Lentogenic Newcastle Disease Virus Strain Research Articles

Deciphering whole genome sequence of a Newcastle disease virus genotype VII.2 isolate from a commercial poultry farm in India

Here we report the isolation and complete genome sequence of virulent Newcastle disease virus (NDV) during the outbreak in 2023. NDV is isolated from the infected egg-laying vaccinated chickens of poultry farms located in Raipur district in Chhattisgarh, India. Next-generation sequencing (NGS) revealed the present NDV isolate has a genome length of 15,118 nucleotides (GenBank: OR185447). The phylogenetic tree illustrates the evolutionary relationship among NDV species and populations indicating the present NDV strain (NDV/Chicken/RPR/01/23) classified as genotype VII in class 2. NDV isolate exhibited 99.19 % and 97.53 % similarities in the nucleotide sequence in comparison with other strains of NDV/Owl/Guwahati/01/20 (GenBank: MZ546197.1) from India and chicken/Banjarmasin/010/10 (GenBank: HQ697254.1) from Indonesia. The present NDV genome is closely aligned with the NDV/Owl/Guwahati/01/20 strain and was clustered in genotype VII in class 2 suggesting possible spillover among the viral strains. The amino acid sequence at the protein Fusion protein (F) cleavage sequence 112RRQKR*F117 which is distinctive with other lentogenic vaccine NDV strain of La Sota and postulated as a major determinant of NDV virulence. Since there is limited information available on the exact cause of the abrupt death of poultry chickens located in Raipur district in Chhattisgarh, India, the present study aims to examine molecular sequencing of circulating NDV strains causing high mortality in this region. The neutralization epitopes of virulent NDV strains will expedite in development next next-generation vaccines to control circulating viral strains across the globe. The derived scientific information on genetic diversity, current taxonomic classification, and antigenic variations that exist within the genome of NDV will help in developing strain-specific vaccines and implementing strict measures.

Genetic characterization and distribution of the virus in chicken embryo tissues infected with Newcastle disease virus isolated from commercial and native chickens in Indonesia.

Background and Aim:Newcastle disease (ND) is a viral infectious disease that affects commercial and native chickens, resulting in economic losses to the poultry industry. This study aimed to examine the viral strains circulating in commercial and native chickens by genetic characterization and observe the distribution of Newcastle disease virus (NDV) in chicken embryonic tissue.Materials and Methods:ND was detected using a quantitative reverse transcription-polymerase chain reaction. Genetic characterization of the fusion (F) and hemagglutinin-neuraminidase (HN) genes from the eight NDVs was performed using specific primers. The sequence was compared with that of other NDVs from GenBank and analyzed using the MEGA-X software. The distribution of NDV in chicken embryos was analyzed based on lesions and the immunopositivity in immunohistochemistry staining.Results:Based on F gene characterization, velogenic NDV strains circulating in commercial and native chickens that showed varying clinical symptoms belonged to genotype VII.2. Lentogenic strains found in chickens without clinical symptoms were grouped into genotype II (unvaccinated native chickens) and genotype I (vaccinated commercial chickens). Amino acid variations in the HN gene, namely, the neutralization epitope and antigenic sites at positions 263 and 494, respectively, occurred in lentogenic strains. The NDV reaches the digestive and respiratory organs, but in lentogenic NDV does not cause significant damage, and hence embryo death does not occur.Conclusion:This study showed that velogenic and lentogenic NDV strains circulated in both commercial and native chickens with varying genotypes. The virus was distributed in almost all organs, especially digestive and respiratory. Organ damage in lentogenic infection is not as severe as in velogenic NDV. Further research is needed to observe the distribution of NDV with varying pathogenicity in chickens.

Prevalence of Newcastle Disease Virus in Wild and Migratory Birds in Haryana, India.

