products were digested with EcoNI and Bsu36I and cloned between the EcoNI and Bsu36I sites of the pCIneoHN plasmid. The resulting plasmids were named pCIneoHN1 (HN2e) and pCIneoHN1 (HN4e), respectively. The transient expression study showed that the altered proteins are properly expressed and transported to the cell surface as shown by the haemoadsorption study using chicken erythrocytes.

Moreover, the monoclonal antibody 6D4, directed against the linear HN epitope of the NDV virus and consisting of (or at least including amino acids 346-354) does not interact with the altered HN proteins.

The plasmids pCIneoHN1 (HN2e) and pCIneoHN1 (HN4e) were digested with NarI and Spel, and fragments containing the altered HN genes were cloned between the NarI and Spel sites of pGEM-HN1 / 2NS and pGEMHN1 / 4NS, respectively. The resulting plasmids were named pNDFL-HN (HN2e) Cm and pNDFL-HN (HN4e) Cm, respectively. The Cm gene was removed therefrom by Xbal cleavage and ligation. The generated plasmids were named pNDFL-HN (HN2e) and pNDFL-HN (HN4e), respectively.

Results

Nucleotide sequence of the 3 'and 5' ends of the genome of the NDV LaSota strain.

A sequence of the probable 3 'end of the NDV genome has been published (Ishida et al., 1986) for a different NDV strain (D26) than that used here (LaSota). Yusoff et al. (1987) published the sequence of the L gene and a relatively large non-coding segment downstream of the L gene of the NDV Beaudette C strain. However, as the present work shows, this sequence does not completely cover the 5 'end of the viral genome, preventing the generation of an infectious copy of the virus. The 3 'and 5' ends of the genome of RNA viruses bearing negative strand RNA play a key role in replication and transcription (Lamb and Kolakofsky, 1996). Thus, to obtain a full-length NDV cDNA that can be used to generate infectious viruses by reverse genetics (Conzelmann, 1996), it is necessary to obtain the correct 3 'and 5' ends of the viral genome. Thus, we determined the exact nucleotide sequence of the RNA genome of the LaSota strain by the 3 'and 5' RACE method (rapid amplification of cDNA ends). The 5 'end was obtained by PCR after ligation of the single-stranded anchor primer (ALG3) into the single-stranded cDNA obtained by reverse transcribing the 5' end of genomic RNA. Using primers complementary to the anchored primer (ALG4) and specific for NDV, a PCR product containing the 5 'end was obtained.

To clone the 3 'end of NDV, the single-stranded ALG3-anchored primer was ligated to the 3' end of the viral RNA with T4 RNA ligase and amplified by PCR using the ALG4 and NDV-specific primer (method I). Alternatively, the 3 'and 5' ends of the NDV RNA were ligated together with T4 RNA ligase and the resulting concatamer RNA was used as a template for RT-PCR using the

Using NDV-specific primers flanking the ligation site (method II). The 3 'and 5' Race products were cloned into the T vector pBluescriptII-TSK (Ichikira and Kurosawa, 1993) or into pGEM4Z, and many independent clones were obtained and sequenced. The results are shown in Table 2. To allow for direct comparison of the 3 'and 5' ends, the sequences are shown as DNA and the 3 'end of the genomic strand is shown as the 5' end of the antigenomic strand. On the genomic RNA level, the 3 'end sequence is 3'-UGGUUUGUCUUAG while the 5' end sequence is UUUAGAAACAAACCA-5 '. The 3 'end sequence is almost identical to the published 3' sequence of the NDV D26 strain (Ishida et al., 1986). In contrast, the 5 'end sequence shows that the LaSota NDV strain contains 64 additional nucleotides compared to the published sequence of the L gene of the Beaudette C strain (Yusoff et al., 1987). Fig. 6.

