Mutations in the Methyltransferase Motifs of L Protein Attenuate Newcastle Disease Virus by Regulating Viral Translation and Cell-to-Cell Spread.

The large (L) polymerase proteins of most nonsegmented, negative-stranded (NNS) RNA viruses have conserved methyltransferase motifs, (G)-G-G-D and K-D-K-E, which are important for the stabilization and translation of mRNA. However, the function of the (G)-G-G-D and K-D-K-E motifs in the NNS RNA virus Newcastle disease virus (NDV) remains unclear. We observed G-G-D and K-D-K-E motifs in all NDV genotypes. By using the infection cloning system of NDV rSG10 strain, recombinant NDVs with a single amino acid mutated to alanine in one motif (G-G-D or K-D-K-E) were rescued. The intracerebral pathogenicity index and mean death time assay results revealed that the G-G-D motif and K-D-K-E motif attenuate the virulence of NDV to various degrees. The replication, transcription, and translation levels of the K-D-K-E motif-mutant strains were significantly higher than those of wild-type virus owing to their altered regulation of the affinity between nucleocapsid protein and eukaryotic translation initiation factor 4E. When the infection dose was changed from a multiplicity of infection (MOI) of 10 to an MOI of 0.01, the cell-to-cell spread abilities of G-G-D- and K-D-K-E-mutant strains were reduced, according to plaque assay and dynamic indirect immunofluorescence assay results. Finally, we found that NDV strains with G-G-D or K-D-K-E motif mutations had less pathogenicity in 3-week-old specific-pathogen-free chickens than wild-type NDV. Therefore, these methyltransferase motifs can affect virulence by regulating the translation and cell-to-cell spread abilities of NDV. This work provides a feasible approach for generating vaccine candidates for viruses with methyltransferase motifs. IMPORTANCE Newcastle disease virus (NDV) is an important pathogen that is widespread globally. Research on its pathogenic mechanism is an important means of improving prevention and control efforts. Our study found that a deficiency in its methyltransferase motifs (G-G-D and K-D-K-E motifs) can attenuate NDV and revealed the molecular mechanism by which these motifs affect pathogenicity, which provides a new direction for the development of NDV vaccines. In addition to the (G)-G-G-D and K-D-K-E motifs of many nonsegmented, negative-stranded RNA viruses, similar motifs have been found in dengue virus, Zika virus, Japanese encephalitis virus (JEV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This suggests that such motifs may be present in more viruses. Our finding also provides a molecular basis for the discovery and functional study of (G)-G-G-D and K-D-K-E motifs of other viruses.

Newcastle Disease Virus Entry into Chicken Macrophages via a pH-Dependent, Dynamin and Caveola-Mediated Endocytic Pathway That Requires Rab5.

The cellular entry pathways and the mechanisms of Newcastle disease virus (NDV) entry into cells are poorly characterized. In this study, we demonstrated that chicken interferon-induced transmembrane protein 1 (chIFITM1), which is located in the early endosomes, could limit the replication of NDV in chicken macrophage cell line HD11, suggesting the endocytic entry of NDV into chicken macrophages. Then, we presented a systematic study about the entry mechanism of NDV into chicken macrophages. First, we demonstrated that a low-pH condition and dynamin were required during NDV entry. However, NDV entry into chicken macrophages was independent of clathrin-mediated endocytosis. We also found that NDV entry was dependent on membrane cholesterol. The NDV entry and replication were significantly reduced by nystatin and phorbol 12-myristate 13-acetate treatment, overexpression of dominant-negative (DN) caveolin-1, or knockdown of caveolin-1, suggesting that NDV entry depends on caveola-mediated endocytosis. However, macropinocytosis did not play a role in NDV entry into chicken macrophages. In addition, we found that Rab5, rather than Rab7, was involved in the entry and traffic of NDV. The colocalization of NDV with Rab5 and early endosome suggested that NDV virion was transported to early endosomes in a Rab5-dependent manner after internalization. Of particular note, the caveola-mediated endocytosis was also utilized by NDV to enter primary chicken macrophages. Moreover, NDV entered different cell types using different pathways. Collectively, our findings demonstrate for the first time that NDV virion enters chicken macrophages via a pH-dependent, dynamin and caveola-mediated endocytosis pathway and that Rab5 is involved in the traffic and location of NDV. IMPORTANCE Although the pathogenesis of Newcastle disease virus (NDV) has been extensively studied, the detailed mechanism of NDV entry into host cells is largely unknown. Macrophages are the first-line defenders of host defense against infection of pathogens. Chicken macrophages are considered one of the main types of target cells during NDV infection. Here, we comprehensively investigated the entry mechanism of NDV in chicken macrophages. This is the first report to demonstrate that NDV enters chicken macrophages via a pH-dependent, dynamin and caveola-mediated endocytosis pathway that requires Rab5. The result is important for our understanding of the entry of NDV in chicken macrophages, which will further advance the knowledge of NDV pathogenesis and provide useful clues for the development of novel preventive or therapeutic strategies against NDV infection. In addition, this information will contribute to our further understanding of pathogenesis with regard to other members of the Avulavirus genus in the Paramyxoviridae family.

