Phylogenetic analysis, genetic diversity, and epidemiology of pigeon paramyxovirus type 1 in China.

Pigeon paramyxovirus type 1 (PPMV-1) poses significant economic challenges to the pigeon industry in China. However, information about the prevalence, genetic diversity, and epidemiology of PPMV-1 in China is still lacking. In this study, we isolated six strains of PPMV-1 from Hubei and Zhejiang provinces in 2022. All six isolates were found to belong to subgenotype VI.2.1.1.2.2. Five of them were identified as mesogenic and one as lentogenic. Multiple mutations were observed in the F and HN proteins of these isolates. Comprehensive analysis of global PPMV-1 strains highlighted the dominance of genotype VI, showing that VI.2.1.1.2.2 has been the dominant subgenotype since 2011. We also identified 36 host-specific amino acid substitutions that are unique to PPMV-1 in comparison to chicken-origin NDVs. The data reported here contribute to our understanding of the epidemiology, genetic diversity, and prevalence of PPMV-1 and serve as a valuable reference for the prevention and control of PPMV-1.

Cocirculation of Newcastle Disease Virus Genotypes XII and VII Along with Nonvirulent Forms Characterized in Domestic Birds from Peru.

Newcastle disease virus (NDV) is one of the most important pathogens affecting poultry, given its impact on health and production systems worldwide, despite widespread vaccination. Over the past 20 years, NDV has caused severe outbreaks of disease in Peru. These outbreaks primarily affected gamecocks and broiler chickens, with an additional reported case in commercial layers. Therefore, our objective was to identify and characterize the virus responsible for these cases in Peru. We analyzed 14 suspected clinical cases in domestic birds for NDV detection, isolation, and genetic characterization. Among these cases, seven involved gamecocks, with six genotype XII isolates and one genotype VII isolate, representing the first report of NDV genotype VII isolate from fighting roosters in Peru. Additionally, among the six cases in broiler chickens, we detected four genotype XII isolates and three genotype II isolates, including one sample containing both genotypes XII and II. Furthermore, a genotype I viral isolate was identified in a laying hen. Hence, we concluded that two divergent, highly virulent NDV genotypes, genotypes XII and VII, along with avirulent forms such as genotypes I and II are circulating among domestic birds in Peru. Genetic analysis indicates that these viruses are evolving locally within avian species and offers the basis necessary for vaccine adaptation to circulating viruses. Our results highlight the cocirculation of multiple virulent and nonvirulent NDV genotypes in domestic birds in Peru, underscoring the potential role of gamecocks as a viral source of virulent NDV strains in the country and the occurrence of outbreaks in poultry farms.

Cocirculación de los genotipos XII y VII del virus de la enfermedad de Newcastle junto con formas no virulentas caracterizadas en aves domésticas del Perú. El virus de la enfermedad de Newcastle (NDV) es uno de los patógenos más importantes que afectan a la avicultura, dado su impacto en la salud y los sistemas de producción en todo el mundo, a pesar de la vacunación generalizada. Durante los últimos 20 años, el virus de la enfermedad de Newcastle ha causado graves brotes de enfermedades en el Perú. Estos brotes afectaron principalmente a gallos de pelea y pollos de engorde, con un caso adicional reportado en aves de postura comerciales. Por lo tanto, nuestro objetivo fue identificar y caracterizar el virus responsable de estos casos en el Perú. Se analizaron 14 casos cl'inicos sospechosos en aves domésticas para la detección, aislamiento y caracterización genética del virus de Newcastle. Entre estos casos, siete involucraron gallos de pelea, con seis aislamientos del genotipo XII y un aislado del genotipo VII, lo que representa el primer informe de aislamiento del genotipo VII del virus de Newcastle de gallos de pelea en Perú. Además, entre los seis casos en pollos de engorde, se detectaron cuatro aislados del genotipo XII y tres aislados del genotipo II, incluida una muestra que con-ten'ia ambos genotipos XII y II. Además, se identificó un aislado viral de genotipo I en una gallina de postura. Por lo tanto, se concluye que dos genotipos divergentes y altamente virulentos del virus de Newcastle, los genotipos XII y VII, junto con formas avirulentas como los genotipos I y II, están circulando entre las aves domésticas en el Perú. El análisis genético indica que estos virus están evolucionando localmente dentro de las especies aviares y ofrece las bases necesarias para realizar adaptaciones de las vacunas contra los virus circulantes. Nuestros resultados resaltan la cocirculación de múltiples genotipos del virus de Newcastle virulentos y no virulentos en aves domésticas en Perú, subrayando el papel potencial de los gallos de pelea como fuente viral de cepas virulentas del virus de Newcastle en el pa'is y la aparición de brotes en granjas av'icolas.