Newcastle disease virus (NDV) can infect approximately 250 avian species and causes highly contagious Newcastle disease (ND) in domestic poultry, leading to huge economic losses. There are three different pathotypes of NDV, i.e., lentogenic, mesogenic, and velogenic. Wild resident (wild) and migratory birds are natural reservoirs of NDV and are believed to play a key role in transmitting the virus to domestic poultry. The present study was conducted to determine the prevalence of NDV in wild and migratory birds in the state of Haryana, India, during two migratory seasons (2018-19 and 2019-20). In total 1379 samples (1368 choanal swabs and 11 tissue samples) were collected from live (n = 1368) or dead birds (n = 4) belonging to 53 different avian species. These samples belonged to apparently healthy (n = 1338), sick (n = 30), and dead (n = 4) birds. All samples were tested for NDV by real-time reverse transcription-PCR using M gene specific primers and probe. Of the 1379 samples, 23 samples from wild birds [Columba livia domestica (n = 12, 52.17%), Pavo cristatus (n = 9, 39.13%), and Psittaciformes (n = 2, 8.69%)] were found positive for NDV. Only one of the 23 samples (from P. cristatus) was positive for F gene, indicating it to be a mesogenic/velogenic strain. These results indicate that both lentogenic and velogenic strains of NDV are circulating in wild birds in Haryana and that further studies are needed to characterize NDV strains from wild/migratory birds and domestic poultry to determine the extent of virus transmission among these populations. This study considers the disease transmission risk from domestic pigeons and parrots to commercial poultry and vice versa, and the results emphasize the need for strict biosecurity strategies to protect commercial poultry in the region.

Molecular differentiation between velogenic isolates and lentogenic LaSota strain of Newcastle disease virus

Objective: Isolation, serological and molecular identification and differentiation between velogenic and lentogenic of Newcastle disease virus (NDV) strains. Design: Descriptive study. Procedures: In this study, A total of 24 Pooled samples were collected from Dakahlia, Egypt, from 2017 to 2018 .The virus was isolated in 10 days old (ECEs) and serologically identified in third egg passage allantoic fluid by Haemagglutination (HA), Haemagglutination inhibition test (HIT) and agar gel precipitation test (AGPT). Molecular confirmation was done by reverse transcriptase-polymerase chain reaction (RT-PCR). Genetic diversity between velogenic and lentogenic NDV strains was done by RT-PCR amplification of 254bp F gene fragment from velogenic NDVs and sequence analysis of 362bp NDV F gene fragment. Results: Out of 24 tested samples, 20 samples were collected from allontoic fluid of 3rd passage positive by HA test, 18 samples by HIT, 16 samples by AGPT and 18 isolates were positive with RT-PCR amplification of 362bp F gene fragment. LaSota strain gave positive results in HA, HIT, AGPT and RT-PCR amplification of 362bp fragment. Interestingly, 254bp fragment failed to be amplified from LaSota strain. The phylogenic analysis revealed that our isolates were related to Egyptian velogenic genotype VII NDVs. The deduced amino acid sequence of the F protein cleavage site of our isolates was characteristic of velogenic NDVs (112R-R-Q-K-R↓F117) while LaSota strain had characteristic lentogenic NDVs cleavage site (112G-R-Q-G-R↓L117). Conclusion and clinical relevance: RT-PCR and sequence analysis in this study can be used for differentiation between velogenic and lentogenic NDV strains.

Genetic Basis of Response of Ghanaian Local Chickens to Infection With a Lentogenic Newcastle Disease Virus.

