Search

Kenyan poultry consists of ~80% free-range indigenous chickens kept in small flocks (~30 birds) on backyard poultry farms (BPFs) and they are traded via live bird markets (LBMs). Newcastle disease virus (NDV) was detected in samples collected from chickens, wild farm birds, and other domestic poultry species during a 2017-2018 survey conducted at 66 BPFs and 21 LBMs in nine Kenyan counties. NDV nucleic acids were detected by rRT-PCR L-test in 39.5% (641/1621) of 1621 analyzed samples, of which 9.67% (62/641) were NDV-positive by both the L-test and a fusion-test designed to identify the virulent virus, with a majority being at LBMs (64.5%; 40/62) compared to BPFs (25.5%; 22/62). Virus isolation and next-generation sequencing (NGS) on a subset of samples resulted in 32 complete NDV genome sequences with 95.8-100% nucleotide identities amongst themselves and 95.7-98.2% identity with other east African isolates from 2010-2016. These isolates were classified as a new sub-genotype, V.3, and shared 86.5-88.9% and 88.5-91.8% nucleotide identities with subgenotypes V.1 and V.2 viruses, respectively. The putative fusion protein cleavage site ( 113 R-Q-K-R↓F 117 ) in all 32 isolates, and a 1.86 ICPI score of an isolate from a BPF chicken that had clinical signs consistent with Newcastle disease, confirmed the high virulence of the NDVs. Compared to genotypes V and VI viruses, the attachment (HN) protein of 18 of the 32 vNDVs had amino acid substitutions in the antigenic sites. A time-scaled phylogeographic analysis suggests a west-to-east dispersal of the NDVs via the live chicken trade, but the virus origins remain unconfirmed due to scarcity of continuous and systematic surveillance data. This study reveals the widespread prevalence of vNDVs in Kenyan backyard poultry, the central role of LBMs in the dispersal and possibly generation of new virus variants, and the need for robust molecular epidemiological surveillance in poultry and non-poultry avian species.

Newcastle disease (ND) is a major constraint to Kenya's poultry production, which is comprised of approximately 80% indigenous chickens (ICs; caged and free-range system) and 20% exotic chickens (intensive system). This study analyzed cases reported as suspected ND in Kenya between 2005 and 2015. Of the suspected 332 ND reported cases from the three production systems in 27 locations within six Kenyan Agro-Ecological Zones (AEZs), 140 diagnosed as infected with avian orthoavulavirus 1 (AOaV-1; formerly Newcastle disease virus) were present in every year in all AEZs. The numbers of AOaV-1-positive cases differed significantly (p < 0.05) between the production systems across the years depending on the season, climate, and location. In the free-range system, both ambient temperatures and season associated significantly (p = 0.001 and 0.02, respectively) with the number of cases, while in the intensive and caged systems, the positive cases correlated significantly with season and relative humidity, respectively (p = 0.05). Regardless of the production systems, the numbers of clinically sick birds positively correlated with the ambient temperatures (r = 0.6; p < 0.05). Failure to detect AOaV-1 in 58% of the ND cases reported, and mortalities exceeding the observed numbers of clinically sick birds suggest deficiencies in the current ND reporting and diagnostic system. Intensive farmers were the slowest in reporting the cases and diagnostic deficiencies were most evident by failure to test the exposure of ICs to natural infection with AOaV-1 and for the AOaV-1-negative cases lack of testing for other pathogens and/or AOaV-1 variants. This study indicates a need for improved surveillance and diagnostics in Kenyan domestic poultry.