Genomic Diversity and Evolutionary Insights of Avian Paramyxovirus-1 in Avian Populations in Pakistan.

The virulent form of Avian paramyxovirus-1 (APMV-1), commonly known as Newcastle Disease Virus (NDV), is a pathogen with global implications for avian health, affecting both wild and domestic bird populations. In Pakistan, recurrent Newcastle Disease (caused by NDV) outbreaks have posed significant challenges to the poultry industry. Extensive surveillance in Pakistan over 20 years has demonstrated a dynamic genetic diversity among circulating APMV-1 strains, emphasizing the potential necessity for customized vaccination strategies and continuous surveillance. In this study, 13 APMV-1-positive isolates harboring four different APMV-1 genotypes circulating throughout Pakistan were identified. These included the highly virulent genotypes VII and XIII, genotype XXI, commonly associated with Columbiformes, and genotype II, hypothesized to have been detected following vaccination. These findings underscore the intricate interplay of mutational events and host-immune interactions shaping the evolving NDV landscape. This study advances our understanding of the evolutionary dynamics of APMV-1 in Pakistan, highlighting the need for tailored vaccination strategies and continuous surveillance to enable effective APMV-1 management in avian populations, further emphasizing the importance of globally coordinated strategies to tackle APMV-1, given its profound impact on wild and domestic birds.

Isolation and Genetic Characterization of Genotype VII Velogenic Pathotype Newcastle Disease Virus from Commercial Chicken Farms in Central Ethiopia, Distinct from the Local Vaccine Strains.

Newcastle disease (ND) is caused by virulent strains of avian paramyxovirus type 1, also known as Newcastle disease virus (NDV). Despite vaccination, the frequency of reported outbreaks in Ethiopia has increased. From January to June 2022, an active outbreak investigation was conducted in six commercial chicken farms across areas of central Ethiopia to identify the circulating NDV strains. Thirty pooled tissue specimens were collected from chickens suspected of being infected with NDV. A questionnaire survey of farm owners and veterinarians was also carried out to collect information on the farms and the outbreak status. NDV was isolated using specific-pathogen-free (SPF)-embryonated chicken eggs and detected using haemagglutination and the reverse transcriptase-polymerase chain reaction (RT-PCR). The genotype and virulence of field NDV isolates were determined using phylogenetic analysis of fusion (F) protein gene sequences and the mean death time (MDT) test in SPF-embryonated chicken eggs. The questionnaire results revealed that ND caused morbidity (23.1%), mortality (16.3%), case fatality (70.8%), and significant economic losses. Eleven of thirty tissue specimens tested positive for NDV using haemagglutination and RT-PCR. The MDT testing and sequence analysis revealed the presence of virulent NDV classified as genotype VII of class II velogenic pathotype and distinct from locally used vaccine strains (genotype II). The amino acid sequences of the current virulent NDV fusion protein cleavage site motif revealed 112RRQKR↓F117, unlike the locally used avirulent vaccine strains (112GRQGR↓L117). The epidemiological data, MDT results, cleavage site sequence, and phylogenetic analysis all indicated that the present NDV isolates were virulent. The four NDV sequences were deposited in GenBank with accession numbers F gene (PP726912-15) and M gene (PP726916-19). The genetic difference between avirulent vaccine strains and circulating virulent NDV could explain the low level of protection provided by locally used vaccines. Further studies are needed to better understand the circulating NDV genotypes in different production systems.