The role of live chicken markets as a source of replication and dissemination of Newcastle disease virus in chickens, northwest Ethiopia.

Newcastle disease (ND) is perceived to be the major constraint in village chickens of Ethiopia causing huge economic loss. Village chickens are mobile and pass through markets, and live chicken markets are a highly productive source of ND virus replication, maintenance, and spread. However, in northwest of Ethiopia, there is a dearth of information on the role of live chicken markets in the maintenance and spread of ND in the village chickens. Therefore, a total of 480 apparently healthy chickens in the 4 live chicken markets were sampled with the aim to detect and estimate ND virus infection. Tracheal and cloacal swabs were collected from each bird and processed for virus isolation in 9- to 11-day-old embryonated chicken eggs, and hemagglutination inhibition (HI) assay was performed on all sera samples. The overall infection rate of ND virus was reported to be 39.2% (95% CI: 34.8-43.5). Of all chickens, 34.6% (95% CI: 30.3-38.9) had mean HI titer ≥4 log2, which was considered as protective. The mean hemagglutination titer for the ND virus was reported to be 6.0 log2, and mean antibody titer was reported to be 6.2 log2, with no statistically significant variation among the markets (P > 0.05). Newcastle disease occurrence was detected in all seasons of the year in the live bird markets, with the highest prevalence (55.8%) during the prerainy dry season (April and May), showing evidence for climatic and socioeconomic aspects as a risk factor in the occurrence of ND in indigenous chicken. In vivo virulence tests, mean death time of the embryo, and the intracerebral pathogenicity index revealed the presence of all pathotypes of ND virus strains: velogenic, mesogenic, and lentogenic. Apparently, healthy appearing birds were reported to be reservoirs of velogenic ND virus strains that could initiate endemicity of ND cycles in the village setting. Hence, it is strongly recommended to implement appropriate prevention and control measures to mitigate the economic loss caused by the disease.

Evolutionary dynamics and transmission patterns of Newcastle disease virus in China through Bayesian phylogeographical analysis.

The Chinese poultry industry has experienced outbreaks of Newcastle disease (ND) dating back to the 1920s. However, the epidemic has exhibited a downtrend in recent years. In this study, both observational and genetic data [fusion (F) and haemagglutinin-neuraminidase genes (HN)] were analyzed, and phylogeographic analysis based on prevalent genotypes of Newcastle disease virus (NDV) was conducted for better understanding of the evolution and spatiotemporal dynamics of ND in China. In line with the observed trend of epidemic outbreaks, the effective population size of F and HN genes of circulating NDV is no longer growing since 2000, which is supported by 95% highest posterior diversity (HPD) intervals. Phylogeographic analysis indicated that the two eastern coastal provinces, Shandong and Jiangsu were the most relevant hubs for NDV migration, and the geographical regions with active NDV diffusion seemed to be constrained to southern and eastern China. The live poultry trade may play an important role in viral spread. Interestingly, no migration links from wild birds to poultry received Bayes factor support (BF > 3), while the migration links from poultry to wild birds accounted for 64% in all effective migrations. This may indicate that the sporadic cases of ND in wild bird likely spillover events from poultry. These findings contribute to predictive models of NDV transmission, and potentially help in the prevention of future outbreaks.

The 3D8 single chain variable fragment protein suppresses Newcastle disease virus transmission in transgenic chickens.