Background: Newcastle disease (ND), which is caused by infections of poultry species with virulent strains of Avian orthoavulavirus-1, also known as avian paramyxovirus 1 (APMV-1), and formerly known as Newcastle disease virus (NDV), may cause neurological signs and encephalitis. Neurological signs are often the only clinical signs observed in birds infected with neurotropic strains of NDV. Experimental infections have shown that the replication of virulent NDV (vNDV) strains is in the brain parenchyma and is possibly confined to neurons and ependymal cells. However, little information is available on the ability of vNDV strains to infect subset of glial cells (astrocytes, oligodendrocytes, and microglia). The objective of this study was to evaluate the ability of NDV strains of different levels of virulence to infect a subset of glial cells both in vitro and in vivo. Thus, neurons, astrocytes and oligodendrocytes from the brains of day-old White Leghorn chickens were harvested, cultured, and infected with both non-virulent (LaSota) and virulent, neurotropic (TxGB) NDV strains. To confirm these findings in vivo, the tropism of three vNDV strains with varying pathotypes (SA60 [viscerotropic], TxGB [neurotropic], and Tx450 [mesogenic]) was assessed in archived formalin-fixed material from day-old chicks inoculated intracerebrally., Results: Double immunofluorescence for NDV nucleoprotein and cellular markers showed that both strains infected at least 20% of each of the cell types (neurons, astrocytes, and oligodendrocytes). At 24 h post-inoculation, TxGB replicated significantly more than LaSota. Double immunofluorescence (DIFA) with markers for neurons, astrocytes, microglia, and NDV nucleoprotein detected the three strains in all three cell types at similar levels., Conclusion: These data indicate that similar to other paramyxoviruses, neurons and glial cells (astrocytes, oligodendrocytes, and microglia) are susceptible to vNDV infection, and suggest that factors other than cellular tropism are likely the major determinant of the neurotropic phenotype.

Our prior work has shown that live poultry vaccines have been intermittently isolated from wild birds sampled during field surveillance studies for Newcastle disease virus (NDV). Thus, we experimentally investigated the susceptibility of four native agriculturally associated wild bird species to the NDV LaSota vaccine and evaluated the shedding dynamics, potential transmission from chickens, and humoral antibody responses. To test susceptibility, we inoculated wild-caught, immunologically NDV-naïve house finches ( Haemorhous mexicanus ; n = 16), brown-headed cowbirds ( Molothrus ater ; n = 9), northern cardinals ( Cardinalis cardinalis ; n = 6), and American goldfinches ( Spinus tristis ; n = 12) with 0.1 ml (10 6.7 mean embryo infectious doses [EID 50/ml ]) of NDV LaSota vaccine via the oculo-nasal route. To test transmission between chickens and wild birds, adult specific-pathogen-free white leghorn chickens were inoculated similarly and cohoused in separate isolators with two to five wild birds of the species listed above. This design resulted in three treatments: wild bird direct inoculation (five groups) and wild bird exposure to one (two groups) or two inoculated chickens (six groups), respectively. Blood and oropharyngeal and cloacal swabs were collected before and after infection with the live vaccine. All wild birds that were directly inoculated with the LaSota vaccine shed virus as demonstrated by virus isolation (VI). Cardinals were the most susceptible species based on shedding viruses from 1 to 11 days postinoculation (dpi) with titers up to 10 4.9 EID 50 /ml. Although LaSota viruses were shed by all inoculated chickens and were present in the drinking water, most noninoculated wild birds cohoused with these chickens remained uninfected for 14 days as evidenced by VI. However, one American goldfinch tested positive for vaccine transmission by VI at 7 dpi and one house finch tested positive for vaccine transmission by real-time reverse-transcription PCR at 13 dpi. Only one directly inoculated cowbird (out of three) and two cardinals (out of two) developed NDV-specific hemagglutination inhibition antibody titers of 16, 16, and 128, respectively. No clinical signs were detected in the chickens or the wild birds postinoculation.

Five, class II, virulent Newcastle disease virus (vNDV) isolates of different genotypes from different host species were evaluated for their ability to infect, cause disease, and transmit to naïve chickens. Groups of five birds received a low, medium, or high dose, by the oculonasal route, of one of the following vNDV: three chicken-origin, one cormorant-origin, and one pigeon-origin. Three naïve birds were added to each group at two days post-inoculation (DPI) to evaluate transmission. Virus shedding was quantified from swabs (2/4/7 DPI), and seroconversion was evaluated at 14 DPI. All inoculated and contact birds in the chicken-origin vNDV groups succumbed to infection, displaying clinical signs typical of Newcastle disease and shed virus titers above 6 log 10 EID 50 /ml. Birds receiving a high and medium dose of the cormorant virus showed primarily neurological clinical signs with 80% and 60% mortality, respectively. The chickens showing clinical disease shed virus at titers below 4 log 10 EID 50 /ml, and the remaining bird in the high dose group seroconverted with a high HI titer. For the pigeon-origin virus, no clinical signs were observed in any of the birds, but all 5 chickens in the high challenge dose and one bird in the medium challenge group shed virus at mean titers of 3.1 and 2.2 log 10 EID 50 /ml, respectively. Overall, the chicken-origin viruses infected chickens and efficiently transmitted to naïve birds, while the cormorant- and pigeon-origin viruses infected chickens only at the higher doses and did not transmit to other birds., (Published by Elsevier B.V.)

