Newcastle Disease Virus Vaccine Strain Research Articles

Molecular characterization of chicken-derived genotype VIId Newcastle disease virus isolates in China during 2005–2012 reveals a new length in hemagglutinin–neuraminidase

Newcastle disease (ND) is one of the most important diseases of poultry, and causes severe economic losses in the global poultry industry. Although all Newcastle disease virus (NDV) isolates belong to a single serotype, significant genetic diversity has been described between different NDV isolates. Here, we report the molecular characterization of 23 virulent genotype VIId NDV isolates of class II circulating in China. Phylogenetic construction and analysis revealed the existence of distinctly genomic and amino acid differences that clearly distinguished these isolates from other typical NDV genotypes and vaccine strains. We also report a new 582-amino-acid hemagglutinin–neuraminidase in genotype VII NDV strains. This is believed to be the first study to investigate systematically the most predominant NDV strains, and provides more information on the genetic nature of genotype VIId NDV of class II circulating in China.

Comparative Immunogenicity of HIV-1 gp160, gp140 and gp120 Expressed by Live Attenuated Newcastle Disease Virus Vector

The development of a vaccine against human immunodeficiency virus-1 (HIV-1) capable of inducing broad humoral and cellular responses at both the systemic and mucosal levels will be critical for combating the global AIDS epidemic. We previously demonstrated the ability of Newcastle disease virus (NDV) as a vaccine vector to express oligomeric Env protein gp160 and induce potent humoral and mucosal immune responses. In the present study, we used NDV vaccine strain LaSota as a vector to compare the biochemical and immunogenic properties of vector-expressed gp160, gp120, and two versions of gp140 (a derivative of gp160 made by deleting the transmembrane and cytoplasmic domains), namely: gp140L, which contained the complete membrane-proximal external region (MPER), and gp140S, which lacks the distal half of MPER. We show that, similar to gp160, NDV-expressed gp140S and gp120, but not gp140L, formed higher-order oligomers that retained recognition by conformationally sensitive monoclonal antibodies. Immunization of guinea pigs by the intranasal route with rLaSota/gp140S resulted in significantly greater systemic and mucosal antibody responses compared to the other recombinants. Immunization with rLaSota/140S, rLaSota/140L rLaSota/120 resulted in mixed Th1/Th2 immune responses as compared to Th1-biased immune responses induced by rLaSota/160. Importantly, rLaSota/gp140S induced neutralizing antibody responses to homologous HIV-1 strain BaL.26 and laboratory adapted HIV-1 strain MN.3 that were stronger than those elicited by the other NDV recombinants. Additionally, rLaSota/gp140S induced greater CD4+ and CD8+ T-cell responses in mice. These studies illustrate that rLaSota/gp140S is a promising vaccine candidate to elicit potent mucosal, humoral and cellular immune responses to the HIV-1 Env protein.

Transcriptional response of chicken embryo cells to Newcastle disease virus (D58 strain) infection

Newcastle disease virus (NDV), the causative agent of Newcastle disease (ND) in chicken causes significant economic loss for the poultry industry worldwide. The mechanism involved in host response to NDV infection is not well understood. For better understanding of the virus-host interaction; transcriptional profile of some genes of chicken embryo (CE) cells infected with NDV vaccine strain D58 was established using quantitative RT-PCR SYBR Green method. The relative standard curve method was used to measure the level of transcripts of the cellular genes against an endogenous control (β actin) gene. Among the genes studied, IFN α, IFN γ, MHC I and DDX 1 were up-regulated while IL 6 was down regulated. The expression of viral genes (M and F) in the infected CE cells was also confirmed by relative quantification. The host cellular genes involved in pro-inflammatory response, interferon-regulated proteins and the cellular immune response were affected by NDV infection, indicating involvement of complex signaling pathways of host cell responses to the infection. Thus, this study contributes to the understanding of the pathogenesis of ND and provides an insight into the virus-host interaction.