Replication of NDV minigenomes by helper virus

To determine whether the 3 'and 5' ends of NDV function in replication and transcription, minigenomes consisting of the 3 'end of NDV (nucleotides 1-119), a reporter gene encoding secreted alkaline phosphatase (SEAP), and the 5' end of NDV were constructed. (nucleotides 1497315186) (Figure 2). Such genomes were cloned in both orientations into the pOLTV5 transcription vector, generating the plasmids pOLTV535 and pOLTV553, respectively (details on construction see Materials and Methods). The plasmid pOLTV5 (Fig. 1) contains a promoter for T7 RNA polymerase, followed by single restriction sites for StuI and Smal, an autocatalytic ribozyme from hepatitis delta virus (HDV), and a transcription termination signal from bacteriophage T7 (Pattnaik et al., 1992) . In vivo or in vitro transcription of plasmid pOLTV535 with T7 RNA polymerase produces antigenomic RNA (ie [+] - RNA), while transcription of plasmid pOLTV553 results in genomic RNA (ie [-] - RNA). (Fig. 5).

To investigate whether RNA minigenomes produced with the plasmids pOLTV535 and pOLTV553 could be replicated and expressed with NDV as a helper virus, we used CER cells expressing T7 RNA polymerase either constitutively (CER-C9 cells, see Materials and Methods) or after infection recombinant avipox virus FPEFLT7pol (Britton et al., 1995; henceforth referred to as FPV-T7) expressing T7 RNA polymerase. CER-C9 cells and FPV-T7 infected CER cells were transfected with the minigenomic plasmids pOLTV535 or pOLTV553 and after incubation for 3 hours at 37 ° C they were infected with NDV or left uninfected. Approximately 24 hours after infection, a sample of the medium was taken and tested for SEAP activity. The results show that the expression of SEAP in cells infected with FPV-T7 and transfected with pOLTV535 was very high. This is not surprising since transcription by T7 RNA polymerase produces antigenomic [+] - RNA, caped by avipox enzymes and efficiently translated by the host cells. In cells transfected with pOLTV553, transcription by T7 RNA polymerase produces genomic [-] - RNA that must be converted into [+] - RNA by a helper virus to be translated into SEAP protein (Fig. 5). In both cases, no increase in expression was observed in NDV infected cells compared to non-NDV infected cells. In contrast, SEAP expression in NDV infected cells was consistently approximately two-fold lower than in uninfected cells (results not shown). For cells transfected with pOLTV535 this can be explained by the very high initial expression level by the transcript generated by T7 RNA polymerase. However, for cells transfected with pOLTV553, with the efficiency of SEAP expression dependent on the conversion of genomic [-] - RNA to antigenomic [+] - RNA or mRNA by the viral polymerase complex, we would expect an increase in SEAP expression after NDV infection.

We believe that the minigenomes may not be expressed and replicated by NDV for two reasons. First, the RNA size of the minigenomes does not meet the so-called Rules of the six (Calain and Roux, 1993; Kolakofsky et al., 1998). According to this principle, paramyxovirus genomes are replicated efficiently only when their length is a multiple of 6 nucleotides. Second, two additional G residues present at the 5 'end of the RNA minigenomes may interfere with proper replication and / or transcription by the viral polymerase complex. To test whether genomic replication is dependent on the rule of six, we cloned a series of short, auto-complementary oligonucleotides increasing 1 nucleotide in length into a single ClaI site of plasmids pOLTV535 and pOLTV553 (Fig. 2). The resulting plasmids (pOLTV535N0 to -N5 and pOLTV553N0 to -N5) differ in size by 1 nucleotide, so one of them should yield an RNA genome capable of satisfying the rule of six. Plasmids were transfected with CER-C9 and CER cells infected with FPV-T7 as described above. The results show that only the plasmids pOLTV535N3 and pOLTV553N3 increased SEAP activity after NDV infection. Length

The RNA minigenomes produced by these plasmids were calculated as 6n + 2. Since two additional G residues are present at the 5 'end of the minigenomic RNAs, this result suggests that only the size of the RNA sequence located between the true 3' and 5 'ends of the minigenomic RNAs matters in principle six. This was verified by the construction of minigenomic plasmids in which the beginning of T7 RNA polymerase transcription was altered such that the first nucleotide incorporated into the RNA was the first nucleotide of the 3 'or 5' end of the NDV (see Materials and Methods). Transfection with such plasmids showed that only the minigenomic RNA produced by the plasmids pOLTV535N3 and pOLTV553N3 is replicated by the helper virus (results not shown). These findings indicate that NDV replication is closely dependent on the rule of six. Moreover, this indicates that the presence of two additional G residues at the 5 'end of the minigenomic RNA does not hinder proper replication. Similar results were obtained with minigenomic plasmids (or DI plasmids) for other paramyxoviridae (Pattnaik et al., 1992; Harty and Palese, 1995).