Virulent Newcastle disease viruses (NDV) have been present in Mexico since 1946, and recently, multiple outbreaks have been reported in the country. Here, we characterized eleven NDV isolated from apparently healthy wild birds and backyard chickens in three different locations of Jalisco, Mexico in 2017. Total RNA from NDV was reverse-transcribed, and 1285 nucleotides, which includes 3/4 of the fusion gene, was amplified and sequenced using a long-read MinION sequencing method. The sequences were 99.99-100% identical to the corresponding region obtained using the Illumina MiSeq. Phylogenetic analysis using MinION sequences demonstrated that nine virulent NDV from wild birds belonged to sub-genotypes Vc and VIn, and two backyard chicken isolates were of sub-genotype Vc. The sub-genotype Vc viruses had nucleotide sequence identity that ranged from 97.7 to 98% to a virus of the same sub-genotype isolated from a chicken in Mexico in 2010. Three viruses from pigeons had 96.3-98.7% nucleotide identity to sub-genotype VIn pigeon viruses, commonly referred to as pigeon paramyxovirus, isolated in the USA during 2000-2016. This study demonstrates that viruses of sub-genotype Vc are still present in Mexico, and the detection of this sub-genotype in both chickens and wild birds suggests that transmission among these species may represent a biosecurity risk. This is the first detection and complete genome sequencing of genotype VI NDV from Mexico. In addition, the utilization of an optimized long-read sequencing method for rapid virulence and genotype identification using the Oxford nanopore MinION system is demonstrated.

Highly virulent Newcastle disease virus (NDV) causes Newcastle disease (ND), which is a threat to poultry production worldwide. Effective disease management requires approaches to accurately determine sources of infection, which involves tracking of closely related viruses. Next-generation sequencing (NGS) has emerged as a research tool for thorough genetic characterization of infectious organisms. Previously formalin-fixed paraffin-embedded (FFPE) tissues have been used to conduct retrospective epidemiological studies of related but genetically distinct viruses. However, this study extends the applicability of NGS for complete genome analysis of viruses from FFPE tissues to track the evolution of closely related viruses. Total RNA was obtained from FFPE spleens, lungs, brains, and small intestines of chickens in 11 poultry flocks during disease outbreaks in Pakistan. The RNA was randomly sequenced on an Illumina MiSeq instrument and the raw data were analyzed using a custom data analysis pipeline that includes de novo assembly. Genomes of virulent NDV were detected in 10/11 birds: eight nearly complete (> 95% coverage of concatenated coding sequence) and two partial genomes. Phylogeny of the NDV complete genome coding sequences was compared to current methods of analysis based on the full and partial fusion genes and determined that the approach provided a better phylogenetic resolution. Two distinct lineages of sub-genotype VIIi NDV were identified to be simultaneously circulating in Pakistani poultry. Non-targeted NGS of total RNA from FFPE tissues coupled with de novo assembly provided a reliable, safe, and affordable method to conduct epidemiological and evolutionary studies to facilitate management of ND in Pakistan.

Over the last decade the US broiler industry has fought long-lasting outbreaks of infectious laryngotracheitis (ILTV). Previously, nine genotypes (I-IX) of ILTVs have been recognized using the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method with three viral alleles (gB, gM and UL47/gG). In this study, the genotyping system was simplified to six genotypes by amplicon sequencing and examining discriminating single nucleotide polymorphisms (SNPs) within these open reading frames. Using phylogenomic analysis of 27 full genomes of ILTV, a single allele (ORF A/ORF B) was identified containing SNPs that could differentiate ILTVs into genotypes congruent with the phylogenetic partitioning. The allelic variations allowed for the cataloging of the 27 strains into 5 genotypes: vaccinal TCO, vaccinal CEO, virulent CEO-like, virulent US and virulent US backyard flocks from 1980 to 1990, correlating with the PCR-RFLP genotypes I/ II/ III (TCO), IV (CEO), V (virulent CEO-like), VI (virulent US) and VII/VIII/IX (virulent US backyard flock isolates). With the unique capabilities of third generation sequencing, we investigated the application of Oxford Nanopore MinION technology for rapid sequencing of the amplicons generated in the single-allele assay. This technology was an improvement over Sanger-based sequencing of the single allele amplicons due to a booster amplification step in the MinION sequencing protocol. Overall, there was a 90% correlation between the genotyping results of the single-allele assay and the multi-allele assay. Surveillance of emerging ILTV strains could greatly benefit from real-time amplicon sequencing using the single-allele assay and MinION sequencing. RESEARCH HIGHLIGHTS A multi-allelic assay identified nine ILTV genotypes circulating in the US Single-allele genotyping is congruent with whole genome phylogenetic partitioning US ILTV strains can be grouped into five genotypes using the single-allele assay The single-allele assay can be done using MinION sequencing of barcoded amplicons.

