The full genome characterization of avian encephalomyelitis virus, Iran: a vertical transmission case.

Avian encephalomyelitis (AE) is an important infectious poultry disease worldwide that is caused by avian encephalomyelitis virus (AEV). The causative virus can be transmitted both horizontally and vertically. In the present study, an AEV suspected outbreak with typical neurological signs occurred in broilers. Histopathological examination, RT-PCR assay and full genome sequencing were applied to confirm the presence of AEV. Phylogenetic analysis of the full genome sequence showed that the detected AEV strain at 7055 nucleotide length is classified in cluster I and is closely related to vaccinal USA and China originated isolates. Although, the outbreaks of AE in progeny of vaccinated breeders have been reported previously, the source of infection was unknown. Based on the results obtained in this study, the outbreaks are vaccine-originated. This study provides the first whole genome analysis of AEV from Iran and reveals that the AEV possesses a hepatitis C virus-like internal ribosome entry site.

Avian Influenza Virus and Avian Paramyxoviruses in Wild Waterfowl of the Western Coast of the Caspian Sea (2017-2020).

The flyways of many different wild waterfowl pass through the Caspian Sea region. The western coast of the middle Caspian Sea is an area with many wetlands, where wintering grounds with large concentrations of birds are located. It is known that wild waterfowl are a natural reservoir of the influenza A virus. In the mid-2000s, in the north of this region, the mass deaths of swans, gulls, and pelicans from high pathogenicity avian influenza virus (HPAIV) were noted. At present, there is still little known about the presence of avian influenza virus (AIVs) and different avian paramyxoviruses (APMVs) in the region's waterfowl bird populations. Here, we report the results of monitoring these viruses in the wild waterfowl of the western coast of the middle Caspian Sea from 2017 to 2020. Samples from 1438 individuals of 26 bird species of 7 orders were collected, from which 21 strains of AIV were isolated, amounting to a 1.46% isolation rate of the total number of samples analyzed (none of these birds exhibited external signs of disease). The following subtypes were determined and whole-genome nucleotide sequences of the isolated strains were obtained: H1N1 (n = 2), H3N8 (n = 8), H4N6 (n = 2), H7N3 (n = 2), H8N4 (n = 1), H10N5 (n = 1), and H12N5 (n = 1). No high pathogenicity influenza virus H5 subtype was detected. Phylogenetic analysis of AIV genomes did not reveal any specific pattern for viruses in the Caspian Sea region, showing that all segments belong to the Eurasian clades of classic avian-like influenza viruses. We also did not find the amino acid substitutions in the polymerase complex (PA, PB1, and PB2) that are critical for the increase in virulence or adaptation to mammals. In total, 23 hemagglutinating viruses not related to influenza A virus were also isolated, of which 15 belonged to avian paramyxoviruses. We were able to sequence 12 avian paramyxoviruses of three species, as follows: Newcastle disease virus (n = 4); Avian paramyxovirus 4 (n = 5); and Avian paramyxovirus 6 (n = 3). In the Russian Federation, the Newcastle disease virus of the VII.1.1 sub-genotype was first isolated from a wild bird (common pheasant) in the Caspian Sea region. The five avian paramyxovirus 4 isolates obtained belonged to the common clade in Genotype I, whereas phylogenetic analysis of three isolates of Avian paramyxovirus 6 showed that two isolates, isolated in 2017, belonged to Genotype I and that an isolate identified in 2020 belonged to Genotype II. The continued regular monitoring of AIVs and APMVs, the obtaining of data on the biological properties of isolated strains, and the accumulation of information on virus host species will allow for the adequate planning of epidemiological measures, suggest the most likely routes of spread of the virus, and assist in the prediction of the introduction of the viruses in the western coastal region of the middle Caspian Sea.

Putative Novel Avian Paramyxovirus (AMPV) and Reidentification of APMV-2 and APMV-6 to the Species Level Based on Wild Bird Surveillance (United States, 2016-2018).