Preparation and Efficacy of a Live Newcastle Disease Virus Vaccine Encapsulated in Chitosan Nanoparticles

BackgroundNewcastle disease (ND) is a highly contagious viral disease of poultry caused by pathogenic strains of the Newcastle disease virus (NDV). Live NDV vaccines are administered by drinking water, eyedrops or coarse aerosol spray. To further enhance mucosal immune responses, chitosan nanoparticles were developed for the mucosal delivery of a live NDV vaccine.Methodology/Principal FindingsA lentogenic live-virus vaccine (strain LaSota) against NDV encapsulated in chitosan nanoparticles were developed using an ionic crosslinking method. Chitosan nanoparticles containing the lentogenic live-virus vaccine against NDV (NDV-CS-NPs) were produced with good morphology, high stability, a mean diameter of 371.1 nm, an encapsulation rate of 77% and a zeta potential of +2.84 mV. The Western blotting analysis showed that NDV structural proteins were detected in NDV-CS-NPs. The virus release assay results of NDV-CS-NPs indicated that NDV was released from NDV-CS-NPs. Chickens immunized orally or intranasally with NDV-CS-NPs were fully protected whereas one out of five chickens immunized with the LaSota live NDV vaccine and three out of five chickens immunized with the inactivated NDV vaccine were dead after challenge with the highly virulent NDV strain F48E9.Conclusions/SignificanceNDV-CS-NPs induced better protection of immunized specific pathogen free chickens compared to the live NDV vaccine strain LaSota and the inactivated NDV vaccine. This study lays a foundation for the further development of mucosal vaccines and drugs encapsulated in chitosan nanoparticles.

Characterization of Live LaSota Vaccine Strain–Induced Protection in Chickens upon Early Challenge with a Virulent Newcastle Disease Virus of Heterologous Genotype

Newcastle disease (ND) is a major threat to the international poultry industry, causing bird mortality, reduction in growth and egg production, and trade restrictions. The primary strategy available to the poultry industry to control virulent Newcastle disease virus (NDV), the causative agent of ND, is vaccination. LaSota and other commonly used live-virus NDV vaccine strains were developed in the 1950s and 1960s and show a great degree of genetic divergence from currently circulating NDV strains. In order to characterize protective immunity induced by LaSota against a heterologous NDV strain, we vaccinated groups of specific-pathogen-free (SPF) chickens with LaSota (virus titers ranging from 10(2) to 10(8) egg infective dose 50 [EID50] in 10-fold increments) and challenged the birds 14 days later with ZJ1 strain, an NDV strain that was isolated in the year 2000 from geese in China. We monitored multiple parameters of immunity, including serum antibody titers, antigen-specific lymphocyte proliferation, and splenic cytokine expression and determined that SPF birds vaccinated with an adequate titer of LaSota strain live vaccine are fully protected from morbidity and mortality due to challenge with ZJ1 strain NDV, and we concluded that in the absence of interfering maternal antibody, protection due to vaccination increases with vaccine titer until a threshold titer is reached, beyond which, little or no further benefit can be elucidated.

The different expression of immune-related cytokine genes in response to velogenic and lentogenic Newcastle disease viruses infection in chicken peripheral blood

Newcastle disease virus (NDV) is an important pathogen hazardous to poultry industry, and the pathogenicity of NDV strains varies with different virulence. Peripheral blood serves as an important producer and carrier of viruses and cytokines in NDV infection. In order to explore the difference of cytokine expression in the peripheral blood between velogenic strain and lentogenic strain infection, NDV virulent strain F48E9 and vaccine strain Lasota were used to infect specific-pathogen-free (SPF) chickens separately, and peripheral blood was collected on 0, 3, 7, 10, 14, and 21days post-infection (d.p.i.). Real-time PCR was then used to detect the expression of six kinds of immune-related cytokine genes. For the F48E9 group, a sharp increase of the expression of interferon-alpha (IFN-α), interferon-gamma (IFN-γ), interleukin-16 and IL-18 was observed on 3d.p.i. before the NDV blood peak (7d.p.i.), followed by a rapid decline to the level lower than that of control group, then the expression of IFN-α increased slowly and reached or exceeded the level of control group in the later phase of the infection, while the expression of IFN-γ, IL-16, and IL-18 fluctuated at the level of control group for the rest of study period. The increase of IL-2 expression was not obvious, and no increase of IL-15 expression was noted. For the Lasota (vaccine) group, the picture was quite different, a sharp increase of IFN-γ (but not IFN-α), IL-2 was observed on 7d.p.i. before the NDV blood peak (10d.p.i.). On the contrary, there was no dramatic increase of IL-16 and IL-18. Interestingly, in contrast to the F48E9 group, there was an increase of IL-15 on day 10d.p.i., but it remained modest. There was also an increase of IFN-α on day 21d.p.i. Our results revealed that infection with NDV strains of different virulence was associated with distinct cytokine expression patterns in peripheral blood, modulation of cytokine responses may play a key role in mediation of NDV pathogenesis.