Genomic Diversity and Geographic Distribution of Newcastle Disease Virus Genotypes in Africa: Implications for Diagnosis, Vaccination, and Regional Collaboration.

The emergence of new virulent genotypes and the continued genetic drift of Newcastle disease virus (NDV) implies that distinct genotypes of NDV are simultaneously evolving in different geographic locations across the globe, including throughout Africa, where NDV is an important veterinary pathogen. Expanding the genomic diversity of NDV increases the possibility of diagnostic and vaccine failures. In this review, we systematically analyzed the genetic diversity of NDV genotypes in Africa using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Information published between 1999 and 2022 were used to obtain the genetic background of different genotypes of NDV and their geographic distributions in Africa. The following genotypes were reported in Africa: I, II, III, IV, V, VI, VII, VIII, XI, XIII, XIV, XVII, XVIII, XX, and XXI. A new putative genotype has been detected in the Democratic Republic of the Congo. However, of 54 African countries, only 26 countries regularly report information on NDV outbreaks, suggesting that this number may be vastly underestimated. With eight different genotypes, Nigeria is the country with the greatest genotypic diversity of NDV among African countries. Genotype VII is the most prevalent group of NDV in Africa, which was reported in 15 countries. A phylogeographic analysis of NDV sequences revealed transboundary transmission of the virus in Eastern Africa, Western and Central Africa, and in Southern Africa. A regional and continental collaboration is recommended for improved NDV risk management in Africa.

Thermostability study of virulent Newcastle disease viruses isolated in Southern Angola.

Newcastle disease (ND) is endemic in Angola. Several outbreaks of ND occurred in small backyard flocks and village chickens with high mortality in the southern provinces of the country, Cunene, Namibe and Huíla, in 2016 and 2018. In those years, 15 virulent ND virus (NDV) strains were isolated and grouped within subgenotype 2 of genotype VII (subgenotype VII.2). We now present a study on the thermostability of the isolates, aiming at the selection of the most thermostable strains that, after being genetically modified to reduce their virulence, can be adapted to the production of vaccines less dependent on cold chain and more adequate to protect native chickens against ND. Heat-inactivation kinetics of haemagglutinin (Ha) activity and infectivity (I) of the isolates were determined by incubating aliquots of virus at 56 °C for different time intervals. The two isolates from Namibe province showed a decrease in infectivity of 2 log10 in ≤ 10 min, therefore belonging to the I-phenotype, but while the NB1 isolate from 2016 maintained the Ha activity up to 30 min and was classified as thermostable virus (I-Ha+), the Ha activity of the 2018 NB2 isolate decreased by 2 log2 in 30 min, being classified as a thermolabile virus (I-Ha-). Of the 13 NDV isolates from Huíla province, 10 isolates were classified as thermostable, eight with phenotype I+Ha+ and 2 with phenotype I-Ha+. The other three isolates from this province were classified as thermolabile viruses (I-Ha-).Contribution: This study will contribute to the control and/or eradication of Newcastle disease virus in Angola. The thermostable viral strains isolated from chickens in the country can be genetically manipulated by reverse genetic technology in order to reduce their virulence and use them as a vaccine in the remote areas of Angola.

Molecular characteristics and phylogenetic analysis of pigeon paramyxovirus type 1 isolates from pigeon meat farms in Shanghai (2009-2012).