Newcastle disease (ND) infects wild birds and poultry species worldwide, severely impacting the economics of the poultry industry. ND is especially problematic in Latin America (Mexico, Colombia, Venezuela, and Peru) where it is either endemic or re-emerging. The disease is caused by infections with one of the different strains of virulent avian Newcastle disease virus (NDV), recently renamed Avian avulavirus 1. Here, we describe the molecular epidemiology of Latin American NDVs, current control and prevention methods, including vaccines and vaccination protocols, as well as future strategies for control of ND. Because the productive, cultural, economic, social, and ecological conditions that facilitate poultry endemicity in South America are similar to those in the developing world, most of the problems and control strategies described here are applicable to other continents.

Newcastle disease (ND) has been defined by the World Organisation for Animal Health as infection of poultry with virulent strains of Newcastle disease virus (NDV). Lesions affecting the neurological, gastrointestinal, respiratory, and reproductive systems are most often observed. The control of ND must include strict biosecurity that prevents virulent NDV from contacting poultry, and also proper administration of efficacious vaccines. When administered correctly to healthy birds, ND vaccines formulated with NDV of low virulence or viral-vectored vaccines that express the NDV fusion protein are able to prevent clinical disease and mortality in chickens upon infection with virulent NDV. Live and inactivated vaccines have been widely used since the 1950's. Recombinant and antigenically matched vaccines have been adopted recently in some countries, and many other vaccine approaches have been only evaluated experimentally. Despite decades of research and development towards formulation of an optimal ND vaccine, improvements are still needed. Impediments to prevent outbreaks include uneven vaccine application when using mass administration techniques in larger commercial settings, the difficulties associated with vaccinating free-roaming, multi-age birds of village flocks, and difficulties maintaining the cold chain to preserve the thermo-labile antigens in the vaccines. Incomplete or improper immunization often results in the disease and death of poultry after infection with virulent NDV. Another cause of decreased vaccine efficacy is the existence of antibodies (including maternal) in birds, which can neutralize the vaccine and thereby reduce the effectiveness of ND vaccines. In this review, a historical perspective, summary of the current situation for ND and NDV strains, and a review of traditional and experimental ND vaccines are presented., (Published by Elsevier B.V.)

Here, we report the circulation of highly related virulent Newcastle disease viruses (NDV) in Bulgaria and Ukraine from 2002 until 2013. All of these NDV isolates have the same virulence-associated cleavage site (" 113 RQKR↓F 117 "), and selected ones have intracerebral pathogenicity index values ranging from 1.61 to 1.96. These isolates are most closely related to viruses circulating in Eastern Europe, followed by viruses isolated in Asia during the same period of time. Interestingly, the majority of the viruses were isolated from backyard poultry, suggesting the possibility of a "domestic" or "urban" cycle of maintenance. The molecular characterization of the nucleotide sequence of the complete fusion protein gene of the studied viruses suggests continued circulation of virulent NDV of sub-genotype VIId in Eastern Europe, with occasional introductions from Asia. Furthermore, the high level of genetic similarity among those isolates suggests that the NDV isolates of sub-genotype VIId from Bulgaria and Ukraine may have been part of a broader epizootic process in Eastern Europe rather than separate introductions from Asia or Africa. The continuous monitoring of backyard poultry flocks for the presence of circulating virulent NDV strains will allow early identification of Newcastle disease outbreaks.