BACKGROUND: The 3D8 single chain variable fragment (scFv) is a mini-antibody sequence that exhibits independent nuclease activity against all types of nucleic acids. In this research, crossing a 3D8 scFv G1 transgenic rooster with wild-type hens produced 3D8 scFv G2 transgenic chickens to evaluate suppression of viral transmission. RESULT: The transgenic chickens were identified using genomic PCR and immunohistochemistry. To evaluate Newcastle disease virus (NDV) protection conferred by 3D8 scFv expression, transgenic, non-transgenic, and specific pathogen-free (SPF) chickens were challenged with virulent NDV by direct injection or aerosol exposure. The three groups of chickens showed no significant differences (p < 0.05) in mean death time after being directly challenged with NDV; however, in contrast to chickens in the non-transgenic and SPF groups, chickens in the transgenic group survived after aerosol exposure. Although the transgenic chickens did not survive after direct challenge, we found that the chickens expressing the 3D8 scFv survived aerosol exposure to NDV. CONCLUSIONS: Our finding suggest that the 3D8 scFv could be a useful tool to prevent chickens from spreading NDV and control virus transmission.

Evolutionary Dynamics and Age-Dependent Pathogenesis of Sub-Genotype VI.2.1.1.2.2 PPMV-1 in Pigeons.

Pigeon paramyxovirus type 1 (PPMV-1) infection causes high morbidity in pigeons, resulting in a significant burden to the poultry industry. In this study, we isolated three PPMV-1 strains from diseased pigeons collected in Guangdong Province, South China, from June 2017 to April 2019. Genetic analysis revealed that these three PPMV-1 strains and most of the PPMV-1 strains isolated from China after 2011 were clustered into sub-genotype VI.2.1.1.2.2. Our Bayesian analysis revealed that the VI.2.1.1.2.2 viruses might have originated in Europe. Phylogeographic analyses revealed that East and South China might have played a key role in seeding the VI.2.1.1.2.2 PPMV-1 epidemic in China. To characterize the effect of age at infection on the outcome of PPMV-1 infection in pigeons, we investigated the pathogenesis and transmission of the pigeon/Guangdong/GZ08/2017 (GZ08) virus in 3-, 6-, and 12-week-old pigeons. Two of six 12-week-old pigeons inoculated with GZ08 survived, and all of the 3- and 6-week-pigeons inoculated with GZ08 died. Moreover, the GZ08 virus could be transmitted to 3-, 6-, and 12-week-old naïve contact pigeons. The lethality of the GZ08 virus through contact with 3-, 6-, and 12-week-old pigeons was 100%, 66.7%, and 0%, respectively, suggesting that the transmissibility of the GZ08 virus was stronger in young pigeons. These findings demonstrated that East and South China was the epicenter for dissemination of VI.2.1.1.2.2 PPMV-1, and age at infection has an impact on the outcome of PPMV-1 infection in pigeons.

Phylogeny, pathogenicity and transmissibility of a genotype XII Newcastle disease virus in chicken and goose.

Although Newcastle disease virus (NDV) has a worldwide distribution, some NDV genotypes have more regional geographical ranges within continents. In this study, we isolated a subgenotype XIIb NDV strain, Goose/CH/GD/E115/2017 (E115), from geese in Guangdong province, Southern China, in 2017. Phylogenetic analysis showed that E115 and six other NDVs from geese in China were grouped under subgenotype XIIb and were distinct from subgenotype XIIa, isolated from chickens in South Africa, and subgenotype XIId, isolated from chickens in Vietnam. To better understand the pathogenicity and transmission of the subgenotype XIIb NDVs from geese in Guangdong province, we inoculated chickens and geese with 106 EID50 of the E115 virus. Eight hours after inoculation, three naïve chickens and three naïve geese were co-housed with the infected chickens or geese to assess intraspecific and interspecific horizontal transmission of the E115 virus. The E115 virus induced significant clinical signs without mortality in chickens, while it was not pathogenic to geese. Intraspecific and interspecific horizontal transmission of the E115 virus was observed among chickens and geese via direct contact. Furthermore, although the current vaccines provided complete protection against disease in chickens after challenging them with the E115 virus, the virus could also be transmitted from vaccinated chickens to naïve contact chickens. Collectively, our findings highlight the need for avoiding the mixing of different bird species to reduce cross-species transmission and for surveillance of NDV in waterfowl.

Isolation and genetic characterization of virulent strains of avian paramyxovirus-1 from multiple avian species in Azad Jammu and Kashmir 2017-2018.