Newcastle disease (ND) is a global threat to domestic poultry, especially in rural areas of Africa and Asia, where the loss of entire backyard local chicken flocks often threatens household food security and income. To investigate the genetics of Ghanaian local chicken ecotypes to Newcastle disease virus (NDV), in this study, three popular Ghanaian chicken ecotypes (regional populations) were challenged with a lentogenic NDV strain at 28 days of age. This study was conducted in parallel with a similar study that used three popular Tanzanian local chicken ecotypes and after two companion studies in the United States, using Hy-line Brown commercial laying birds. In addition to growth rate, NDV response traits were measured following infection, including anti-NDV antibody levels [pre-infection and 10 days post-infection (dpi)], and viral load (2 and 6 dpi). Genetic parameters were estimated, and two genome-wide association study analysis methods were used on data from 1,440 Ghanaian chickens that were genotyped on a chicken 600K Single Nucleotide Polymorphism (SNP) chip. Both Ghana and Tanzania NDV challenge studies revealed moderate to high (0.18 – 0.55) estimates of heritability for all traits, except viral clearance where the heritability estimate was not different from zero for the Tanzanian ecotypes. For the Ghana study, 12 quantitative trait loci (QTL) for growth and/or response to NDV from single-SNP analyses and 20 genomic regions that explained more than 1% of genetic variance using the Bayes B method were identified. Seven of these windows were also identified as having at least one significant SNP in the single SNP analyses for growth rate, anti-NDV antibody levels, and viral load at 2 and 6 dpi. An important gene for growth during stress, CHORDC1 associated with post-infection growth rate was identified as a positional candidate gene, as well as other immune related genes, including VAV2, IL12B, DUSP1, and IL17B. The QTL identified in the Ghana study did not overlap with those identified in the Tanzania study. However, both studies revealed QTL with genes vital for growth and immune response during NDV challenge. The Tanzania parallel study revealed an overlapping QTL on chromosome 24 for viral load at 6 dpi with the US NDV study in which birds were challenged with NDV under heat stress. This QTL region includes genes related to immune response, including TIRAP, ETS1, and KIRREL3. The moderate to high estimates of heritability and the identified QTL suggest that host response to NDV of local African chicken ecotypes can be improved through selective breeding to enhance increased NDV resistance and vaccine efficacy.

In Vitro Oncolytic activity of non-virulent Newcastle Disease Virus LaSota Strain against Mouse mammary adenocarcinoma

Newcastle disease virus (NDV) is a wide-spectrum anti-tumor agent. The oncolytic selectivity of NDV, a family of Paramyxoviridae, depends on the differential type of inducing different death pathways. This work was conducted to further understand the oncolytic effect of LaSota strain. A mouse breast cancer model (Murine mammary adenocarcinoma cell line AMN3) was used in this study. Methyl Thiazolyl Tetrazolium (MTT) viability assay tested different NDV multiplicity of infection (MOI) values on mouse mammary adenocarcinoma cells incubated for 72 hours post-infection. The IC50 values and anti-tumor activity of LaSota strain against AMN3 cell line were determined. Following Hematoxylin and Eosin Stain, we examined the morphological modifications of IC50 along with 10 MOI values of NDV. The induction of NDV apoptosis in AMN3 cells was investigated using the technique of staining acridine orange and propidium iodide (AO / PI). Immunocytochemistry assay was performed using anti-NDV mAbs and caspases 8 and 9 to study NDV replication and apoptosis induction mechanisms. The lentogenic LaSota NDV strain, a live vaccine, demonstrated the oncolytic effect on mammary cancer cells of the AMN3 mouse and showed that LaSota strain triggered a dose-dependent increase in infected cells’ apoptosis relative to untreated mammary cancer cells. The immunocytochemistry study showed that NDV infected cells were positive for virus infection and that caspase9 in mouse mammary cancer cells after LaSota strain infection was significantly enhanced compared to caspase 8. In conclusions, NDV LaSota strain had oncolytic effects by destroying tumor cells and triggering the intrinsic apoptosis pathways in mouse mammary cancer cells. However, the mechanisms of the in vivo anti-tumor activity need to be better understood.

A class Ⅰ lentogenic newcastle disease virus strain confers effective protection against the prevalent strains