Packaging of NDV minigenomes by helper viruses

To determine whether the minigenomic RNA could be packaged by the helper virus NDV, the medium from the transfected cells was transferred to fresh monolayers and after adsorption for 1 hour, the cultures were washed three times with PBS and incubated in complete medium. After 24 hours incubation, the SEAP activity of the medium was measured. The results show that SEAP activity was only present in cells treated with the medium from cells transfected with pOLTV553N3 (Table 4). This indicates that minigenomic RNA can be packaged in NDV envelopes and the resulting particles can infect cells. Further, the result shows that the packaging is replication dependent and that only RNA molecules in complex with the viral NP, P and L proteins are packaged into virus-like particles.

Replication of NDV minigenomes by plasmids expressing the NP, P and L proteins

To determine whether the minigenomic RNAs could also be replicated by plasmids encoding the basic proteins NP, P and L, we cotransfected cells infected with FPV-T7. Cells were transfected with a combination of plasmids consisting of a minigenomic plasmid and plasmids pCIneoNP, -P and -L, respectively (c). As a negative control, plasmid pCIneoL (c), encoding the essential L protein, was replaced with the vector plasmid pCIneo. The results (Table 5) show that indeed the plasmids encoding the NP, P and L proteins are capable of replicating minigenomic RNAs. Further, the results show that, similar to the replication of minigenomes by the helper virus, replication by the NP, P, and L proteins is dependent on the rule of six.

Nucleotide sequence of the complete genome of the NDV strain of Lasota

Subgenomic cDNA stretches covering the entire genome of NDV were obtained by RT-PCR (Fig. 4). To minimize the number of PCR errors, a correction property enzyme mixture (Expand Long Template, Boehringer Mannheim) was used in combination with a small number of PCR cycles (15 cycles). A 3UIT primer, complementary to the 3 'end of the NDV RNA was used for reverse transcription and gene specific primers were used for PCR. To identify possible PCR errors, three independent reactions were performed and their products were used to generate three independent sets of subgenomic cDNA. Ranging in length from about 4 to 7 kDNAs were cloned into pGEM-T. The nucleotide sequences of the two sets were determined using primers derived from the publicly available NDV sequence, or designed from an NDV sequence established during this sequencing project (Table 1). Remaining sites of concern were identified by sequencing a third set of cDNA clones. The genome of the LaSota NDV strain consists of 15,186 nucleotides (Fig. 3), making it the smallest of all paramyxovirus genomes whose complete sequence has been determined by then (Kolakofsky et al., 1998).

Construction of a full-length NDV cDNA clone on the pOLTV5 transcription plasmid

To construct a full-length NDV cDNA clone of the LaSota strain, overlapping cDNA clones covering the entire NDV genome were ligated at common restriction sites according to the strategy outlined in Figure 4. The entire NDV cDNA was ligated into the minigenomic plasmid pOLTV535 (see above) obtained from the transcription plasmid pOLTV5.

As can be seen in Figure 4B, the final step in fusing the complete NDV cDNA was to clone an approximately 8.8 kb ClaI segment from pGEM-B onto p535-DI containing the NDV sequences flanking the Clal site on both sides (i.e. nucleotides 1-3521 and 12355-15186). This step turned out to be quite difficult as we repeatedly failed to obtain the correct clones. Thus, the ClaI fragment of pGEM-B was marked with the chloramphenicol resistance gene (Cm) from plasmid pACYC184. A Clal fragment bearing the Cm gene was isolated and cloned into the Clal site of p535-DI, and transformants were selected for resistance to both Cm. Since transformants grew poorly, anti-24

The biotic treatment was reduced to 15pg / ml Cm and 10pg / ml Km and the incubation temperature was lowered from 37 ° C to 32 ° C. Finally, the Cm gene was removed from the plasmid by digestion with BsiWI followed by recirculation with T4 DNA ligase. The resulting plasmid consisting of the full length NDV cDNA cloned between the Smal and Stul sites of the pOLTV5 transcription plasmid was named pNDFL +.