The majority of pigeon paramyxovirus type 1 (PPMV-1) strains are generally non-pathogenic to chickens; however, they can induce severe illness and high mortality rates in pigeons, leading to substantial economic repercussions. The genomes of 11 PPMV-1 isolates from deceased pigeons on meat pigeon farms during passive monitoring from 2009 to 2012 were sequenced and analyzed using polymerase chain reaction and phylogenetic analysis. The complete genome lengths of 11 isolates were approximately 15,192 nucleotides, displaying a consistent gene order of 3'-NP-P-M-F-HN-L-5'. ALL isolates exhibited the characteristic motif of 112RRQKRF117 at the fusion protein cleavage site, which is characteristic of velogenic Newcastle disease virus. Moreover, multiple mutations have been identified within the functional domains of the F and HN proteins, encompassing the fusion peptide, heptad repeat region, transmembrane domains, and neutralizing epitopes. Phylogenetic analysis based on sequences of the F gene unveiled that all isolates clustered within genotype VI in class II. Further classification identified at least two distinct sub-genotypes, with seven isolates classified as sub-genotype VI.2.1.1.2.2, whereas the others were classified as sub-genotype VI.2.1.1.2.1. This study suggests that both sub-genotypes were implicated in severe disease manifestation among meat pigeons, with sub-genotype VI.2.1.1.2.2 displaying an increasing prevalence among Shanghai's meat pigeon population since 2011. These results emphasize the value of developing pigeon-specific vaccines and molecular diagnostic tools for monitoring and proactively managing potential PPMV-1 outbreaks.

Spillover of Newcastle disease virus to Himalayan Griffon vulture: a possible food-based transmission.

The Newcastle disease virus (NDV) affects wild and domesticated bird species, including commercial poultry. Although the diversity of NDV in domestic chickens is well documented, limited information is available about Newcastle disease (ND) outbreaks in other bird species. We report an annotated sequence of NDV/Vulture/Borjuri/01/22, an avirulent strain of NDV reported from Borjuri, Northeast India, in Himalayan Griffon vulture. The complete genome is 15,186 bases long with a fusion protein (F) cleavage site 112GRQGR↓L117. The phylogenetic analysis based on the F protein gene and the whole genome sequence revealed that the isolate from the vulture belongs to genotype II, sharing significant homology with vaccine strain LaSota. The study highlights the possible spillover of the virus from domestic to wild species through the food chain.

Molecular Identification and Phylogenetic Study Based on the Fusion Gene of Newcastle Disease Virus Isolated from Broiler Poultry Farms in Markazi Province, Iran.

Newcastle disease (ND) is an economically significant and extremely spreadable viral illness affecting a wide variety of avian species. ND can rapidly spread within poultry farms and result in considerable economic losses for the global poultry industry. This disease is endemic in Iran, and despite intensive vaccination efforts in the poultry industry, outbreaks of ND occur unexpectedly. This study aimed to isolate the Newcastle disease virus (NDV) from poultry farms with breathing problems in Markazi province, Iran, and investigate the evolutionary relationship and molecular characteristics of the isolates during 2017-2019. To this end, tissue samples (lung, brain, and trachea) were taken from 42 broiler farms exhibiting respiratory symptoms. The samples were inoculated into 9-11-day-old embryonated eggs, and the virus was isolated from 20 (47.6%) of the 42 farms. Subsequently, RT-PCR was used to amplify partial fusion gene sequences from the new isolates. The amplified products were sequenced and compared phylogenetically to the standard pilot dataset (125 selected sequences) generated by the NDV consortium. As determined by phylogenetic analysis, all nine isolates belonged to subgenotype VII.1.1 of genotype VII and were highly similar to isolates from other parts of Iran and China. Moreover, all isolates possessed a polybasic cleavage site motif (112RRQKRF117), characteristic of virulent strains. Furthermore, the present isolates shared a high nucleotide identity (96%) with viruses previously isolated from other provinces of Iran, as determined by BLAST searches and multiple alignments. In addition, they shared a high degree of sequence similarity but were distinct from the existing NDV vaccines. Therefore, the genetic dissimilarity between current vaccine strains and circulating NDVs must be considered in vaccination programs.