The traditional vaccine strains, such as LaSota, do not completely prevent the shedding of NDV. An ideal vaccine which could not only prevent the clinical signs, but significantly reduce the shedding of NDV is urgently needed for the eradication of ND. In this study, an NDV isolate APMV-1/Chicken/China (SC)/PT3/2016 (hereafter referred as PT3) was identified as a class Ⅰ NDV and a lentogenic strain. The antigenic relationship between PT3 and 3 other NDV strains, including vaccine strain LaSota and 2 prevalent genotype Ⅶd and Ⅵb strains were analyzed. The protective efficacy of PT3 and LaSota against challenge with genotype Ⅶd and Ⅵb strains were assessed. The antigenic analysis result showed that 4 strains belong to the single serotype and the PT3 antiserum exhibited the highest HI titer against 3 other NDV strains. The results of protective efficacy showed that both of LaSota and PT3 could provide 100% survivability for infected chickens. However, PT3 performed better in inducing higher humoral responses and reducing virus shedding than the LaSota strain. Lentogenic strains from Class I NDV appear to be promising vaccine candidates for the control of ND, and allows for the easy discrimination of field NDV and vaccine strains.

Cormorants as Potential Victims and Reservoirs of Velogenic Newcastle Disease Virus (Orthoavulavirus-1) in Central Asia.

Virulent strains of avian orthoavulavirus 1, historically known as Newcastle disease virus (NDV), are widespread and cause high levels of mortality in poultry worldwide. Wild birds may play an important role in the maintenance of Avian orthoavulavirus 1 in nature. Prior to 2014, most of the lentogenic NDV strains isolated from Central Asia were obtained from the avian order Anseriformes (ducks and geese). Wild birds were monitored from 2014-2016 to detect the circulation of NDV. A total of 1522 samples belonging to 73 avian species were examined, and 26 positive samples were identified. The isolates of Avian orthoavulavirus 1 belonged to three genotypes: viruses from doves (Columbiformes) and cormorants (Suliformes) were attributed to the velogenic genotypes VI and XIII, respectively, while the isolate from poultry belonged to lentogenic genotype I. The isolation of Avian orthoavulavirus 1 from doves may confirm their role as a reservoir of pigeon paramyxoviruses (antigenic variant of the genotype VI NDV) in nature and indicates the potential threat of introduction of velogenic strains into the poultry population. Our study describes an epizootic scenario in Kazakhstan among cormorants with mortality among juveniles of up to 3 wk of age and isolation of the NDV from apparently healthy birds. These observations may support the idea that cormorants are one of the potential reservoirs and victims of velogenic Avian orthoavulavirus 1 in Central Asia. The seasonal migrations of cormorants may partially contribute to viral dissemination throughout the continent; however, this hypothesis needs more evidence.

A novel recombinant attenuated Newcastle disease virus expressing H9 subtype hemagglutinin protected chickens from challenge by genotype VII virulent Newcastle disease virus and H9N2 avian influenza virus

Newcastle disease virus (NDV) and H9 subtype avian influenza virus (AIV) are two avian pathogens across the globe. Inasmuch as most poultry flocks worldwide are vaccinated with a live low-virulence or attenuated NDV vaccine, we embarked on the development of vaccine prototypes that would have dual specificities and would allow a single immunization against both avian influenza (AI) and Newcastle disease (ND). Therefore, in the present work, a cloned full-length copy of the genome of the lentogenic NDV strain rmNA-1 was selected as a backbone vector to construct three chimeric NDVs that expressed (i) the ORF encoding the HA, (ii) the ectodomain of HA fused with the transmembrane domain and cytoplasmic tail regions derived from the NDV F protein and (iii) the ectodomain of HA fused with a short GS linker and the GCN4 sequences, and designated as rmNA-H9, rmNA-H9F, and rmNA-H9 (ECTO), respectively. rmNA-H9, rmNA-H9F, and rmNA-H9 (ECTO) stably expressed the modified HA gene for 10 egg passages and the three recombinants were found innocuous to chickens. The insertion of the chimeric HA-F, rather than HA-ECTO or ORF of HA, resulted in a recombinant virus with enhanced incorporation of the HA protein into the viral surface. A single immunization of SPF chickens with the three recombinants induced NDV- and AIV H9-specific antibodies, and protected chickens against a challenge with a lethal dose of velogenic NDV or AIV H9N2. Remarkably, non-shedding of influenza virus and higher levels of H9 subtype HI titers were observed 7 days post challenge (dpc) in rmNA-H9F vaccinated chickens, than other recombinants. Furthermore, a prime-boost vaccination of chickens with rmNA-H9F induced higher levels of NDV- and H9- HI and secretory IgA, as well as reduced viral shedding and virus-induced gross lesions, compared with the commercial vaccine. Therefore, the recombinant rmNA-H9F is a promising bivalent vaccine candidate against NDV and H9 subtype AIV in chickens.