Avian paramyxoviruses (APMVs) (subfamily Avulavirinae) have been isolated from over 200 species of wild and domestic birds around the world. The International Committee on Taxonomy of Viruses (ICTV) currently defines 22 different APMV species, with Avian orthoavulavirus 1 (whose viruses are designated APMV-1) being the most frequently studied due to its economic burden to the poultry industry. Less is known about other APMV species, including limited knowledge on the genetic diversity in wild birds, and there is a paucity of public whole-genome sequences for APMV-2 to -22. The goal of this study was to use MinION sequencing to genetically characterize APMVs isolated from wild bird swab samples collected during 2016 to 2018 in the United States. Multiplexed MinION libraries were prepared using a random strand-switching approach using 37 egg-cultured, influenza-negative, hemagglutination-positive samples. Forty-one APMVs were detected, with 37 APMVs having complete polymerase coding sequences allowing for species identification using ICTV's current Paramyxoviridae phylogenetic methodology. APMV-1, -4, -6, and -8 viruses were classified, one putative novel species (Avian orthoavulavirus 23) was identified from viruses isolated in this study, two putative new APMV species (Avian metaavulavirus 24 and 27) were identified from viruses isolated in this study and from retrospective GenBank sequences, and two putative new APMV species (Avian metaavulavirus 25 and 26) were identified solely from retrospective GenBank sequences. Furthermore, coinfections of APMVs were identified in four samples. The potential limitations of the branch length being the only species identification criterion and the potential benefit of a group pairwise distance analysis are discussed. IMPORTANCE Most species of APMVs are understudied and/or underreported, and many species were incidentally identified from asymptomatic wild birds; however, the disease significance of APMVs in wild birds is not fully determined. The rapid rise in high-throughput sequencing coupled with avian influenza surveillance programs have identified 12 different APMV species in the last decade and have challenged the resolution of classical serological methods to identify new viral species. Currently, ICTV's only criterion for Paramyxoviridae species classification is the requirement of a branch length of >0.03 using a phylogenetic tree constructed from polymerase (L) amino acid sequences. The results from this study identify one new APMV species, propose four additional new APMV species, and highlight that the criterion may have insufficient resolution for APMV species demarcation and that refinement or expansion of this criterion may need to be established for Paramyxoviridae species identification.

Avian Orthoavulavirus 1 Vaccination Failure in Minnesota Turkeys.

Minnesota is the leading state in number of turkeys produced in the United States. Turkey flocks in the field are usually vaccinated several times with live avian orthoavulavirus 1 (AOAV-1) vaccines starting as early as 2 wk of age (WOA). During the years 2018-2019, many turkey flocks were diagnosed with low-virulence AOAV-1 infection around 9 WOA that led to respiratory disease, although they were previously vaccinated. This study was designed to investigate the immunity against AOAV-1 in Minnesota turkey flocks in the field and experimentally after vaccination. We reviewed antibody titers against AOAV-1 from turkey flocks tested by ELISA at Minnesota Poultry Testing Laboratory (n = 1292). Up to 9 WOA, more than 85% of the field flocks tested had unprotective antibody titers against AOAV-1. However, commercial poults at 3 WOA experimentally vaccinated by eye-drop method had an ELISA geometric mean titer of 6011 at 7 WOA. Oropharyngeal virus shedding after vaccination was 10%, 70%, 80%, and 40% at 1, 3, 5, and 7 days postvaccination, respectively. This study demonstrates that experimentally vaccinated turkeys respond very well to AOAV-1 vaccine when properly administered. However, there is clear vaccination failure in the field, where vaccine is commonly administered in drinking water, a method that is more susceptible to failure because of many variables in this procedure. We recommend choosing the most effective method of vaccine administration. Given the high incidence of inadequate immunity induced in commercial turkeys on mass application of live AOAV-1 vaccines in water, alternative application methods and subsequent monitoring of the serologic antibody response must be undertaken to ensure a proper immune response.