An Outbreak in Pigeons Caused by the Subgenotype VI.2.1.2 of Newcastle Disease Virus in Brazil.

Newcastle disease virus (NDV) can infect over 250 bird species with variable pathogenicity; it can also infect humans in rare cases. The present study investigated an outbreak in feral pigeons in São Paulo city, Brazil, in 2019. Affected birds displayed neurological signs, and hemorrhages were observed in different tissues. Histopathology changes with infiltration of mononuclear inflammatory cells were also found in the brain, kidney, proventriculus, heart, and spleen. NDV staining was detected by immunohistochemistry. Twenty-seven out of thirty-four tested samples (swabs and tissues) were positive for Newcastle disease virus by RT-qPCR test, targeting the M gene. One isolate, obtained from a pool of positive swab samples, was characterized by the intracerebral pathogenicity index (ICPI) and the hemagglutination inhibition (HI) tests. This isolate had an ICPI of 0.99, confirming a virulent NDV strain. The monoclonal antibody 617/161, which recognizes a distinct epitope in pigeon NDV strains, inhibited the isolate with an HI titer of 512. A complete genome of NDV was obtained using next-generation sequencing. Phylogenetic analysis based on the complete CDS F gene grouped the detected isolate with other viruses from subgenotype VI.2.1.2, class II, including one previously reported in Southern Brazil in 2014. This study reports a comprehensive characterization of the subgenotype VI.2.1.2, which seems to have been circulating in Brazilian urban areas since 2014. Due to the zoonotic risk of NDV, virus surveillance in feral pigeons should also be systematically performed in urban areas.

Pathotyping of Newcastle Disease Virus: a Novel Single BsaHI Digestion Method of Detection and Differentiation of Avirulent Strains (Lentogenic and Mesogenic Vaccine Strains) from Virulent Virus.

We provide a novel single restriction enzyme (RE; BsaHI) digestion approach for detecting distinct pathotypes of Newcastle disease virus (NDV). After scanning 4,000 F gene nucleotide sequences in the NCBI database, we discovered a single RE (BsaHI) digestion site in the cleavage site. APMV-I "F gene" class II-specific primer-based reverse transcriptase PCR was utilized to amplify a 535-bp fragment, which was then digested with the RE (BsaHI) for pathotyping avian NDV field isolates and pigeon paramyxovirus-1 isolates. The avirulent (lentogenic and mesogenic strains) produced 189- and 346-bp fragments, respectively, but the result in velogenic strains remained undigested with 535-bp fragments. In addition, 45 field NDV isolates and 8 vaccine strains were used to confirm the approach. The sequence-based analysis also agrees with the data obtained utilizing the single RE (BsaHI) digestion approach. The proposed technique has the potential to distinguish between avirulent and virulent strains in a short time span, making it valuable in NDV surveillance and monitoring research. IMPORTANCE The extensive use of the NDV vaccine strain and the existence of avirulent NDV strains in wild birds makes it difficult to diagnose Newcastle Disease virus (NDV). The intracerebral pathogenicity index (ICPI) and/or sequencing-based identification, which are required to determine virulent NDV, are time-consuming, costly, difficult, and cruel techniques. We evaluated 4,000 F gene nucleotide sequences and discovered a restriction enzyme (RE; BsaHI) digestion technique for detecting NDV and vaccine pathotypes in a short time span, which is cost-effective and useful for field cases as well as for large-scale NDV monitoring and surveillance. The data acquired using the single RE BsaHI digestion technique agree with the sequence-based analysis.

A Pigeon-Derived Sub-Genotype XXI.1.2 Newcastle Disease Virus from Bangladesh Induces High Mortality in Chickens.