More effective vaccines are needed to control avian diseases. The use of chicken interferon gamma (chIFNγ) during vaccination is a potentially important but controversial approach that may improve the immune response to antigens. In the present study, three different systems to co-deliver chIFNγ with Newcastle disease virus (NDV) antigens were evaluated for their ability to enhance the avian immune response and their protective capacity upon challenge with virulent NDV. These systems consisted of: 1) a DNA vaccine expressing the Newcastle disease virus fusion (F) protein co-administered with a vector expressing the chIFNγ gene for in ovo and booster vaccination, 2) a recombinant Newcastle disease virus expressing the chIFNγ gene (rZJ1*L/IFNγ) used as a live vaccine delivered in ovo and into juvenile chickens, and 3) the same rZJ1*L/IFNγ virus used as an inactivated vaccine for juvenile chickens. Co-administration of chIFNγ with a DNA vaccine expressing the F protein resulted in higher levels of morbidity and mortality, and higher amounts of virulent virus shed after challenge when compared to the group that did not receive chIFNγ. The live vaccine system co-delivering chIFNγ did not enhanced post-vaccination antibody response, nor improved survival after hatch, when administered in ovo, and did not affect survival after challenge when administered to juvenile chickens. The low dose of the inactivated vaccine co-delivering active chIFNγ induced lower antibody titers than the groups that did not receive the cytokine. The high dose of this vaccine did not increase the antibody titers or antigen-specific memory response, and did not reduce the amount of challenge virus shed or mortality after challenge. In summary, regardless of the delivery system, chIFNγ, when administered simultaneously with the vaccine antigen, did not enhance Newcastle disease virus vaccine immunogenicity.

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., (© The Author(s) 2015.)

Infectious laryngotracheitis is a highly contagious disease of chickens responsible for significant economic losses for the poultry industry worldwide. The disease is caused by Gallid herpesvirus-1 (GaHV-1) commonly known as the infectious laryngotracheitis virus. Although characterized by their potential to regain virulence, chicken embryo origin (CEO) vaccines are the most effective vaccines against laryngotracheitis as they significantly reduce the replication of challenge virus in the trachea and conjunctiva. Knowledge on the nature of protective immunity elicited by CEO vaccines is very limited. Therefore, elucidating the origin of the immune responses elicited by CEO vaccination is relevant for development of safer control strategies. In this study the transcription levels of key host immune genes (IFN-γ, IFN-β, IL-1β, IL-6, IL-8, IL-18) and viral genes (ICP4, ICP27, UL46, UL49), as well as viral genome loads in trachea were quantified at 6 and 12 hours post-challenge of CEO vaccinated and non-vaccinated chickens. Immediately after challenge a significant increase in IFN-γ gene expression was followed by a significant reduction in viral replication. In contrast to the rapid induction of IFN-γ, expression of the pro-inflammatory cytokines (IL-1β, IL-6, IL-8) and type I IFN β was either slightly reduced or remained at basal levels. These suggest that the former cytokines may not play important roles during immediate early responses induced by ILTV challenge in either vaccinated or non-vaccinated chickens. Overall, these results suggest that the rapid expression of IFN-γ may induce pathways of antiviral responses necessary for blocking early virus replication.

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 (10(6.9) 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 (10(5.3-5.5) 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.