Generation of infectious NDV from full-length cDNA

To generate infectious NDV completely from the cloned cDNA of the plasmid, pNDFL + was used for cotransfection with the plasmids pCIneoNP, -P, -L (c), as described above for the minigenomic plasmids. CER and CEF cell transfections were controlled by using the minigenomic plasmid pOLTV553N3 and measuring SEAP expression. For the negative control, pCIneoL (c) was replaced with pCIneo. After cotransfection, cells were incubated for 3 to 6 days in medium containing 5% allantoic fluid. The addition of allantoic fluid is necessary as CER and CEF cells lack the proteases required for cleavage of the LaSota strain F protein. Cleavage of the F proteins is required for the spread from cell to cell of the virus and the formation of infectious viruses. After three days of incubation, we performed immunostaining of the fixed monolayer cultures with a monoclonal antibody against the F protein. The results showed that the antibody stained cells were only present in the cultures co-transfected with pNDFL (+), pCIneoNP, -P, -L (c). This indicates that these cells were replicating and expressing the genome. No staining was observed for cells in which pCIneoL (c) was replaced by pCIneo transfection.

To recover infectious viruses, supernatant from the transfected CEF cultures was injected into the cavity of the nucleating egg allantoic. After four days, the amniotic fluid was collected, examined for hemagglutination and passed into eggs. The results show that only the supernatant from cells transfected with the pNDFL (+), pCIneoNP, -P, -L (c) combination gave a positive reaction in the hemagglutination test. The amniotic fluid positive for haemagglutination was then tested in a haemagglutination inhibition test with the monoclonal antibodies 7B7, 8C11, 5A1, 7D4, and 4D6 (Long et al., 1986), which can be used to distinguish NDV strains. The test results indicated that the NDV strain recovered from the inoculated eggs showed the same reactivity as the starting LaSota strain. The virus recovered from inoculated eggs was named NDFL to distinguish it from the starting LaSota strain.

Generation of genetically altered NDVs from full-length cDNAs

To show unequivocally that the cotransfection system can be used to recover infectious virus from cloned full-length NDV cDNAs, a genetic tag was introduced into the pNDFL (+) plasmid. For this, the amino acid sequence of the protease cleavage site in the Fo protein was changed from the LaSota strain (GRRQGR | L) to the consensus sequence of the virulent NDV strains (GRRQROR | F) by PCR-mutagenesis (see Materials and Methods for details). The resulting plasmid, pNDFL + ^ w], was used to generate virus using the above-described co-transfection system. An infectious virus, named NDFLjF '⁷'] was recovered from the allantoic fluid of the embryonic eggs into which it was inoculated with cotransfected CEF cells. In the HI test, all monoclonal antibodies, including 7D4, specific for the LaSota strain, showed the same reactivity for the newly generated virus as the starting LaSota strain. The nucleotide sequence of the segment encoding the protease cleavage site of the F protein was determined by RT-PCR. The results show that the nucleotide sequence contains exactly the same nucleotide changes as introduced by the mutagenizing primer used to alter the original LaSota sequence. This shows that the virus was obtained from the pNDFL + [F''j plasmid and that NDV (genetically altered) could be obtained entirely from cloned full-length NDV cDNA.

The protease cleavage site in the Fo NDV protein is a key determinant of virulence

It is generally recognized that the amino acid sequence of the protease cleavage site in the Fo protein is a key determinant of the virulence of various NDV strains. The creation of a genetically modified LaSota strain, in which the amino acid sequence of the protease cleavage site has been changed from that of a lentogenic (non-virulent) strain to that of a multigeneous (virulent) strain of NDV, offers the only possibility to verify this assumption. We therefore determined the intracerebral pathogenicity index (ICPI) of the newly produced NDFL virus [F '' ''] and compared it with the rate for NDFL and the starting LaSota strain (clone E13-1). The ICPI of the NDFL [F "" "] strain was 1.3, which is far above the values ​​for the NDFL strains (ICPI = 0.0) or clone E13-1 (ICPI = 0.3). These results indicate that, as expected, the virulence of NDV is largely determined by the amino acid sequence of the protease cleavage site in the Fo protein.