Newcastle disease virus (NDV) is a significant pathogen of poultry; however, variants also affect other species, including pigeons. While NDV is endemic in Bangladesh, and poultry isolates have been recently characterized, information about viruses infecting pigeons is limited. Worldwide, pigeon-derived isolates are commonly of low to moderate virulence for chickens. Here, we studied a pigeon-derived NDV isolated in Bangladesh in 2010. To molecularly characterize the isolate, we sequenced its complete fusion gene and performed a comprehensive phylogenetic analysis. We further studied the biological properties of the virus by estimating mean death time (MDT) and by experimentally infecting 5-week-old naïve Sonali chickens. The studied virus clustered in sub-genotype XXI.1.2 with NDV from pigeons from Pakistan isolated during 2014-2018. Deduced amino acid sequence analysis showed a polybasic fusion protein cleavage site motif, typical for virulent NDV. The performed in vivo pathogenicity testing showed a MDT of 40.8 h, and along with previously established intracerebral pathogenicity index of 1.51, these indicated a velogenic pathotype for chickens, which is not typical for pigeon-derived viruses. The experimental infection of chickens resulted in marked neurological signs and high mortality starting at 7 days post infection (dpi). Mild congestion in the thymus and necrosis in the spleen were observed at an advanced stage of infection. Microscopically, lymphoid depletion in the thymus, spleen, and bursa of Fabricius were found at 5 dpi, which progressed to severe in the following days. Mild to moderate proliferation of glial cells was noticed in the brain starting at 2 dpi, which gradually progressed with time, leading to focal nodular aggregation. This study reports the velogenic nature for domestic chickens of a pigeon-derived NDV isolate of sub-genotype XXI.1.2. Our findings show that not all pigeon-derived viruses are of low virulence for chickens and highlight the importance of biologically evaluating the pathogenicity of NDV isolated from pigeons.

Virulence during Newcastle Disease Viruses Cross Species Adaptation.

The hypothesis that host adaptation in virulent Newcastle disease viruses (NDV) has been accompanied by virulence modulation is reviewed here. Historical records, experimental data, and phylogenetic analyses from available GenBank sequences suggest that currently circulating NDVs emerged in the 1920-1940's from low virulence viruses by mutation at the fusion protein cleavage site. These viruses later gave rise to multiple virulent genotypes by modulating virulence in opposite directions. Phylogenetic and pathotyping studies demonstrate that older virulent NDVs further evolved into chicken-adapted genotypes by increasing virulence (velogenic-viscerotropic pathotypes with intracerebral pathogenicity indexes [ICPIs] of 1.6 to 2), or into cormorant-adapted NDVs by moderating virulence (velogenic-neurotropic pathotypes with ICPIs of 1.4 to 1.6), or into pigeon-adapted viruses by further attenuating virulence (mesogenic pathotypes with ICPIs of 0.9 to 1.4). Pathogenesis and transmission experiments on adult chickens demonstrate that chicken-adapted velogenic-viscerotropic viruses are more capable of causing disease than older velogenic-neurotropic viruses. Currently circulating velogenic-viscerotropic viruses are also more capable of replicating and of being transmitted in naïve chickens than viruses from cormorants and pigeons. These evolutionary virulence changes are consistent with theories that predict that virulence may evolve in many directions in order to achieve maximum fitness, as determined by genetic and ecologic constraints.

A New Variant Among Newcastle Disease Viruses Isolated in the Democratic Republic of the Congo in 2018 and 2019.