Despite intensive vaccination, endemicity of Avian paramyxoviruses-1 (APMV-1) is a significant problem in developing countries in Africa, Middle East, and Asia. Given the importance of APMV-1 in poultry and multiple non-poultry avian species, it is important to continue surveillance programs, routine monitoring and characterization of field isolates in the region where viruses are endemic. The purpose of this study was to pathotyped and genetically characterized 21 APMV-1s isolated from multiple avian species reared in different regions of Azad Jammu and Kashmir (AJK). Phylogenetic analysis based on complete fusion (F) gene sequences showed that 17 APMV-1 isolates obtained from commercial poultry and backyard birds belonged to sub-genotype VIIi. Though, one pigeon-origin APMV-1 isolate was clustered in sub-genotype VIg and three in recently designated new sub-genotype VIm of genotype VI. The pigeon-origin isolates had the following two motifs 113-RKKR↓F-117 and 113-RQRR↓F-117, while all other isolates had the polybasic amino acid sequence 113-RQKR↓F-117 at the F-cleavage site, which is characteristic of virulent APMV-1 strains. These results are consistent with the five viruses that had intracerebral pathogenicity indices (ICPIs) of between 1.50 and 1.73, corresponding to a velogenic pathotype. The APMV-1s isolated from commercial poultry and backyard birds in this study showed low nucleotide distance (0.3-0.9%) and genetically closely related (> 97%) to viruses repeatedly isolated (2011-2017) from multiple avian species in other states of Pakistan. Strengthened surveillance programs in both commercial poultry and backyard flocks are needed to better assess the commercial-backyard bird interface and form a basis for evidence-based measures to limit and prevent APMV-1 transmission.

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.

Cormoranes como posibles víctimas y reservorios del virus velogénicos de la enfermedad de Newcastle (Orthoavulavirus-1) en Asia Central. Las cepas virulentas del Orthoavulavirus aviar 1, históricamente conocido como virus de la enfermedad de Newcastle (NDV), están muy extendidas y causan altos niveles de mortalidad en avicultura en todo el mundo. Las aves silvestres pueden desempeñar un papel importante en el mantenimiento del Orthoavulavirus aviar 1 en la naturaleza. Antes del año 2014, la mayoría de las cepas de Newcastle lentogénicas aisladas de Asia Central se obtenían del orden aviar Anseriformes (patos y gansos). Las aves silvestres fueron monitoreadas entre los años 2014 y 2016 para detectar la circulación de virus de Newcastle. Se examinaron un total de 1522 muestras pertenecientes a 73 especies de aves, y se identificaron 26 muestras positivas. Los aislamientos de Orthoavulavirus aviar 1pertenecían a tres genotipos: los virus de palomas (Columbiformes) y de cormoranes (Suliformes) se atribuyeron a los genotipos velogénicos VI y XIII, respectivamente, mientras que los aislamientos de aves comerciales pertenecieron al genotipo lentogénico I. El aislamiento del Orthoavulavirus aviar 1 lentogénico de las palomas puede confirmar su papel como reservorio de los paramixovirus de paloma (variantes antigénicas del genotipo VI del virus de Newcastle) en la naturaleza e indica la amenaza potencial de la introducción de cepas velogénicas en la población avícola. Este estudio describe un escenario epizoótico en Kazajstán entre cormoranes con mortalidad de aves jóvenes de hasta tres semanas de edad y aislamiento del virus de Newcastle de aves aparentemente sanas. Estas observaciones pueden apoyar la idea de que los cormoranes son uno de los reservorios potenciales y también víctimas del Orthoavulavirus aviar 1 velogénico en Asia Central. Las migraciones estacionales de cormoranes pueden contribuir parcialmente a la diseminación viral en todo el continente; Sin embargo, esta hipótesis requiere de más evidencia.

Evaluation of transmission potential and pathobiological characteristics of mallard originated Avian orthoavulavirus 1 (sub-genotype VII.2) in commercial broilers.

Newcastle disease (ND), caused by Avian orthoavulavirus 1 (AOAV-1), affects multiple avian species around the globe. Frequent disease outbreaks are not uncommon even in vaccinates despite routine vaccination and, in this regards, viruses of diverse genotypes originating from natural reservoirs (migratory waterfowls) play an important role in a disease endemic setting. Though genomic characterization of waterfowl originated viruses has been well-elucidated previously, there is a paucity of data on clinico-pathological assessment of mallard-originated sub-genotype VII.2 in commercial chickens. Hence, the current study was designed to evaluate its transmission potential, tissue tropism and micro- and macroscopic lesions in commercial broilers. Based on complete genome and complete F gene, phylogenetic analysis clustered the study isolate within genotype VII and sub-genotype VII.2 in close association with those reported previously from multiple avian species worldwide. The study strain was found to be velogenic on the basis of typical residue pattern in the F-protein cleavage site (112R-RQ-K-R↓F117), sever disease induction in chicken, tissue tropism and subsequent clinico-pathological characteristics. Giving a clear evidence of horizontal transmission, a 100% mortality was observed by 4th and 6th day post infection (dpi) in chickens challenged with the virus and those kept with the challenged birds (contact birds), respectively. The observed clinical signs, particularly the greenish diarrhea, and macroscopic lesions such as pinpoint hemorrhages in proventriculus and caecal tonsils were typical of the infection caused by an AOAV-1 in chickens. The virus exhibited a broad tissue tropism where genomic RNA corresponding to study virus was detected in all of the tissues collected from recently mortile and necropsied birds. The study concludes that mallard-originated Avian orthoavulavirus 1 is highly velogenic to commercial chicken and therefore ascertain continuous disease monitoring and surveillance of migratory/aquatic fowls to better elucidate infection epidemiology and subsequent potential impacts on commercial poultry.