PL 197 722 B1

Introduction of a serological marker

The NDV, F and HN envelope glycoproteins are the most immunogenic proteins in the virus. When infected, both F and HN elicit a strong antibody neutralizing response. The induction of such a neutralizing antibody response is the basis for the effectiveness of vaccination with non-virulent NDV strains (such as the popularly used LaSota strain).

However, the humoral response to the NDV strains used for vaccines is indistinguishable from the humoral response to environmental virulent NDV strains. Thus, contamination with virulent virus from the environment cannot be followed serologically. This situation is disadvantageous as environmental virus infections are masked by the vaccine and clinical signs caused by environmental viruses may be overlooked or even attributed to the vaccine. Since effective discrimination of vaccination from infection is crucial for the eradication of NDV, we decided to create genetically engineered NDV strains that can serve as a vaccine and serologically differentiate from environmental NDV strains (so-called marker vaccines). For a marker vaccine to be produced, the virus must be genetically altered such that one or more immunodominant epitopes of one of the (main) antigens are deleted or altered. Removal of an important portion (one or more) of the protein can lead to the loss of the biological function of the protein. Therefore, we decided to change one of the main immunogenic proteins of the NDV envelope in such a way that the biological function of the protein is maintained, with a changed antibody repertoire responding to the altered protein from the original. For the reasons detailed below, we have chosen to modify the NDV HN protein as one embodiment of the invention. NDV infection begins with the fusion of the virion envelope with the plasma membrane of the host cell. The F and HN proteins are required for this process. It has been shown that the F and HN proteins interact physically with each other and that this interaction is required for membrane fusion (Deng et al., 1995). Further, the interaction has been shown to be type-specific, i.e. the F and HN proteins must be from the same virus in order to exhibit fusion activity. The interacting domains of the NDV HN protein have been located in the so-called core region of the protein, containing the first 92 amino acids of the outer domain of the HN protein (Deng et al., 1995).

The hybrid proteins consisting of NDV amino acids 1-141 and amino acids 141-572 3 of the human parainfluenza virus type (hPIV3) have been shown to retain fusion activity when co-expressed with the NDV F protein. This suggests that genetically altered NDV strains having a hybrid HN protein consisting of the NDV core region and the globular HN protein head of various avian paramyxoviruses may be viable. Further, such strains will elicit anti-HN antibodies distinct from the response to NDV. Since the neutralizing humoral response against the F protein is sufficient for effective protection against a provoked viral infection, such genetically altered NDV strains fulfill the two basic requirements of a marker vaccine, i.e. disease protection and serological discrimination.

Hybrid HN genes were constructed, consisting of either fusions or amino acids 1-141 of NDV ^{and 142}-⁵80 ^Frias^ande^goh piaram ^ sowkusa ^typeat ² (AND^PMV2) (designated HN1 / 2¹⁴¹), ^lat^b Ί- amino acids¹⁴³NDV and 144-580 APMV2 designated HN1 / 2¹⁴³). HN hybrid genes were constructed similarly, consisting of either the fusions of either the amino acids 1-141 of NDV and 142-569 of the avian paramyxovirus type 4 (APMV4) (designated HN1 / 4).¹⁴¹)^{, l}at^b Ί- amino acids^{143 NDV and 144}-⁵⁸⁰ AND^PMV4 Marked ^{HN1 / 4143}). ^Hybr^yThese genes were cloned into pCIneo eukaryotic expression vectors and used for co-transfection with a plasmid encoding the NDV F protein. For this purpose, the F protein was changed in such a way that the amino acid sequence of the protease cleavage site between the F1 and F2 proteins was changed from that characteristic of the LaSota strain to the consensus one for virulent NDV strains (F^{in l}, see Materials and Methods). Experiments with co-transfection in CER and QM5 cells indicated that both HN1 / 2 ”1 and HN1 / 2^143, I^k also ^{HN1 / 4141 and HN1 / 4143,} beechy ^fUzj^{ê p}about^dtime ^koe^ks^presj^and with ^Fwt. ^T.en w^yn^ikindicates that the complexes of hybrid HN proteins and F proteins have biological activity. HN1 / 2 hybrid proteins¹⁴³ and HN1 / 4¹⁴¹ was used to replace the original HN gene in a full-length cDNA clone ^pNDFL +^, what did ^pNDFL-HN1 / 2^{143 and pNDFL}-^{HN1 / 4143}. ^ABOUTstatmch ^din^aboutc^{h pl}azm^gowu^alivewe were wished^bluntto generate infectious viruses by using the co-transfection system described above. Viable in^andrus^y re^kohm^binated (marked with ^NDFL- ^{HN1 / 2141} and ^NDFL-^{HN1 / 4143}) mo^withon w^yigold^ex with ^flowsnu allantoic cells of embryonic eggs previously inoculated with supernatant from transfected cultures.