Cleavage site of Newcastle disease virus determines viral fitness in persistent infection cells

Newcastle disease, caused by infection with virulent strains of Newcastle disease virus (NDV), poses a risk for the poultry industry. The virulence of NDV is mainly determined by the cleavage site of F protein. Lentogenic NDV can become velogenic after passages in SPF chicken brain and air sac based on some strains isolated from water birds, because the proportion of virulent-related strains gradually increases. In contrast, this proportion remains unchanged if NDV is passaged via 10-day-old SPF chicken embryos. This information suggests that environmental conditions rather than mutation affect NDV fitness in quasispecies. However, it is unknown how the environment selects virulent-related strains from a viral population. In this study, velogenic and lentogenic NDV marked by green or red fluorescence were used to establish persistent infection (PI) in BHK-21 cells. Monitoring viruses by different methods, we found that, without competition, persistently infected cells harbored lentogenic and velogenic NDV strains similarly in terms of viral release, viral spread and the period of persistent viral infection. In contrast, under competitive co-infection, velogenic NDV became dominant in quasispecies from the fifth passage of PI cells, which resulted in the progressive disappearance of the lentogenic NDV strain. This domination was concomitant with a short-term reduction in the superinfection exclusion and supernatant interference in PI cells resulting in a velogenic virus rebound. We concluded that virulent-related F protein cleavage site facilitates the spread and replication of NDV in conditions under which cells do not secret trypsin-like proteases and do not inhibit free virus infection, resulting in a gradual increase in virulent strains in quasispecies with the number of passages.

Immune responses of mature chicken bone-marrow-derived dendritic cells infected with Newcastle disease virus strains with differing pathogenicity.

Infection of chickens with virulent Newcastle disease virus (NDV) is associated with severe pathology and increased morbidity and mortality. The innate immune response contributes to the pathogenicity of NDV. As professional antigen-presenting cells, dendritic cells (DCs) play a unique role in innate immunity. However, the contribution of DCs to NDV infection has not been investigated in chickens. In this study, we selected two representative NDV strains, i.e., the velogenic NDV strain Chicken/Guangdong/GM/2014 (GM) and the lentogenic NDV strain La Sota, to investigate whether NDVs could infect LPS-activated chicken bone-derived marrow DCs (mature chicken BM-DCs). We compared the viral titres and innate immune responses in mature chicken BM-DCs following infection with those strains. Both NDV strains could infect mature chicken BM-DC, but the GM strain showed stronger replication capacity than the La Sota strain in mature chicken BM-DCs. Gene expression profiling showed that MDA5, LGP2, TLR3, TLR7, IFN-α, IFN-β, IFN-γ, IL-1β, IL-6, IL-18, IL-8, CCL5, IL-10, IL-12, MHC-I, and MHC-II levels were altered in mature DCs after infection with NDVs at all evaluated times postinfection. Notably, the GM strain triggered stronger innate immune responses than the La Sota strain in chicken BM-DCs. However, both strains were able to suppress the expression of some cytokines, such as IL-6 and IFN-α, in mature chicken DCs at 24 hpi. These data provide a foundation for further investigation of the role of chicken DCs in NDV infection.

Different Origins of Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein Modulate the Replication Efficiency and Pathogenicity of the Virus.

To investigate the exact effects of different origins of Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein to the biological characteristics of the virus, we systematically studied the correlation between the HN protein and NDV virulence by exchanging the HN of velogenic or lentogenic NDV strains with the HN from other strains of different virulence. The results revealed that the rSG10 or rLaSota derivatives bearing the HN gene of other viruses exhibited decreased or increased hemadsorption (HAd), neuraminidase and fusion promotion activities. In vitro and in vivo tests further showed that changes in replication level, tissue tropism and virulence of the chimeric viruses were also consistent with these biological activities. These findings demonstrated that the balance among three biological activities caused variation in replication and pathogenicity of the virus, which was closely related to the origin of the HN protein. Our study highlights the importance of the HN glycoprotein in modulating the virulence of NDV and contributes to a more complete understanding of the virulence of NDV.