One year after a virulent Newcastle disease virus (vNDV) outbreak in Pakistan, the causative strain was present in vaccinated chickens of multiple farms despite the existence of high-average NDV-specific antibody titers (>4.75 log2). The data suggest a possible role of vaccinated birds as reservoirs of vNDV., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Newcastle disease (ND) remains a constant threat to poultry producers worldwide, in spite of the availability and global employment of ND vaccinations since the 1950s. Strains of Newcastle disease virus (NDV) belong to the order Mononegavirales, family Paramyxoviridae, and genus Avulavirus, are contained in one serotype and are also known as avian paramyxovirus serotype-1 (APMV-1). They are pleomorphic in shape and are single-stranded, non-segmented, negative sense RNA viruses. The virus has been reported to infect most orders of birds and thus has a wide host range. Isolates are characterized by virulence in chickens and the presence of basic amino acids at the fusion protein cleavage site. Low virulent NDV typically produce subclinical disease with some morbidity, whereas virulent isolates can result in rapid, high mortality of birds. Virulent NDV are listed pathogens that require immediate notification to the Office of International Epizootics and outbreaks typically result in trade embargos. Protection against NDV is through the use of vaccines generated with low virulent NDV strains. Immunity is derived from neutralizing antibodies formed against the viral hemagglutinin and fusion glycoproteins, which are responsible for attachment and spread of the virus. However, new techniques and technologies have also allowed for more in depth analysis of the innate and cell-mediated immunity of poultry to NDV. Gene profiling experiments have led to the discovery of novel host genes modulated immediately after infection. Differences in virus virulence alter host gene response patterns have been demonstrated. Furthermore, the timing and contributions of cell-mediated immune responses appear to decrease disease and transmission potential. In view of recent reports of vaccine failure from many countries on the ability of classical NDV vaccines to stop spread of disease, renewed interest in a more complete understanding of the global immune response of poultry to NDV will be critical to developing new control strategies and intervention programs for the future., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The role of interferon gamma (IFN-γ) expression during Newcastle disease virus (NDV) infection in chickens is unknown. Infection of chickens with highly virulent NDV results in rapid death, which is preceded by increased expression of IFN-γ in target tissues. IFN-γ is a cytokine that has pleiotropic biological effects including intrinsic antiviral activity and immunomodulatory effects that may increase morbidity and mortality during infections. To better understand how IFN-γ contributes to NDV pathogenesis, the coding sequence of the chicken IFN-γ gene was inserted in the genome of the virulent NDV strain ZJ1 (rZJ1-IFNγ), and the effects of high levels of IFN-γ expression during infection were determined in vivo and in vitro. IFN-γ expression did not significantly affect NDV replication in fibroblast or in macrophage cell lines. However, it affected the pathogenesis of rZJ1-IFNγ in vivo. Relative to the virus expressing the green fluorescent protein (rZJ1-GFP) or lacking the IFN-γ insert (rZJ1-rev), expression of IFN-γ by rZJ1-IFNγ produced a marked decrease of pathogenicity in 4-week-old chickens, as evidenced by lack of mortality, decreased disease severity, virus shedding, and antigen distribution. These results suggest that early expression of IFN-γ had a significant protective role against the effects of highly virulent NDV infection in chickens, and further suggests that the level and timing of expression of this cytokine may be critical for the disease outcome. This is the first description of an in vivo attenuation of a highly virulent NDV by avian cytokines, and shows the feasibility to use NDV for cytokine delivery in chicken organs. This approach may facilitate the study of the role of other avian cytokines on the pathogenesis of NDV., (Copyright © 2013. Published by Elsevier Ltd.)

Newcastle disease, one of the most important health problems that affects the poultry industry around the world, is caused by virulent strains of Newcastle disease virus. Newcastle disease virus is considered to be endemic in several countries in the Americas, including Mexico. In order to control Newcastle disease outbreaks and spread, intensive vaccination programs, which include vaccines formulated with strains isolated at least 60 years ago, have been established. These vaccines are dissimilar in genotype to the virulent Newcastle disease viruses that had been circulating in Mexico until 2008. Here, 28 isolates obtained between 2008 and 2011 from different regions of Mexico from free-living wild birds, captive wild birds, and poultry were phylogenetically and biologically characterized in order to study the recent epidemiology of Newcastle disease viruses in Mexico. Here we demonstrate that, until recently, virulent viruses from genotype V continued to circulate and evolve in the country. All of the Newcastle disease viruses of low virulence, mostly isolated from nonvaccinated free-living wild birds and captive wild birds, were highly similar to LaSota (genotype II) and PHY-LMV42 (genotype I) vaccine strains. These findings, together with the discovery of two virulent viruses at the Mexican zoo, suggest that Newcastle disease viruses may be escaping from poultry into the environment.

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.

Little is known about the host response of chickens infected with Newcastle disease virus (NDV) and the relationship between the innate immune response and the severity of clinical disease. Measurement of cytokine responses during infection in vivo can help to elucidate the mechanisms of virus pathogenesis. The transcriptional response of several cytokines from paraffin-embedded, formalin-fixed spleen of chicken naturally infected by NDV velogenic viscerotropic viruses was compared to the responses of atypical velogenic, velogenic neurotropic, and mesogenic strains during the first five days after infection. The RNA expression for IFN-γ and IL-6 was enhanced at day two in the highly virulent velogenic viscerotropic viruses (California and rZJ1 strains) and corresponded with the presence of the virus in tissues. However, in one atypical velogenic viscerotropic virus (Australia strain), two velogenic neurotropic viruses (Turkey ND and Texas GB) and, a mesogenic virus (Anhinga strain) the cytokine responses to infection were delayed or reduced. Increased levels of IFN-β RNA expression were only detected in the velogenic viscerotropic virus infected chickens (California and rZJ1 strains) at 3 days post-infection and one mesogenic strain (Anhinga) early in infection. The RNA expression levels of IL-2 did not increase upon infection with any of the viruses. A pronounced increase of RNA expression levels of IL-6 and IFN-γ was detected simultaneously with infiltration of macrophages and/or lymphoid necrosis in the histopathological analysis of the spleen and cecal tonsils. The differences in the RNA expression levels may help explain possible underlying mechanisms of clinical disease and/or immune responses in birds infected with strains of APMV-1 that cause distinct pathologic changes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Newcastle disease virus (NDV) is an avian paramyxovirus that causes significant economic losses to the poultry industry worldwide. There is limited knowledge about the avian immune response to infection with virulent NDVs, and how this response may contribute to disease. In this study, pathogenesis and the transcriptional host response of chickens to a virulent NDV strain that rapidly causes 100% mortality was characterized. Using microarrays, a strong transcriptional host response was observed in spleens at early times after infection with the induction of groups of genes involved in innate antiviral and pro-inflammatory responses. There were multiple genes induced at 48 h post-infection including: type I and II interferons (IFNs), several cytokines and chemokines, IFN effectors and inducible nitric oxide synthase (iNOS). The increased transcription of nitric oxide synthase was confirmed by immunohistochemistry for iNOS in spleens and measured levels of nitric oxide in serum. In vitro experiments showed strong induction of the key host response genes, alpha IFN, beta interferon, and interleukin 1β and interleukin 6, in splenic leukocytes at 6 h post-infection in comparison to a non-virulent NDV. The robust host response to virulent NDV, in conjunction with severe pathological damage observed, is somewhat surprising considering that all NDV encode a gene, V, which functions as a suppressor of class I IFNs. Taken together, these results suggest that the host response itself may contribute to the pathogenesis of this highly virulent strain in chickens.