The presence of the hybrid HN gene in both recombinants was checked by RTPCR. The haemagglutination inhibition test showed that NDV monoclonal antibodies and polyvalent antisera were unable to inhibit haemagglutination of chicken erythrocytes^pthe virus^y NDFL-HN1 / 2^{143 and NDFL}- ^{HN1 / 4143}. ^Youn^iki these are ashamed ^withsincerely^{py NDFL}-^{HN1 / 2143}

PL 197 722 B1 and NDFL-HN1 / 4¹⁴³ they can be used as vaccines and can be serologically differentiated from classic NDV vaccines.

Expression of a heterologous protein from recombinant NDV

To test whether foreign genes could be inserted into the NDV genome, we constructed a recombinant virus carrying a SEAP reporter gene. The SEAP gene was derived from the pOLTV535 plasmid and was altered to contain typical NDV transcriptional start and stop sites. A DNA fragment containing the SEAP gene and the downstream transcription start and stop sites was inserted into the Xmnl restriction site (nucleotide 109) on the pNDFL + [Fw] plasmid. Infectious virus, designated NDFL-AP, was generated by a co-transfection system and the presence of the SEAP gene was checked by RT-PCR. Cells infected with the NDFL-AP strain expressed high levels of SEAP protein. Using the specific activity of the SEAP protein, we calculated that x% of the proteins expressed in the NDFL-AP infected cells were SEAP protein. These results show that heterologous genes can be expressed at a very high level by recombinant NDV.

Generation of an NDV deletion mutant in a trans-complementing cell line

To abolish the expression of the NDV M protein, a large portion of the M gene was removed by digesting pNDFL + [Fw] BsaAI (nucleotide 3087) followed by partial digestion with HindIII. After the end of the HindIII cleavage was filled with the Klenow fragment of DNA polymerase, the fragment was recirculated with T4 Dna ligase and used to transform E.coli. The resulting plasmid, named pNDFL + [Fw] dM, was used to generate virus by a cotransfection system in transcomplementing CER-M cells expressing the NDV M protein. The supernatant from transfected cultures was passaged three times on CER-M cells and tested for virus. The virus was obtained as evidenced by the positive results of the haemagglutination (HA) and haemagglutination inhibition (IH) tests for the supernatant from the third passage culture. The virus was named NDFL-dM. When infecting cultures of NDFL-dM CER cells, the virus was still able to spread from cell to cell as demonstrated by the anti-protein F monoclonal antibody IPMA assay. As expected, M protein expression could not be demonstrated by the anti-protein M monoclonal antibody IPMA assay. When the supernatant was used to infect CEF or CER-M cells, we were unable to demonstrate the presence of replicating virus in cultures using IPMA. This indicates that the infectious virus cannot arise in non-complementing cells. This was confirmed by the observation that inoculation of the germinal egg with supernatant from infected CEF cells did not result in the generation of progeny detectable by HA or HI tests. The need for better NDV vaccines, and especially marker vaccines, prompted us to develop a reverse genetics system that allows genetic alterations to be made to NDV. In this document we describe the generation of infectious NDV entirely from cloned full-length cDNA. We show that the virulence of NDV can be dramatically altered by changing only 3 nucleotides determining the specificity of the protease cleavage site in protein F. In this case, the protease cleavage site has been changed from a LaSota cleavage site to a consensus cleavage site in virulent NDV strains. In creating this genetically altered NDV strain, we provide formal evidence that the ability to cleave the F protein is a key determinant (but not the only one) of NDV virulence. Using the same reverse genetics approach, one can change the cleavage site as desired in any other amino acid sequence. This could lead to the generation of a series of NDV strains showing the entire spectrum of virulence levels, in vivo.