Detection of specific antigens of Newcastle disease virus using an absorbed Western blotting method.

Newcastle disease virus (NDV) is an economically important poultry pathogen with a worldwide distribution that may infect a wide range of domestic and wild avian species. The identification of different pathotypes of NDVs plays an important role in the diagnosis and development of vaccines to control and eradicate NDV infections. In our previous study, we showed that mono-specific antibodies can differentiate velogenic and lentogenic strains of NDV in Agar Gel Immuno-Diffusion tests. To evaluate the ability of the specific antibodies to detect NDV specific antigens, this study was conducted with a range of NDV isolates. The samples included 9 NDV neuropathogenic/velogenic isolates from diseased chickens collected from poultry farms in central and northern parts of Iran plus La-Sota and B1 vaccine strains. All samples were propagated in embryonated chicken eggs and concentrated and purified by ultra-centrifugation. All samples were subjected to 12.5% SDS-PAGE and Western blotting using the specific antibodies mentioned previously. In SDS-PAGE all velogenic and vaccine strains showed the same electrophoretic pattern. The detected bands included 15, 38, 46, 48, 53, 55, 68, 74 and 220 kDa proteins. In Western blotting analysis, the mono-specific antibodies reacted specifically to the viral proteins with 15, 38, 48, 55, 74 and 220 kDa and non-specifically to the viral protein with 53 kDa. The results suggest that specific anti-NDV antibodies can react specifically to glycoproteins (haemagglutin-neuraminidase and fusion proteins) but not to internal proteins (nucleoprotein or matrix protein) of NDV strains.

Phylogenetic analysis of some Newcastle disease virus isolates from the Sudan.

A reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify 1412 bp of the fusion protein gene (F gene) of four Newcastle disease virus (NDV) isolates; two velogenic (TY-1/90 and DIK-90) and two lentogenic isolates (Dongla 88/1 and GD.S.1). Following sequencing, nucleotide sequences were annotated and 894 bp were compared phylogenetically with those from strains previously reported in the Sudan and the virus strains published on the GenBank. It could be demonstrated that TY-1/90 and DIK-90 strains belong to the genotype VI of NDV and are in close genetic relationship to sub- genotype VIb. TY-1/90 and DIK-90 strains were observed to be genetically unrelated to the earlier Sudanese isolates of 1970/80s and the late of 2000s suggesting a different origin. The close genetic relationship to the European and African pigeon paramyxovirus type 1 (PPMV-1) suggests a common ancestor. Dongola, GD.S.1 strains were classified into genotype II that comprises non-pathogenic lentogenic NDV strains. The present genetic classification of NDV isolates of the Sudan provides valuable information on genotypes of NDV. Further molecular epidemiological investigations of the recent outbreaks of Newcastle disease in the Sudan are needed in order to improve the efficiency of control strategies and vaccine development.

Evolution of Newcastle Disease Virus Quasispecies Diversity and Enhanced Virulence after Passage through Chicken Air Sacs.