Rescue of Newcastle disease virus from cloned cDNA using an RNA polymerase II promoter

A new system was developed to improve the efficiency and simplify the procedure of recovery of Newcastle disease virus (NDV) from cloned cDNA. A full-length cDNA clone of mesogenic NDV vaccine strain Mukteswar was assembled from five subgenomic cDNA fragments and cloned into a plasmid allowing transcription driven by cellular RNA polymerase II. The full-length viral cDNA was flanked by hammerhead ribozyme (HamRz) and hepatitis delta virus ribozyme (HdvRz) sequences, resulted in the synthesis of antigenomic RNA with exact termini. Without supplying T7 RNA polymerase, infectious NDV could be generated efficiently in some eukaryotic cell lines by simultaneous transcription of antigenomic RNA from the full-length plasmid and expression of NP, P and L proteins from helper plasmids introduced by cotransfection. The efficiency of recovery with the conventional T7 promoter system based on BRS-T7 cells and the cytomegalovirus (CMV) promoter system was compared, and the results demonstrate that the new system facilitates the generation of recombinant NDV and more efficient than the T7 rescue system using BRS-T7.

Generation and Evaluation of a Newcastle Disease Virus–Based H9 Avian Influenza Live Vaccine

Infection with H9 avian influenza virus (AIV) and Newcastle disease virus (NDV) are two important causes of egg drop in layer and breeder poultry, leading to severe economic loss in the industry. Currently in China, inactivated H9 AIV vaccine and live attenuated NDV vaccine have to be repeatedly administered to prevent egg drop in layer animals. Using reverse genetics, we constructed a recombinant NDV expressing an H9 AIV hemagglutinin (HA) from an H9N2 field isolate, A/Chicken/ Shandong/2/2007. The HA gene was inserted into the intergenic region between the phosphoprotein (P) and matrix (M) genes of the LaSota NDV vaccine strain. The recombinant virus stably expressing the HA gene, rL-H9, was found to be innocuous after intracerebral inoculation of 1-day-old chickens. A single dose of 10(6) 50% egg infectious dose of the recombinant virus intranasally inoculated into chickens induced high levels of NDV- and AIV H9-specific hemagglutination-inhibition antibody. Complete protection from clinical disease and mortality against challenge with a lethal dose of velogenic NDV was observed in chickens and 90% of chickens were protected from clinical disease, mortality, and virus shedding against challenge with homologous H9N2 AIV. Our results suggest that recombinant NDV is suitable as a potential bivalent live attenuated vaccine against both NDV and H9 AIV infection in poultry.

Immunization of Chickens with Newcastle Disease Virus Expressing H5 Hemagglutinin Protects against Highly Pathogenic H5N1 Avian Influenza Viruses

BackgroundHighly-pathogenic avian influenza virus (HPAIV) and Newcastle disease virus (NDV) are the two most important poultry viruses in the world. Natural low-virulence NDV strains have been used as vaccines over the past 70 years with proven track records. We have previously developed a reverse genetics system to produce low-virulent NDV vaccine strain LaSota from cloned cDNA. This system allows us to use NDV as a vaccine vector for other avian pathogens.Methodology/Principal FindingHere, we constructed two recombinant NDVs (rNDVs) each of which expresses the hemagglutinin (HA) gene of HPAIV H5N1strain A/Vietnam/1203/2004 from an added gene. In one, rNDV (rNDV-HA), the open reading frame (ORF) of HA gene was expressed without modification. In the second, rNDV (rNDV-HAF), the ORF was modified so that the transmembrane and cytoplasmic domains of the encoded HA gene were replaced with those of the NDV F protein. The insertion of either version of the HA ORF did not increase the virulence of the rNDV vector. The HA protein was found to be incorporated into the envelopes of both rNDV-HA and rNDV-HAF. However, there was an enhanced incorporation of the HA protein in rNDV-HAF. Chickens immunized with a single dose of either rNDV-HA or rNDV-HAF induced a high titer of HPAIV H5-specific antibodies and were completely protected against challenge with NDV as well as lethal challenges of both homologous and heterologous HPAIV H5N1.Conclusion and SignificanceOur results suggest that these chimeric viruses have potential as safe and effective bivalent vaccines against NDV and. HPAIV. These vaccines will be convenient and affordable, which will be highly beneficial to the poultry industry. Furthermore, immunization with these vaccines will permit serological differentiation of vaccinated and avian influenza field virus infected animals.