As already mentioned above, it has been shown that apart from cleavage of the HN and F proteins, also other viral factors can influence its pathogenicity. Changes in transcription and translation can modulate viral initiation and spread between cells and / or cytotoxicity. The availability of the infectious NDV cDNA allows for the systematic introduction of sequence changes involved in transcription and replication. This could lead to the design of new NDV vaccines that combine optimal immunogenicity with almost no virulence.

Safety is one of the most important characteristics of live vaccines. However, for many live vaccines, including NDV, immunogenicity is often inversely related to virulence. Thus, further attenuation of live vaccines without loss of immunogenicity is one of the most desirable changes for which genetic modification can be used.

With this in mind, it is worth mentioning that the elimination of Sendai virus V protein expression has been shown to result in significantly reduced pathogenicity in vivo in mice (Kato et al., 1997). Like the Sendai virus, NDV also produces protein V through a mechanism called RNA editing (Steward

PL 197 722 B1 et al., 1993). It can be predicted that deletion of NDV V protein expression may also result in in vivo attenuation of the phenotype.

In addition to altering NDV virulence, we have shown that it is possible to alter the antigenic pattern of NDV in such a way as to generate strains serologically distinguishable from ambient NDV strains. These so-called marker vaccines are an invaluable tool for determining the number of NDV cases in herds worldwide. Moreover, the widespread use of such marker vaccines could eventually lead to the complete elimination of NDV through intensive screening and disposal of infected herds. In this description we show that foreign genes can be inserted into the NDV genome. These foreign genes can be expressed at very high levels in infected cells. This shows that NDV can be used as a vaccine vector for the expression of antigens from other (poultry) pathogens. Several properties make NDV an ideal vaccine vector for vaccination against respiratory and intestinal diseases. 1) NDV can be easily grown to high titers in embryonic eggs. 2) mass breeding of NDV in embryonic eggs is relatively cheap. 3) NDV vaccines are relatively stable and can be easily administered by mass administration methods such as addition to drinking water or aerosolized spraying. 4) the natural route of NDV infection is through the digestive or respiratory system, as for many other poultry pathogens. 5) NDV can induce local immunization despite the presence of circulating maternal antibodies.

Finally, we show that viable NDV deletion mutants can be created by using transcomplementing cell lines. An NDV deletion mutant incapable of expressing the matrix protein (M) involved in the budding of NDV from within the cell membrane was created. We show that a phenotypically complementary NDV strain unable to express the M protein can still infect cells and spread from cell to cell. However, the mutant virus is unable to produce infectious progeny viruses in non-complementary cells. This indicates that phenotypically complementing NDV deletion mutants can be used as safe self-limiting vaccines that cannot spread into the environment. Such a non-transmissive vaccine combines the most important advantage of live vaccines, i.e. efficacy, with the most important advantage of dead vaccines, i.e. safety.

Brief description of the drawings

Figure 1

The pOLTV5 transcription vector is derived from the transcription vector described by Pattnaik et al. (1992). Details of the structure in the text. The plasmid contains the T7 DNA-dependent RNA polymerase promoter (marked in bold) followed by unique restriction sites for StuI and Smal and the autocatalytic ribozyme from hepatitis delta virus (HDV). DNA fragments can be cloned between the StuI and Smal sites and can be transcribed in vitro or in vivo with T7 polymerase. The 5 'end of the resulting transcripts contain two additional G residues not encoded by the insert. Due to the activity of the ribozyme, the 3 'end of the transcripts corresponds exactly to the last nucleotide of the insert.

Figure 2

Structure of the minigenomic plasmids pOLTV535 (Fig. 2A) and pOLTV553 (Fig. 2B). The minigenomic plasmids are based on the pOLTV5 transcription plasmid (cf. Fig. 1) and contain a 3 '(nucleotides 1-119) or 5' (nucleotides 14970-15186) region of the NDV LaSota strain, flanking the gene encoding secreted alkaline phosphatase (SEAP). Transcription of pOLTV535 by T7 RNA polymerase produces antigenomic RNA (or [+] RNA) while transcription of pOLTV553 results in genomic RNA (or [-] - RNA). Start (S) and end (E) sites of transcription are marked. The start codon of the SEAP gene is underlined. Also shown is the sequence of the inserts (N0-N5) in the ClaI site, giving minigenomic plasmids differing from each other by 1 nucleotide in length (pOLTV535N0-N5 and pOLTV553N0-5, respectively).