It has been reported that lentogenic Newcastle disease virus (NDV) isolates have the potential to become velogenic after their transmission and circulation in chickens, but the underlying mechanism is unclear. In this study, a highly velogenic NDV variant, JS10-A10, was generated from the duck-origin lentogenic isolate JS10 through 10 consecutive passages in chicken air sacs. The velogenic properties of this selected variant were determined using mean death time (MDT) assays, intracerebral pathogenicity index (ICPI), the intravenous pathogenicity index (IVPI), histopathology, and the analysis of host tissue tropism. In contrast, JS10 remained lentogenic after 20 serial passages in chicken eggs (JS10-E20). The JS10, JS10-A10, and JS10-E20 genomes were sequenced and found to be nearly identical, suggesting that both JS10-A10 and JS10-E20 were directly generated from JS10. To investigate the mechanism for virulence enhancement, the partial genome covering the F0 cleavage site of JS10 and its variants were analyzed using ultradeep pyrosequencing (UDPS) and the proportions of virulence-related genomes in the quasispecies were calculated. Velogenic NDV genomes accumulated as a function of JS10 passaging through chicken air sacs. Our data suggest that lentogenic NDV strains circulating among poultry might be a risk factor to future potential velogenic NDV outbreaks in chickens. An avirulent isolate, JS10, was passaged through chicken air sacs and embryos, and the pathogenicity of the variants was assessed. A virulent variant, JS10-A10, was generated from consecutive passage in air sacs. We developed a deep-sequencing approach to detect low-frequency viral variants across the NDV genome. We observed that virulence enhancement of JS10 was due to the selective accumulation of velogenic quasispecies and the concomitant disappearance of lentogenic quasispecies. Our results suggest that because it is difficult to avoid contact between natural waterfowl reservoirs and sensitive poultry operations, circulating lentogenic NDV strains may represent a potential reservoir for emergent velogenic NDV strains that could cause outbreaks in chickens.

Previous infection with virulent strains of Newcastle disease virus reduces highly pathogenic avian influenza virus replication, disease, and mortality in chickens.

Highly pathogenic avian influenza virus (HPAIV) and Newcastle disease virus (NDV) are two of the most important viruses affecting poultry worldwide and produce co-infections especially in areas of the world where both viruses are endemic; but little is known about the interactions between these two viruses. The objective of this study was to determine if co-infection with NDV affects HPAIV replication in chickens. Only infections with virulent NDV strains (mesogenic Pigeon/1984 or velogenic CA/2002), and not a lentogenic NDV strain (LaSota), interfered with the replication of HPAIV A/chicken/Queretaro/14588-19/95 (H5N2) when the H5N2 was given at a high dose (106.9 EID50) two days after the NDV inoculation, but despite this interference, mortality was still observed. However, chickens infected with the less virulent mesogenic NDV Pigeon/1984 strain three days prior to being infected with a lower dose (105.3–5.5 EID50) of the same or a different HPAIV, A/chicken/Jalisco/CPA-12283-12/2012 (H7N3), had reduced HPAIV replication and increased survival rates. In conclusion, previous infection of chickens with virulent NDV strains can reduce HPAIV replication, and consequently disease and mortality. This interference depends on the titer of the viruses used, the virulence of the NDV, and the timing of the infections. The information obtained from these studies helps to understand the possible interactions and outcomes of infection (disease and virus shedding) when HPAIV and NDV co-infect chickens in the field.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0237-5) contains supplementary material, which is available to authorized users.

Neuropathogenic Capacity of Lentogenic, Mesogenic, and Velogenic Newcastle Disease Virus Strains in Day-Old Chickens.

Strains of Newcastle disease virus (NDV) have different abilities to elicit neurologic signs. To determine the capacity of different NDV strains to replicate and cause lesions in the brain, independently of their peripheral replication, 1-day-old chickens were inoculated in the subdural space with 7 NDV strains of different virulence (4 velogenic, 2 mesogenic, 1 lentogenic). Velogenic strains induced severe necrotizing and heterophilic ventriculitis and meningitis, as well as edema of the neuroparenchyma, and replicated extensively in the nervous tissue by day 2 postinfection, as demonstrated by immunohistochemistry, when all infected birds died. Clinical signs, microscopic lesions, and viral replication were delayed (days 3 and 4 postinfection) with mesogenic strains. Velogenic and mesogenic NDV strains replicated mainly in neurons, and immunolabeling was first detected in surface-oriented areas (periventricular and submeningeal), possibly as a reflection of the inoculation route. The lentogenic NDV strain did not cause death of infected birds; replication was confined to the epithelium of the ependyma and choroid plexuses; and lesions consisted of lymphoid aggregates limited to the choroid plexuses. Results show that extensive NDV replication in the brain is typical of velogenic and mesogenic, but not lentogenic, NDV strains. In addition, this study suggests that differences in the rate of NDV replication in nervous tissue, not differences in neurotropism, differentiate velogenic from mesogenic NDV strains. This study indicates that intracerebral inoculation might be used as an effective method to study the mechanisms of NDV neuropathogenesis.