Newcastle disease virus vaccine strains: immunogenicity is not influenced by ICPI

Intracerebral pathogenicity index (ICPI) and mean death time (MDT) were determined using commercial live vaccines against Newcastle disease available in Brazil. The ICPI profiles obtained for B1 vaccine strains were nonvirulent and varied from 0 to 0.19, and their MDT was 104-116 hours. The LaSota strains had an ICPI varying between 0.02 and 0.37 and MDT from 92 to 116 hours. ICPI and MDT for the Clone 30 were 0.11 and 104 hours, respectively. For Ulster vaccines, ICPI and MDT were 0 and >150 hours; for VG-GA was 0.03 and 140 hours; and for C2, 0.04 and >144 hours. Eye drop vaccination and IM challenge, at the 1st week and the 4th week, respectively, resulted in highest protection for B1 (95-100%) and LaSota (90-100%) strains. The variability in vaccine ICPI did not interfere with immune response and all vaccines provided similar protection. All vaccines were considered non virulent and were classified as lentogenic according to the immunobiological product standards.

WITHDRAWN: Natural recombination between Newcastle disease virus vaccine strains and circulating viruses.

This article has been retracted.

Response of chickens to oral vaccination with Newcastle disease virus vaccine strain I 2 coated on maize offal

Thermostable Newcastle disease (ND) vaccine virus strain I2 was investigated for its efficacy as foodborne vaccine, using maize offal as the vehicle. Immune response to vaccination and resistance to challenge were assessed by standard methods. Results showed that following primary vaccination, 40 (64.5%) out of the 62 birds produced detectable haemagglutination inhibiting (HI) antibody, but only 4 (6.5%) produced HI (log2) antibody titre
3.0 regarded as protective with a geometric mean titre (GMT) of 3.1. After a booster dose, 49 (79.0%) seroconverted and 20 (32.3%) had HI (log2) titres
3.0 with GMT of 4.9. When challenged all vaccinated birds survived while all control (unvaccinated) birds died. Prechallenge HI antibody titre of 50 vaccinated birds selected for challenge showed that 13 (26.0%) had titres
3.0 and GMT = 4.5, while post-challenge, 31 (62.0%) had HI (log2)
3.0 with GMT of 7.2. Using Student t test analysis of significance, the birds were observed to show 70% HI antibody production at a P
0.3 and 3 degree of freedom (df), and 70% secondary immune response on challenge at 4df. It is therefore concluded that the vaccine could be effective for protection of village chickens as food-borne vaccine provided the carrier foods are adequately treated.

Evaluating Viral Interference Between Infectious Bronchitis Virus and Newcastle Disease Virus Vaccine Strains Using Quantitative Reverse Transcription–Polymerase Chain Reaction

The potential for infectious bronchitis virus (IBV) and Newcastle disease virus (NDV) replication interference was evaluated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Fourteen-day-old broiler chickens were inoculated via eyedrop with live commercial vaccine strains of IBV and NDV alone or in combination to directly evaluate IBV and NDV replication in the trachea at 1, 3, and 5 days after vaccination. Commercial NDV vaccine strains used were B1, VG/GA, and C2. The vaccine strains of IBV tested were Massachusetts (Mass) and Arkansas (Ark). The NDV + Mass vaccines used were commercially manufactured combined products. The NDV + Ark vaccines used were commercial vaccines manufactured as single entity products that were administered by eyedrop to opposite eyes of each chicken. As measured by qRT-PCR, the replication of NDV strains B1, VG/GA, and C2 did not interfere with the growth of IBV Mass and Ark strain vaccines in the combined vaccine treatment groups. Combination vaccinations using B1 and VG/GA did not interfere with IBV immunity based on challenge or serum antibody production. In the C2 + Mass vaccination trial, IBV immunity after challenge was reduced, but it did not seem to be a result of reduced Mass vaccine growth or the ability of the Mass vaccine to induce serum IBV antibody. In contrast, the replication of IBV strains Mass and Ark interfered with the growth of NDV strains B1, VG/GA, and C2 as measured by qRT-PCR. However, interference with NDV replication was not reflected in a reduction in Newcastle disease challenge of immunity findings when combination Mass + NDV products manufactured by vaccine companies were tested. Moreover, NDV immunity was not compromised in two of three trials using single entity vaccines of NDV and Ark IBV vaccines manufactured separately but administered simultaneously. However, in one trial, NDV immunity was decreased where a NDV single entity product (C2) was given with an IBV single entity Ark vaccine. This finding emphasizes the importance of using manufactured combination vaccines whenever possible to avoid potential interference.