Newcastle disease (ND) is a highly transmissible and devastating disease that affects poultry and wild birds worldwide. Comprehensive knowledge regarding the characteristics and epidemiological factors of the ND virus (NDV) is critical for the control and prevention of ND. Effective vaccinations can prevent and control the spread of the NDV in poultry populations. For decades, the Democratic Republic of the Congo (DRC) has reported the impacts of ND on commercial and traditional poultry farming systems. The reports were preliminary clinical observations, and few cases were confirmed in the laboratory. However, data on the phylogenetic, genetic, and virological characteristics of NDVs circulating in the DRC are not available. In this study, the whole-genome sequences of three NDV isolates obtained using the next-generation sequencing method revealed two isolates that were a new variant of NDV, and one isolate that was clustered in the subgenotype VII.2. All DRC isolates were velogenic and were antigenically closely related to the vaccine strains. Our findings reveal that despite the circulation of the new variant, ND can be controlled in the DRC using the current vaccine. However, epidemiological studies should be conducted to elucidate the endemicity of the disease so that better control strategies can be implemented.

Surveillance and Genetic Characterization of Virulent Newcastle Disease Virus Subgenotype V.3 in Indigenous Chickens from Backyard Poultry Farms and Live Bird Markets in Kenya.

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 (113R-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.

Genetic and biological characterization of Newcastle disease viruses circulating in Bangladesh during 2010-2017: further genetic diversification of class II genotype XIII in Southcentral Asia.

Newcastle disease virus (NDV) is endemic in Bangladesh and is a major threat to commercial poultry operations. While complete fusion (F) genes are recommended for molecular characterization and classification of NDV isolates, heretofore, only partial F gene data have been available for Bangladeshi NDVs. To this end, we obtained the full-length F gene coding sequences of 11 representative NDVs isolated in Bangladesh between 2010 and 2017. In addition, one of the viruses (MK934289/chicken/Bangladesh/C161/2010) was used in an experimental infection of chickens to establish the viral pathotype and study gross and microscopic lesions. Phylogenetic analysis provided evidence that all studied Bangladeshi isolates belong to genotype XIII.2 of class II NDVs. Six of the viruses were isolated between 2010 and 2017 and grouped together with isolates from neighbouring India during 2013-2016. Another four Bangladeshi isolates (2010-2016) formed a separate monophyletic branch within XIII.2 and showed high nucleotide distance from the isolates from India and the other six Bangladeshi viruses within the sub-genotype; however, none of these groups fulfils all classification criteria to be named as a separate sub-genotype. The eleventh Bangladeshi virus studied here (C162) was genetically more distant from the remaining isolates. It out-grouped the viruses from sub-genotypes XIII.2.1 and XIII.2.2 and showed more than 9.5 % nucleotide distance from all genotype XIII sub-genotypes. This isolate may represent an NDV variant that is evolving independently from the other viruses in the region. The experimental infection in chickens revealed that the tested isolate (C161) is a velogenic viscerotropic virus. Massive haemorrhages, congestion and necrosis in different visceral organs, and lymphoid depletion in lymphoid tissues, typical for infection with velogenic NDV, were observed. Our findings demonstrate the endemic circulation of sub-genotype XIII.2 in Southcentral Asia and further genetic diversification of these viruses in Bangladesh and neighbouring India. This constant evolution of the viruses may lead to the establishment of new genetic groups in the region. Additional historical and prospective virus and surveillance data from the region and neighbouring countries will allow a more detailed epidemiological inference.

Phylodynamic analyses of class I Newcastle disease virus isolated in China.

Newcastle disease virus (NDV), the pathogen of Newcastle disease, has caused significant losses to the poultry industry worldwide. However, owing to its avirulence, class I NDVs have not been studied as much as class II NDVs. We aimed to epidemiologically monitor the spread of class I NDVs in China. We isolated 104 class I NDV strains from poultry in live poultry markets (LPMs) of Guangdong Province, south China, between January 2016 and December 2018. Genetic analysis revealed that all 104 isolates and most of the strains isolated from China were clustered into genotype 1.1.2 of class I NDVs. Bayesian analysis revealed that, although the United States may be the source, east and south China may be the epicentres of class I NDVs in China. In addition, in China, class I NDVs are presumably transmitted by chickens and domestic ducks as the virus is mostly prevalent in these birds. These novel findings demonstrated that class I NDVs are prevalent in south China, and it is important to perform routine surveillance and limit the numbers of different birds in different areas of LPMs to decrease the risk of intra- and interspecies transmission of NDVs.