Virulent Newcastle disease viruses from chicken origin are more pathogenic and transmissible to chickens than viruses normally maintained in wild birds.

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 log10 EID50/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 log10 EID50/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 log10 EID50/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.

Cross-species transmission of poultry pathogens in backyard farms: ducks as carriers of chicken viruses.

In backyard farms of Lao People's Democratic Republic, mixed-species rearing of poultry is a breeding-ground for cross-species transmission. Here, the epidemiology of viruses circulating among backyard poultry in Vientiane Province was assessed to guide future control strategies. Oral/tracheal and cloacal swabs, collected from 605 poultry (308 ducks, 297 chickens) between 2011 and 2015, were screened by PCR for Newcastle disease virus (NDV), coronavirus (CoV) and chicken anaemia virus (CAV). Chicken sera were screened for anti-NDV antibodies by ELISA. Statistical and phylogenetic analyses revealed transmission patterns and relationships. Closely related strains co-circulated in chickens and ducks. While CoV RNA was detected in oral/tracheal swabs of 9.3% of the chickens and 2.4% of the ducks, rates were higher in faecal swabs of both species (27.3% and 48.2%). RNA of infectious bronchitis virus (IBV) and duck CoV was found in faecal swabs of chickens (19.7% and 7.1%) and ducks (4.1% and 44.1%). Moreover, DNA of the generally chicken-specific CAV was detected in oral/tracheal swabs of chickens (18.1%) and, sporadically, of ducks (2.4%). Despite serological evidence of NDV circulation or vaccination (86.9%), NDV RNA was not detected. We found a high prevalence and indication for cross-species transmission of different CoV strains in backyard poultry. Interestingly, ducks served as biological, or at least mechanical, carriers of viral strains closely related not only to IBV, but also to CAV. Bird containment and poultry species separation could be first steps to avoid cross-species transmission and emergence of novel strains with broad host range and enhanced pathogenicity. RESEARCH HIGHLIGHTS High rates of avian viruses were detected by PCR in backyard poultry from Lao PDR. Diverse coronavirus and chicken anemia virus strains co-circulated. Phylogenetic analyses suggested virus transmission between chickens and ducks. Serological evidence of Newcastle disease was found, but viral RNA was not detected.

Comparison between class I NDV and class II NDV in aerosol transmission under experimental condition.

Recent epidemiological surveys have shown that class I Newcastle disease virus (NDV) is widely distributed in China. However, little is currently known about its transmission. Therefore, in this study, we compared the transmission of class I and class II NDV. Specific-pathogen-free chickens were divided into a class I NDV inoculation group and an aerosol-exposed infection group and kept in 2 separate isolators (A and B, respectively) that were connected with an airtight plastic pipe. After inoculation, air samples were collected regularly with an All-Glass Impinger-30 (Liaoyang, China), and the airborne virus contents were analyzed using the plaque count method. In addition, oral and cloacal swabs were collected regularly to detect virus shedding using quantitative reverse transcription PCR. Similar trials were conducted simultaneously with class II NDV in isolators C and D. We consistently detected class I NDV aerosols in both isolators A and B up to 40 D post-inoculation (dpi). The aerosol concentration reached a maximum of 13.81 × 103 plague-forming units per cubic meter of air at 18 dpi and was significantly higher than that of class II NDV at 21 and 24 dpi. We also detected class I virus shedding from 2 to 40 dpi in the inoculated chickens and from 7 to 40 D post-aerosol-exposed infection in the aerosol-exposed chickens. This phenomenon may explain why class I NDV has been the primary epidemic strain of NDV in recent years.

Investigation of suspected Newcastle disease (ND) outbreaks in Egypt uncovers a high virus velogenic ND virus burden in small-scale holdings and the presence of multiple pathogens.