Newcastle Disease Virus-Based Live Attenuated Vaccine Completely Protects Chickens and Mice from Lethal Challenge of Homologous and Heterologous H5N1 Avian Influenza Viruses

H5N1 highly pathogenic avian influenza virus (HPAIV) has continued to spread and poses a significant threat to both animal and human health. Current influenza vaccine strategies have limitations that prevent their effective use for widespread inoculation of animals in the field. Vaccine strains of Newcastle disease virus (NDV), however, have been used successfully to easily vaccinate large numbers of animals. In this study, we used reverse genetics to construct a NDV that expressed an H5 subtype avian influenza virus (AIV) hemagglutinin (HA). Both a wild-type and a mutated HA open reading frame (ORF) from the HPAIV wild bird isolate, A/Bar-headed goose/Qinghai/3/2005 (H5N1), were inserted into the intergenic region between the P and M genes of the LaSota NDV vaccine strain. The recombinant viruses stably expressing the wild-type and mutant HA genes were found to be innocuous after intracerebral inoculation of 1-day-old chickens. A single dose of the recombinant viruses in chickens induced both NDV- and AIV H5-specific antibodies and completely protected chickens from challenge with a lethal dose of both velogenic NDV and homologous and heterologous H5N1 HPAIV. In addition, BALB/c mice immunized with the recombinant NDV-based vaccine produced H5 AIV-specific antibodies and were completely protected from homologous and heterologous lethal virus challenge. Our results indicate that recombinant NDV is suitable as a bivalent live attenuated vaccine against both NDV and AIV infection in poultry. The recombinant NDV vaccine may also have potential use in high-risk human individuals to control the pandemic spread of lethal avian influenza.

Positive identification of Newcastle disease virus vaccine strains and detection of contamination in vaccine batches by restriction site analysis of the matrix protein gene.

Twelve vaccine batches prepared from avirulent vaccine strains of Newcastle disease virus produced by seven manufacturers were identified by analysis of the matrix (M) protein gene with restriction enzymes MboI and HinfI. The analyses have revealed the presence of the strain indicated by the manufacturers (namely B-1, LaSota or Ulster 2C), except in one case when the vaccine contained strain V4 Queensland instead of VGGA as indicated. In addition, several batches of both monovalent and combined vaccines containing strain LaSota of the same company consistently disclosed contamination with strain B-1. The mixed nature of the preparations was verified not only by the dual patterns of restriction fragments but also by separating the two components and identifying them individually. Restriction analysis of the M gene, by allowing positive identification of each of the lentogenic vaccine strains, should provide an improvement in controlling vaccine batches by revealing homologous contaminants or exchange of the vaccine strain.

On the origins and relationships of Newcastle disease virus vaccine strains Hertfordshire andMukteswar, and virulent strain Herts'33

The origins and relationships of Newcastle disease virus (NDV) vaccine strains Hertfordshire OI) andMukteswar, and the virulent Herts'33 were studied using partial sequence analysis of the fusion protein gene.The mesogenic strain H was obtained by egg passages of a field virus isolated in England in 1933 (later knownas Herts'33). Different lines of the strain Herts'33, however, divided into two distinct groups: genotype IV, and ahitherto undescribed lineage, which comprised the Weybridge line (Herts'33/56). Vaccine strain H and the twoclusters comprising viruses designated Herts'33 displayed 6.5 to 6.8% and 15.6 to 16.3% mutational distances,respectively, which precluded parent-offspring relationships with either of them. In contrast, the different linesof the vaccine strain Mukteswar, which was reportedly derived from an Indian field isolate in the mid-1940s,showed 98.9 to 100% sequence similarity to strain H. It is therefore probable that the two vaccines were derivedfrom the same virus stock.

Correlation of haemagglutinin-neuraminidase and fusion protein content with protective antibody response after immunisation with inactivated Newcastle disease vaccines

The correlation between the antigen content of inactivated Newcastle disease (ND) oil emulsion–vaccines and the serological response after immunisation was studied. The haemagglutinin-neuraminidase (HN) and fusion (F) proteins of Newcastle disease virus (NDV) were quantified in 33 inactivated oil-adjuvanted ND vaccines using isopropylmyristate (IPM)-extraction and antigen ELISAs. These commercial vaccines differed in NDV–vaccine strain, the method applied to inactivate the vaccine virus, the vaccine valence and the composition of the oil emulsions. Large differences, up to 100-fold, in the antigen quantities present in different vaccines were found. The NDV–HN content and the NDV–F content of the vaccines both highly correlated with the haemagglutination-inhibiting (HI) antibody titres after immunisation. These correlation’s were found over a 10,000-fold range of applied antigen. The antigen content of the oil emulsion–vaccines also highly correlated with virus neutralising antibody titres. The presence of serum HI antibody titres in individual specific pathogens free (SPF)-chickens after immunisation with inactivated ND vaccines was highly indicative for clinical protection against challenge with the virulent NDV-Herts strain. Our results are the first to demonstrate that the protective serological response after immunisation highly correlates with the antigen content of oil-adjuvanted vaccines.