Artículo regular­Fracaso de la vacunación contra el Orthoavulavirus aviar 1 en pavos de Minnesota. Minnesota es el estado líder en número de pavos producidos en los Estados Unidos. Las parvadas de pavos en el campo generalmente se vacunan varias veces con vacunas vivas con Orthoavulavirus Aviar 1 (AOAV-1) comenzando desde las 2 semanas de edad (WOA). Durante los años 2018­2019, muchas parvadas de pavos fueron diagnosticadas con infección por Orthoavulavirus Aviar 1 de baja virulencia alrededor de las nueve semanas de edad que condujeron a una enfermedad respiratoria, aunque las aves fueron vacunadas previamente. Este estudio fue diseñado para investigar la inmunidad contra Orthoavulavirus Aviar 1 en parvadas de pavos de Minnesota en el campo y experimentalmente después de la vacunación. Se revisaron los títulos de anticuerpos contra Orthoavulavirus Aviar 1 de parvadas de pavos analizados por ELISA en el Laboratorio de Diagnóstico Avícola de Minnesota (n = 1292). Hasta las nueve semanas de edad, más del 85% de las parvadas de campo analizadas tenían títulos de anticuerpos no protectores contra Orthoavulavirus Aviar 1. Sin embargo, los pavipollos comerciales a las tres semanas de edad vacunados experimentalmente por el método de gota ocular tenían un título medio geométrico de ELISA de 6011 a las siete semanas de edad. La diseminación del virus orofaríngeo después de la vacunación fue del 10%, 70%, 80% y 40% a los 1, 3, 5 y 7 días después de la vacunación, respectivamente. Este estudio demuestra que los pavos vacunados experimentalmente respondieron muy bien a la vacuna con Orthoavulavirus Aviar 1 cuando se administra correctamente. Sin embargo, existe un claro fracaso de la vacunación en el campo, donde la vacuna se administra comúnmente en el agua potable, un método que es más susceptible al fracaso debido a muchas variables en este procedimiento. Se recomienda elegir el método de administración de vacunas más eficaz. Considerando la alta incidencia de inmunidad inadecuada inducida en pavos comerciales con la aplicación masiva de vacunas vivas con Orthoavulavirus Aviar 1 en agua, se deben llevar a cabo métodos de aplicación alternativos y monitoreo posterior de la respuesta de anticuerpos serológicos para asegurar una respuesta inmune adecuada.

Recovery of Recombinant Avian Paramyxovirus Type-3 Strain Wisconsin by Reverse Genetics and Its Evaluation as a Vaccine Vector for Chickens.

A reverse genetic system for avian paramyxovirus type-3 (APMV-3) strain Wisconsin was created and the infectious virus was recovered from a plasmid-based viral antigenomic cDNA. Green fluorescent protein (GFP) gene was cloned into the recombinant APMV-3 genome as a foreign gene. Stable expression of GFP by the recovered virus was confirmed for at least 10 consecutive passages. APMV-3 strain Wisconsin was evaluated against APMV-3 strain Netherlands and APMV-1 strain LaSota as a vaccine vector. The three viral vectors expressing GFP as a foreign protein were compared for level of GFP expression level, growth rate in chicken embryo fibroblast (DF-1) cells, and tissue distribution and immunogenicity in specific pathogen-free (SPF) day-old chickens. APMV-3 strain Netherlands showed highest growth rate and GFP expression level among the three APMV vectors in vitro. APMV-3 strain Wisconsin and APMV-1 strain LaSota vectors were mainly confined to the trachea after vaccination of day-old SPF chickens without any observable pathogenicity, whereas APMV-3 strain Netherlands showed wide tissue distribution in different body organs (brain, lungs, trachea, and spleen) with mild observable pathogenicity. In terms of immunogenicity, both APMV-3 strain-vaccinated groups showed HI titers two to three fold higher than that induced by APMV-1 strain LaSota vaccinated group. This study offers a novel paramyxovirus vector (APMV-3 strain Wisconsin) which can be used safely for vaccination of young chickens as an alternative for APMV-1 strain LaSota vector.

Genomic and biological characteristics of Avian Orthoavulavirus-1 strains isolated from multiple wild birds and backyard chickens in Pakistan.