Highly contagious Newcastle disease (ND) is associated with devastating outbreaks with highly variable clinical signs among gallinaceous birds. In this study we aimed to verify clinical ND suspicions in poultry holdings in Egypt suffering from respiratory distress and elevated mortality, comparing two groups of ND-vaccinated poultry holdings in three governorates. Besides testing for Newcastle disease virus (NDV), samples were screened for infectious bronchitis virus (IBV) and avian influenza virus (AIV) by RT-qPCR as well as by non-directed cell-culture approach on LMH-cells. Virulent NDV was confirmed only in group A (n = 16) comprising small-scale holdings. Phylogenetic analysis of the fusion protein gene of 11 NDV-positive samples obtained from this group assigned all viruses to genotype 2.VIIb and point to four different virus populations that were circulating at the same time in one governorate, indicating independent epidemiological events. In group B, comprising large commercial broiler farms (n = 10), virulent NDV was not present, although in six farms NDV vaccine-type virus (genotype 2.II) was detected. Besides, in both groups, co-infections by IBV (n = 10), AIV H9 (n = 3) and/or avian reovirus (ARV) (n = 5) and avian astrovirus (AastVs) (n = 1) could be identified. Taken together, the study confirmed clinical ND suspicion in small scale holdings, pointing to inefficient vaccination practices in this group A. However, it also highlighted that, even in an endemic situation like ND in Egypt, in cases of suspected ND vaccine failure, clinical ND suspicion has to be verified by pathotype-specific diagnostic tests. RESEARCH HIGHLIGHTS Velogenic NDV circulates in small-scale poultry holdings in Egypt. Viral transmission occurred among neighbouring farms and over long distances. Co-infections with multiple pathogens were identified. Pathotype specific diagnostic tests are essential to verify ND suspicions.

Global phylodynamic analysis of avian paramyxovirus-1 provides evidence of inter-host transmission and intercontinental spatial diffusion.

BACKGROUND: Avian avulavirus (commonly known as avian paramyxovirus-1 or APMV-1) can cause disease of varying severity in both domestic and wild birds. Understanding how viruses move among hosts and geography would be useful for informing prevention and control efforts. A Bayesian statistical framework was employed to estimate the evolutionary history of 1602 complete fusion gene APMV-1 sequences collected from 1970 to 2016 in order to infer viral transmission between avian host orders and diffusion among geographic regions. Ancestral states were estimated with a non-reversible continuous-time Markov chain model, allowing transition rates between discrete states to be calculated. The evolutionary analyses were stratified by APMV-1 classes I (n = 198) and II (n = 1404), and only those sequences collected between 2006 and 2016 were allowed to contribute host and location information to the viral migration networks. RESULTS: While the current data was unable to assess impact of host domestication status on APMV-1 diffusion, these analyses supported the sharing of APMV-1 among divergent host taxa. The highest supported transition rate for both classes existed from domestic chickens to Anseriformes (class I:6.18 transitions/year, 95% highest posterior density (HPD) 0.31-20.02, Bayes factor (BF) = 367.2; class II:2.88 transitions/year, 95%HPD 1.9-4.06, BF = 34,582.9). Further, among class II viruses, domestic chickens also acted as a source for Columbiformes (BF = 34,582.9), other Galliformes (BF = 34,582.9), and Psittaciformes (BF = 34,582.9). Columbiformes was also a highly supported source to Anseriformes (BF = 322.0) and domestic chickens (BF = 402.6). Additionally, our results provide support for the diffusion of viruses among continents and regions, but no interhemispheric viral exchange between 2006 and 2016. Among class II viruses, the highest transition rates were estimated from South Asia to the Middle East (1.21 transitions/year; 95%HPD 0.36-2.45; BF = 67,107.8), from Europe to East Asia (1.17 transitions/year; 95%HPD 0.12-2.61; BF = 436.2) and from Europe to Africa (1.06 transitions/year, 95%HPD 0.07-2.51; BF = 169.3). CONCLUSIONS: While migration appears to occur infrequently, geographic movement may be important in determining viral diversification and population structure. In contrast, inter-order transmission of APMV-1 may occur readily, but most events are transient with few lineages persisting in novel hosts.

Muscovy ducks infected with velogenic Newcastle disease virus (genotype VIId) act as carriers to infect in-contact chickens.