Differentiation of velogenic, mesogenic and lentogenic strains of Newcastle disease virus by multiplex RT‐PCR

SummaryA multiplex RT‐PCR technique has been developed for differentiation of velogenic, mesogenic and lentogenic pathotypes of Newcastle disease virus (NDV), using a set of three oligonucleotide primers designed from NDV genomic RNA (P1, P2 and P3). The primer pair P1 and P2 generated a RT‐PCR product of 204 bp, only with RNA from velogenic and mesogenic strains, whereas the P1 and P3 generated a 364 bp product only with RNA from mesogenic and lentogenic strains. Thirty four NDV strains, including some reference strains (known pathotypes), NDV field isolates and NDV vaccine strains, as well as other avian virus strains, were tested with multiplex RT‐PCR. All reference strains tested were differentiated in agreement with their intracerebral pathogenicity index (ICPI) values or with the pathotypes known in previous reports. The nucleotide sequence analysis of RT‐PCR products for four NDV strains was fully in agreement with the RT‐PCR characterisations of these strains. The RT‐PCR results of other avian RNA viruses further confirmed the reliability and specificity of this technique. However, the RT‐PCR failed to detect some other avian NDV, which may not originate from chicken. This multiplex RT‐PCR technique is simple and easy to perform. It could be applied not only to determine the origin of NDV, but also may be used diagnostically in molecular epidemiological analysis of ND and for prediction of pathotypes of NDV isolates.

An in vitro and in vivo evaluation of the virulence of Newcastle disease virus and vaccines for the chicken reproductive tract

The virulence of two vaccine strains and two field strains of Newcastle disease virus (NDV) for the female reproductive tract of chickens was assessed using oviduct organ cultures (OOC) prepared from precociously-induced oviducts in young chicks by oestrogen treatment. Ciliostasis, haemagglutination and virus isolation from infected OOC supernatants, histopathology and immunoperoxidase test results indicated the pathogenic nature of both vaccine and virulent NDVs for the precocious oviducts. The virulent viruses, mesogenic and lentogenic vaccines caused damage in that order of magnitude and the uterus had a higher susceptibility than oviducts. One virulent and the mesogenic strain of NDV were used for in vivo trials. The pathogenicity was assessed in oestrogen-treated infected chickens using histopathology and immunoperoxidase test. The vaccine virus produced transient damage up to 6 days post-infection, while the damage with the virulent isolate persisted for at least 9 days post-infection. This technique could be a pointer to possible variations in virulence of NDV vaccine and field strains, and warrants further investigation. The potential value of OOC from young chickens for testing the possibility of NDV vaccines causing damage by themselves and offering protection against damage of the reproductive tract caused by virulent isolates is emphasized.

Newcastle disease outbreaks in western China were caused by the genotypes VIIa and VIII

Twelve Newcastle disease virus (NDV) strains were isolated from chickens involved in outbreaks of Newcastle disease (ND) in western China (Shaanxi, Gansu, Xinjiang, Qinghai and Guangxi provinces) between 1979 and 1999. All strains were determined to be velogenic by plaque formation, the mean death time (MDT) of embryonated eggs, and the intracerebral pathogenicity index (ICPI). For preparation of virus RNA, the acid guanidinium-thiocyanate method was used. A 908 bp fragment of nucleotide was amplified by RT-PCR starting from the N terminal of the F gene and the PCR segments were cloned into the PGEM-T vector and sequenced. The similarities of the nucleotide sequences (1–519 bp) and predicted amino acid sequences of the F gene (1–125) were analyzed by comparing the 12 NDV isolates with the NDV vaccine strains Lasota, B1, H1 and V4, with classical NDV strains and recent epizootic strains. Phylogenetic analysis demonstrated that all strains were of two novel genotypes; the NDV strains that caused the outbreak of ND in western China during 1998–1999 was of the genotype VIIa, whereas the strains from the Qinghai province (1979–1985) were of genotype VIII, which has been found predominately in southern Africa.