Strains of Newcastle disease Virus, Circulating among Sick Poultry Flocks in Nsukka-Nigeria

Aim: To study prevalence of the different strains of Newcastle disease virus (NDV) circulating among sick poultry flocks, NDV detected from disease outbreaks in Nsukka-Nigeria were characterized into the three main NDV strains. Materials and Methods: NDV was detected from 63 cases of poultry disease that manifested clinical signs and lesions suggestive of Newcastle disease (ND), by the haemagglutination test. The detected NDV samples were characterized by a combination of their ability to agglutinate mammalian erythrocytes and their erythrocyte elution time (EET). Results: Fifty three out of 63 (84%) NDV samples detected from the ND cases belonged to Lentogenic strains while 10 (16%) were Mesogenic strains. None was of the Velogenic strains. Conclusion: Rates of circulation of Lentogenic, Mesogenic and Velogenic NDV strains among sick poultry flocks in Nsukka- Nigeria suggest that the practice of using live NDV vaccines for prophylaxis may be an important factor in epizootiology of Newcastle disease in the country.

Genomic characterisation of a lentogenic Newcastle disease virus strain HX01 isolated from sick pigs in China

This paper describes the complete genome sequence of HX01, an isolate of the Newcastle disease virus (NDV) collected from a swine disease outbreak. The genome is 15,186 nt long and consists of six genes in the order of 3'-NP-P-M-F-HN-L-5'. This genome has the same length as the old NDV genotypes (I-IV), whereas the new NDV genotypes (V-IX) are 15,192 nt long. Compared with the genomic sequences of the reference NDV strains, the HX01 genome is highly similar to the genome of other NDV strains. However, some unique features of the HN gene were found in HX01. HX01 possesses the motif (112)G-R-Q-G-R-L(117) at the fusion protein cleavage site, which is typical of lentogenic strains. Pathogenicity tests based on the mean death time and the intracerebral pathogenicity index also revealed the isolate's lentogenic character. Phylogenetic analysis based on the variable region of the F gene (nt 47-420) revealed that HX01 was clustered to genotype II within class II NDV. Genetically, HX01 has a high similarity with the La Sota vaccine strain based on the single gene or complete genomic but is far different from the prevalent genotype VIId NDV which circulates in fowls and waterfowls in mainland China.

Genomic characterization of the first class I Newcastle disease virus isolated from the mainland of China

The complete genomic sequence of Newcastle disease virus (NDV) strain NDV08-004, isolated from domestic ducks in China, was determined in this study. The genome is 15198 nucleotides (nt) in length, follows the "rule of six" and contains a 55-nt leader sequence at the 3' end and a 114-nt trailer sequence at the 5' end. Compared with the full genome sequences of Class II NDV strains, the NDV08-004 isolate has a 12-nt insertion (TGGGAGACGGGG) in the phosphoprotein gene between nucleotides 2381 and 2382 of the genome (numbered according to the genomic sequence of the La Sota strain, which consists of 15186 nt). Strain NDV08-004 has the motif (112)E-Q-Q-E-R-L(117) at the cleavage site of the fusion protein, which is typical of lentogenic NDV strains, and this is in agreement with the results of pathogenic tests based on the mean death time (MDT) and the intracerebral pathogenicity index (ICPI). Phylogenetic analysis based on the full genome revealed that all the NDV strains studied could be divided into two distinct clades, namely class I and class II, and the NDV08-004 isolate characterized in this study was grouped in class I. Further phylogenetic analysis based on a 374-bp fragment of the F gene in class I strains of NDV demonstrated that NDV08-004 belongs to genotype 3, and should be therefore similar to strains obtained from live bird markets in Hong Kong in recent years.