This work was designed to study the dynamics of transmission of Newcastle disease virus (NDV), genotype VIId, from Muscovy ducks (Cariana moscata) infected either by intramuscular (IM) or intranasal (IN) inoculation, to in-contact broiler chickens (Gallus gallus). IM-infected Muscovy ducks (G1d) exhibited only 5% mortality, and the concentration of virus shed from the cloaca was greater and for longer period than virus shed from the trachea. In contrast, IN-infected ducks (G2d) exhibited no mortality, and virus shedding from the trachea was higher than that from the cloaca starting from 4 days post infection (dpi) and continued up to 16 dpi, while in IM-infected ducks (G1d), tracheal shedding stopped at 11 dpi. Chickens in contact with IM-infected and IN-infected ducks, G1c and G2c, respectively, not only developed severe clinical symptoms and death (80% and 20% mortality, respectively), but also shed the virus at higher concentrations than infected ducks. G1c chickens had higher viral shedding titers in both the trachea and cloaca than G2c chickens until 11 dpi. All broiler chickens infected by IM route (G3c) died, while the IN route of infection resulted in lower mortality (70%) in G4c. Generally, all IM-infected birds produced an earlier and higher level of NDV hemagglutination inhibition (HI) antibody titer, along with higher rates and shorter periods of viral shedding than those infected by the intranasal route. Our conclusion is that Muscovy ducks are efficient carriers of NDV-genotype VIId and transmit the virus to contact chickens.

Pathogenicity and transmission of virulent Newcastle disease virus from the 2018-2019 California outbreak and related viruses in young and adult chickens.

In May of 2018, virulent Newcastle disease virus was detected in sick, backyard, exhibition chickens in southern California. Since, the virus has affected 401 backyard and four commercial flocks, and one live bird market in California, and one backyard flock in Utah. The pathogenesis and transmission potential of this virus, along with two genetically related and widely studied viruses, chicken/California/2002 and chicken/Belize/2008, were evaluated in both 3-week- and 62-week-old chickens given a low, medium, or high challenge dose. All three viruses were highly virulent causing clinical signs, killing all the chickens in the medium and high dose groups, and efficiently transmitting to contacts. The three viruses also replicated in the reproductive tract of the adult hens. Virus shedding for all viruses was detected 24 hours after challenge, peaking with high titers at day 4 post challenge. Although not genetically identical, the studied isolates were shown to be phenotypically very similar, which allows the utilization of the available literature in the control of the current outbreak.

Experimental Infection and Transmission of Newcastle Disease Vaccine Virus in Four Wild Passerines.

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 (106.7 mean embryo infectious doses [EID50/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 104.9 EID50/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.

Infección experimental y transmisión del virus de la vacuna contra la enfermedad de Newcastle en cuatro paseriformes silvestres. Nuestras investigaciones anteriores han demostrado que las vacunas vivas utilizadas en avicultura se han aislado de forma intermitente de aves silvestres muestreadas durante los estudios de vigilancia en el campo para el virus de la enfermedad de Newcastle (NDV). Por lo tanto, se investigó experimentalmente la susceptibilidad a la vacuna contra la enfermedad de Newcastle cepa LaSota en cuatro especies de aves silvestres y nativas asociadas con se han asociado con la agricultura y se evaluó la dinámica de transmisión, la transmisión potencial desde el pollo y las respuestas de anticuerpos humorales. Para evaluar la susceptibilidad, se inocularon pinzones mexicanos (Haemorhous mexicanus; n = 16), tordos cabecicafés (Molothrus ater; n = 9), cardenales (Cardinalis cardinalis; n = 6) y jilgueros norteamericanos (Spinus tristis; n = 12), todos de origen silvestre y sin exposición previa al virus de Newcastle. Estas aves se inocularon con 0.1 ml (106.7 dosis medias infecciosas para embrión de pollo [EID50]/ml) de la vacuna de Newcastle cepa LaSota a través de la vía oculonasal. Para determinar la transmisión entre pollos y aves silvestres, se inocularon de igual forma aves adulta tipo Leghorn libres de patógenos específicos y se alojaron en unidades de aislamiento en cohabitación con dos a cinco aves silvestres de las especies mencionadas anteriormente. Este diseño dio como resultado tres tratamientos: inoculación directa de aves silvestres (cinco grupos), exposición de aves silvestres a un pollo inoculado (dos grupos), o exposición a dos pollos inoculados (seis grupos), respectivamente. Se recolectaron muestras de sangre e hisopos de la orofaringe y de la cloaca antes y después de la infección con la vacuna viva. Todas las aves silvestres que se inocularon directamente con la vacuna LaSota eliminaron el virus, como se demostró mediante el aislamiento viral (VI). Los cardenales fueron la especie más susceptible con base en el aislamiento viral de uno a 11 días después de la inoculación con títulos de hasta 104.9 EID50/ml. Aunque todos los pollos inoculados eliminaron el virus LaSota y este virus estaba presente en el agua de bebida, la mayoría de las aves silvestres no inoculadas que cohabitaron con estos pollos permanecieron sin infectar durante 14 días, como lo demuestra el aislamiento viral. Sin embargo, un jilguero norteamericano resultó positivo mediante aislamiento viral a la transmisión de la vacuna a los siete días después de la inoculación y un pinzón mexicano resultó positivo para la transmisión de la vacuna mediante transcripción reversa y PCR en tiempo real a los 13 días después de la inoculación. Solo un tordo cabecicafé inoculado directamente (de un total de tres) y dos cardenales (de un total de dos) desarrollaron títulos de anticuerpos de inhibidores de la hemaglutinación específicos contra la enfermedad de Newcastle de 16, 16 y 128, respectivamente. No se detectaron signos clínicos en los pollos ni en las aves silvestres después de la inoculación.