Complete genome sequence of a subgenotype XXI.1.1 pigeon paramyxovirus type 1 virus (PPMV­1) isolated from Iran in 2018 and phylogenetic analysis of a possible novel, but unassigned, PPMV-1 group isolated in 2014.

Newcastle disease (ND) is one of the most serious infectious and contagious viral diseases in avian species. Recently, several ND outbreaks in pigeon caused by pigeon paramyxovirus serotype-1 (PPMV-1) have been reported from Iran, but unfortunately, phylogenetic studies have been mostly conducted on partial sequence of NDV fusion (F) gene. In addition, a complete genome data of Iranian PPMV-1 strains are not available. In the present study, a PPMV-1, named Avian avulavirus 1/pigeon/Iran/UT-EGV/2018, isolated from an infected pigeon, was subjected to whole-genome sequencing. The isolate showed an MDT of 74 h, thus categorizing it as mesogenic. The phylogenetic analysis based on the F gene sequence revealed the isolate belongs to XXI.1.1 subgenotype (min 0.9 % and max 3 %). To our knowledge, our study is the first study to publish the complete genome of a PPMV-1 from Iran. According to BLAST results, the whole genome of UT-EGV had high homology with some Russian, Egyptian and Ukrainian strains (the highest was 96.55 %). Additionally, we conducted a phylogenetic analysis on five PPMV-1 that we isolated in 2014 to find that they may belong to a completely unreported subgenotype (6 % distance when compared as a group). The information obtained from this study can be useful in preventive measures, including constructing an effective vaccine against PPMV-1 in Iran.

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.

Identification of Newcastle disease virus subgenotype VII.2 in wild birds in Turkey.

BACKGROUND: Newcastle disease viruses (NDVs) can spread across continents via migratory birds. Hence, we investigated the frequency of NDV in both non-migratory and birds migrating on the Black Sea-Mediterranean flyway, in Istanbul, Turkey. Birds were trapped using nets placed around the Kucukcekmece lake Avcilar, Istanbul, in spring seasons of 2016 and 2018. In total, 297 birds belonging to 42 different species were trapped, categorized according to species and sex, and flocked oropharyngeal swabs were collected. In addition, flocked swabs were also collected from 115 mallards caught by hunters around Edirne and from 207 birds which had been treated in the Veterinary Faculty of Istanbul university-Cerrahpasa. Tissue samples were taken from dead wild birds brought by public to Veterinary Faculty. A total of 619 flocked oropharyngeal swabs were pooled into 206 samples. RNA was extracted from swabs and tissue samples. Real-time RT-PCR prob. assay was used to detect NDV-RNA in samples. RESULTS: There was no amplification in real time RT-PCR in samples taken from wild birds caught by traps. However, amplification of NDV-F gene was observed in oropharyngeal swabs taken from 2 waterfowls (Common Moorhen and Mallard), and in tissue samples taken from 2 little owls and 1 common kestrel. Sequencing and phylogenetic analyses of these 5 samples for NDV-F gene showed great similarity with NDV subgenotype VII.2 viruses. Analysis also showed that there is a high similarity with the F gene sequences previously reported from Turkey in 2012 and as well as the sequences from neighbouring countries Bulgaria and Georgia and geographically close country such as Pakistan. Although the strains found in this study are closely related, there is a relatively small degree of molecular divergence within 543 bp of F gene of the Turkish NDV isolate and strains detected in Israel, Pakistan, Iran, United Arab Emirates and Belgium. CONCLUSIONS: Our findings revealed the presence of subgenotype VII.2 of NDVs in wild birds in north west of Turkey and demonstrated some degree of molecular evolution when compared to the earlier NDV-VII.2 isolate in Turkey.