Distribution, character, and severity of lesions were evaluated in tissues from the central nervous system of chickens inoculated with 10 different Newcastle disease virus (NDV) isolates: CA 1083, Korea 97-147, Australia (all velogenic viscerotropic), Texas GB and Turkey North Dakota (both velogenic neurotropic), Nevada cormorant, Anhinga and Roakin (all mesogenic), and B1 and QV4 (lentogenic). Tissues for the present study included archived formalin-fixed, paraffin-embedded brain (all strains) plus spinal cord (two strains). Encephalitis was observed in all velogenic viscerotropic and velogenic neurotropic strains, and in some mesogenic strains. In general, the encephalitic lesions began at 5 days post infection, with more severe lesions occurring around 10 days post infection. At this time point, especially in the grey matter of the brain, cerebellum and spinal cord, there were neuronal necrosis, neuronal phagocytosis, and clusters of cells with microglial morphology. Axonal degeneration and demyelination was also observed. Immunohistochemistry (IHC) for viral nucleoprotein confirmed the presence of virus. In the areas of encephalomyelitis, IHC for CD3 revealed that many of the inflammatory cells were T lymphocytes. IHC using an antibody for glial fibrillar acid protein showed reactive astrogliosis, which was most pronounced at the later time points.

The aim of this study was to evaluate the disease-inducing ability of four chimeric Newcastle disease viruses (NDV) by clinicopathological assessment. The infectious clones were previously generated by insertion of hemagglutinin-neuraminidase (HN) and/or fusion (F) genes from virulent strains (Turkey North Dakota and California 02) into a mesogenic strain (Anhinga) backbone. Groups of 4-week-old chickens were inoculated via eye drop instillation, clinical signs were monitored daily, and necropsies with collection of tissues were performed at 2, 5, 10, and 14 days post infection. Tissue sections were evaluated for histopathology and immunohistochemistry for NDV nucleoprotein. All viruses replicated successfully in the natural host, although viral recovery, seroconversion, and extent of immunohistochemical staining were greatest from birds infected with those viruses containing both F and HN genes from the same virulent virus. There was minimal to no increase in clinicopathologic disease due to infection with the chimeras compared to the recombinant backbone. However, all birds developed histological evidence of encephalitis. The results suggest that the inherent virulence of Turkey North Dakota and California 2002 strains is due to more than the simple presence of their F and HN genes.