Burden of exposure to infectious bursal disease virus, infectious bronchitis virus, Newcastle disease virus, Mycoplasma gallisepticum, and intestinal parasites in introduced broiler chickens on the Galapagos.

Diseases in introduced broilers can possibly spill over to wild birds on the Galapagos. Knowledge about the current burden of exposure to pathogens in broilers on the Galapagos is very limited. The objective of the study reported here was to measure the burden of exposure to infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), Mycoplasma gallisepticum (MG), and intestinal parasites in a sample of broiler chickens on 13 farms on Santa Cruz Island and San Cristobal Island in July 2017. Blood serum samples were tested for detection of antibodies to IBDV, IBV, NDV, and MG by using an IDEXX Enzyme-linked Immunosorbent Assay. In addition, fecal samples and pen bedding environmental samples were processed and analyzed for diagnosis of intestinal parasite eggs under a compound light microscope. The frequency of seropositive broilers to IBDV was 74/130 or 56% (95% CI = 48, 65%), to IBV was 27/130 or 20% (14, 28%), and to NDV was 1/130 or 0.7% (0.1, 4%). All broilers tested negative to MG antibodies. Eimeria spp. infection was common in study broilers. Finally, we observed interaction between broiler chickens and wild birds (finches) inside broiler pens, as well as the presence of backyard chickens inside property limits of study farms. This study produced evidence that exposure to IBDV, IBV, and intestinal parasites in broilers on Santa Cruz Island and San Cristobal Island is important. Study results are relevant because (i) they provide new baseline data on the burden of exposure to avian pathogens in broiler farms, (ii) justify the need to verify standard operating procedures in hatcheries that supply (non-vaccinated) day-old chicks to the Galapagos and (iii) to implement enhanced biosecurity standards on broiler chicken farms to mitigate risk of disease transmission between broilers, backyard poultry, and wild birds on the Galapagos.

Potential of genotype VII Newcastle disease viruses to cause differential infections in chickens and ducks.

Newcastle disease (ND), caused by ND virus (NDV), is one of the most infectious and economically important diseases of the poultry industry worldwide. While infections are reported in a wide range of avian species, the pathogenicity of chicken-origin virulent NDV isolates in ducks remains elusive. In this study, two NDV strains were isolated and biologically and genetically characterized from an outbreak in chickens and apparently healthy ducks. Pathogenicity assessment indices, including the mean death time (MDT), intracerebral pathogenicity index (ICPI) and cleavage motifs in the fusion (F) protein, indicated that both isolates were velogenic in nature. While these isolates carried pathogenic characteristics, interestingly they showed differential pathogenicity in ducks. The chicken-origin isolate caused high (70%) mortality, whereas the duck-origin virus resulted in low (20%) mortality in 4-week-old ducks. Intriguingly, both isolates showed comparable disease pathologies in chickens. Full-genome sequence analysis showed that the virus genome contains 15 192 nucleotides and carried features that are characteristic of velogenic strains of NDV. A phylogenetic analysis revealed that both isolates clustered in class II and genotype VII. However, there were several mutations in the functionally important regions of the fusion (F) and haemagglutinin-neuraminidase (HN) proteins, which may be responsible for the differential pathogenicity of these viruses in ducks. In summary, these results suggest that NDV strains with the same genotype show differential pathogenicity in chickens and ducks. Furthermore, chicken-origin virulent NDVs are more pathogenic for ducks than duck-origin viruses. These findings propose a role for chickens in the evolution of viral pathogenicity and the potential genetic resistance of ducks to poultry viruses.