Figure 3

Amino acid sequence of the NDV genome of the LaSota strain and the resulting amino acid sequence of the NDV genes. The sequence shown corresponds to the antigenomic strand and is shown in the 5'-3 'direction in the form of ssDNA. The figure shows the consensus sequence determined after the complete sequencing of two independent sets of overlapping subgenomic cDNAs occupying the entire cDNA of the NDV genome. The remaining ambiguities (probably due to a PCR error) were resolved by sequencing the appropriate regions of the third independent set of clones. Full-length sequence of the pNDFL + cDNA clone consisting of partially

The overlapping subgenomic cDNA clones (see Figure 4) differs from the NDV consensus sequence at the following positions (consensus sequence in parentheses): nucleotide (nt) 1755, G (A); nt 3766, A (G); nt 5109, G (A); nt 6999, T (C); nt 7056, G (A); nt 9337, G (A); nt 9486, A (T); nt 10195, T (C); nt 13075, A (G). These changes cause three amino acid changes (consensus sequence^{the owl in the aisle}and^asand^e): bi^andLuke^about F. ^- R¹⁸⁹(Q) HN - S protein²⁰⁰ (P) L - N protein³⁶⁹ (AND).

Figure 4 (A) general strategy used to splice full-length genomic cDNA from subgenomic overlapping clones. The cDNA was assembled on plasmid pOLTV535 already having the 3 'and 5' ends of the NDV LaSota strain (cf. FIG. 2). The resulting plasmid, designated pNDFL +, was used to generate infectious NDV.

(B) a cloning procedure to assemble the full-length cDNA genomic from sub-genomic overlapping clones. Cm is the chloramphenicol resistance gene temporarily introduced as a phenotypic marker (details in the text of the description).

(C) the detailed cloning procedure used in generating the genetically altered full-length NDV cDNA. The change consists of three nucleotide changes introduced into the F gene that change the amino acid sequence of the proteolysis site of the F protein (details in the text of the description).

Figure 5 (A) plasmid series pOLTV535

Transcription by T7 RNA polymerase produces antigenomic RNA (or [+] RNA) which can be translated directly into the SEAP protein in a cell. Upon transfection of cells with a helper virus (or cotransfection with plasmids encoding NP, P, L), antigenomic RNA is used by the viral polymerase complex to synthesize genomic RNA (i.e. [-] - RNA). The genomic RNA is then used by the viral polymerase complex to synthesize both mRNA (using specific start [S] and end [E] transcription sites) and antigenomic RNA.

(B) plasmid series pOLTV553

Transcription by T7 RNA polymerase produces genomic RNA (or [-] - RNA) that cannot be translated into SEAP protein. Upon transfection of cells with helper virus (or co-transfection with plasmids encoding NP, P, L), genomic RNA is used by the viral polymerase complex to synthesize both mRNA (using specific start [S] and end [E] transcription sites) and antigenomic RNA .

Figure 6.

Alignment of the nucleic acid sequences of the 5 'end of the NDV LaSota strain and other paramyxoviruses as a comparison of the NDV sequence with four members of the genus Rubulavirus, three members of the genus Paramyxovirus, and three members of the genus Morbillivirus. Sequences are shown from the end of the L gene to the 5 'end (3'-5' CDNA).

NDV, Newcastle disease virus; hPIV2, human parainfluenza type 2 virus, MuV, mumps virus; SV5 and SV41, simian virus 5 and 41, respectively; SeV Sendai virus; bPIV3 and hPIV3 bovine and human parainfluenza virus, respectively; CDV, canine distemper virus; MeV, measles virus; RPV, rinderpest virus. The nucleotide sequences of the complete genomes were obtained as follows (accession number): NDV (AF077761); hPIV2 (x57559); MuV (AB000388); SV5 (AF052755); SV41 (X64275);

bPIV3 (D84095); hPIV3 (Z11575), CDV (L13194); MeV (X16565); RPV (Z30697).

PL 197 722 B1

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