Since the discovery of Newcastle disease virus (NDV) in 1926, nine genotypes of class I viruses and ten of class II have been identified, representing a diverse and continually evolving group of viruses. The emergence of new virulent genotypes from global epizootics and the year-to-year changes observed in the genomic sequence of NDV of low and high virulence implies that distinct genotypes of NDV are simultaneously evolving at different geographic locations across the globe. This vast genomic diversity may be favored by the large variety of avian species susceptible to NDV infection and by the availability of highly mobile wild bird reservoirs. The genomic diversity of NDV increases the possibility of diagnostic failures, resulting in unidentified infections. Constant epidemiological surveillance and pro-active characterization of circulating strains are needed to ensure that the immunological and PCR reagents are effective in identifying NDV circulating worldwide. For example, in the United States, the widely used real-time reverse transcription polymerase chain reaction (RRT-PCR) matrix gene assay for the identification of NDV often fails to detect low virulence APMV-1 from waterfowl, while the RRT-PCR fusion gene assay, used to identify virulent isolates, often fails to detect certain virulent NDV genotypes. A new matrix-polymerase multiplex test that detects most of the viruses currently circulating worldwide and a modified fusion test for the identification of virulent pigeon viruses circulating in the U.S. and Europe have recently been developed. For newly isolated viruses with unknown sequences, recently developed random priming sequencing methods need to be incorporated into the diagnostic arsenal. In addition, the current system of classifying NDV into genotypes or lineages is inadequate. Here, we review the molecular epidemiology and recent diagnostic problems related to viral evolution of NDV and explain why a new system, based on objective criteria, is needed to categorize genotypes., (Published by Elsevier B.V.)

In 2002-2003, velogenic Newcastle Disease Virus outbreaks, closely related to the Mexican isolates, were confirmed in the United States (U.S.) in southern California, Arizona, Nevada, and Texas. In this report, virulent NDVs isolated in Mexico between 1998 and 2006 were subjected to biologic characterization, using standard pathogenicity tests, and to phylogenetic analysis. Chicken embryo mean death time (MDT) test results ranged from 39.7 to 61.5 hours, and intracerebral pathogenicity index (ICPI) values were between 1.59 and 1.94, compared to a possible maximum value of 2.0. These isolates showed a dibasic amino acid motif at the fusion protein cleavage site sequence required for host systemic replication. Phylogenetic analysis indicated that the Mexican virulent NDVs belong to the class II, genotype V viruses and can be clearly divided in two groups as follows: isolates from 1998 to 2001 with close epidemiologic relationship with the latest U.S. NDV outbreaks, and phylogenetically distinct viruses, isolated from 2004 to 2006, which showed higher virulence. The assessment of the evolution of viruses from Mexico and other neighboring countries will aid in the U.S surveillance efforts for early detection of highly virulent NDV.

The Villegas-Glisson/University of Georgia (VG/GA) strain of Newcastle disease virus (NDV) isolated from the intestine of healthy turkeys has been proposed to replicate in the respiratory and intestinal tract of chickens. In the present study, the virus distribution, the mucosal and systemic immune response, the efficacy against lethal challenge and the full genome sequence of the VG/GA strain were compared with the La Sota strain of NDV. The VG/GA strain was detected at different time points in the respiratory and intestinal tract of chickens with a preferential tropism for the latter. Both the VG/GA and La Sota strains induced NDV-specific immunoglobulin A (IgA) at the upper respiratory tract. IgA levels were higher in the trachea for the La Sota strain, while they were higher in the bile and intestine for the VG/GA strain. Positive correlation between virus distribution of the viruses and IgA production was observed. Despite the presence of the maternal antibodies in broilers, early vaccination with the VG/GA strain afforded 95% to 100% protection against lethal challenge, equivalent to the protection conferred by the La Sota strain. Full genome sequence analysis classified the VG/GA strain within class II, genotype II viruses, which also include most of the respirotropic vaccine strains. Differences with the La Sota strain at the nucleotide and amino acid levels that may explain the differential phenotype of the VG/GA were observed; however, verification of the significance of those changes is required. Taken together, these results validate field observations on the efficacy of VG/GA vaccination and demonstrated the unique characteristics of the strain.

A major route of infection of avian influenza virus (AIV) and Newcastle disease virus (NDV) in chickens is through cells of the respiratory epithelium. Here we describe the development of a method for culture of tracheal epithelial cells from chicken embryos as well as their use in studies of infection with avian respiratory viruses such as low-pathogenicity AIV and lentogenic NDV. Positive immunostaining for cytokeratin, the presence of cilia and microvilli, and microarray analysis of transcribed RNA demonstrated that the isolated cells were epithelial in nature. Infection of the epithelial cell cultures with AIV and NDV was demonstrated using immunofluorescence or green fluorescence protein fluorescence microscopy, respectively. Growth curves of AIV and NDV in tracheal epithelial cells revealed that tracheal epithelial cells can fully support AIV and NDV growth and reinfection. This system, which mimics that of the natural infection, will be useful to study the mechanisms of early viral infection and cellular host transcriptional responses.