Circulation of the dominant sub-genotype VII.2 of Avian Orthoavulavirus-1 (AOAV-1) is affecting multiple poultry and non-poultry avian species and causing significant economic losses to the poultry industry worldwide. In countries where ND is endemic, continuous monitoring and characterization of field strains are necessary. In this study, genetic characteristics of eleven AOAV-1 strains were analyzed isolated from wild birds including parakeets (n = 3), lovebird parrot (n = 1), pheasant (n = 1), peacock (n = 1), and backyard chickens (n = 5) during 2015-2016. Genetic characterization (genome size [15,192 nucleotides], the presence of typical cleavage site [112-RRQKRF-117]) and biological assessment (HA log 27 to 29 and intracerebral pathogenicity index [ICPI] value ranging from 1.50 to 1.86) showed virulent AOAV-1. Phylogenetic analysis showed that the studied isolates belonged to sub-genotype VII.2 and genetically very closely related (> 98.9%) to viruses repeatedly isolated (2011-2018) from commercial poultry. These findings provide evidence for the existence of epidemiological links between poultry and wild bird species in the region where the disease is prevalent. The deduced amino acid analysis revealed several substitutions in critical domains of fusion and hemagglutinin-neuraminidase genes. The pathogenesis and transmission potential of wild bird-origin AOAV-1 strain (AW-Pht/2015) was evaluated in 21-day-old chickens that showed the strain was highly virulent causing clinical signs and killed all chickens. High viral loads were detected in different organs of the infected chickens correlating with the severity of lesions developed. The continuous monitoring of AOAV-1 isolates in different species of birds will improve our knowledge of the evolution of these viruses, thereby preventing possible panzootic.

Comparative clinico-pathological assessment of velogenic (sub-genotype VIIi) and mesogenic (sub-genotype VIm) Avian avulavirus 1 in chickens and pigeons.

Newcastle disease (ND), caused by virulent Avian avulavirus 1 (AAvV 1), affects a wide range of avian species worldwide. Recently, several AAvVs of diverse genotypes have emerged with varying genomic and residue substitutions, and subsequent clinical impact on susceptible avian species. We assessed the clinico-pathological influence of two different AAvV 1 pathotypes [wild bird originated-velogenic strain (sub-genotype VIIi, MF437287) and feral pigeon originated-mesogenic strain (sub-genotype VIm, KU885949)] in commercial broiler chickens and pigeons. The velogenic strain caused 100% mortality in both avian species while the mesogenic strain caused 0% and 30% mortality in chickens and pigeons, respectively. Both strains showed tissue tropism for multiple tissues including visceral organs; however, minor variances were observed according to host and pathotype. The observed gross and microscopic lesions were typical of AAvV 1 infection. Utilizing oropharyngeal and cloacal swabs, a comparable pattern of viral shedding was observed for both strains from each of the infected individuals of both avian species. The study concludes a varying susceptibility of chickens and pigeons to different wild bird-originated AAvV 1 pathotypes and, therefore, suggests continuous monitoring and surveillance of currently prevailing strains for effective control of the disease worldwide, particularly in disease-endemic countries.

A comparative genomic and evolutionary analysis of circulating strains of Avian avulavirus 1 in Pakistan.

Newcastle disease, caused by Avian avulavirus 1 (AAvV 1), is endemic to many developing countries around the globe including Pakistan. Frequent epidemics are not uncommon even in vaccinated populations and are largely attributed to the genetic divergence of prevailing isolates and their transmission in the environment. With the strengthening of laboratory capabilities in Pakistan, a number of genetically diverse AAvV 1 strains have recently been isolated and individually characterized in comparison with isolates reported elsewhere in the world. However, there lacks sufficient comparative genomic and phylogenomic analyses of field circulating strains that can elucidate the evolutionary dynamics over a period of time. Herein, we enriched the whole genome sequences of AAvV reported so far (n = 35) from Pakistan and performed comparative genomic, phylogenetic and evolutionary analyses. Based on these analyses, we found only isolates belonging to genotypes VI, VII and XIII of AAvV 1 in a wide range of avian and human hosts. Comparative phylogeny revealed the concurrent circulation of avulaviruses representing different sub-genotypes such as VIg, VIm, VIIa, VIIb, VIIe, VIIf, VIIi, XIIIb and XIIId. We found that the isolates of genotype VII were more closely associated with viruses of genotype XIII than genotype VI. An inter-genotype comparative residue analysis revealed a few substitutions in structurally and functionally important motifs. Putative recombination events were reported for only one of the captive-wild bird (pheasant)-origin isolates. The viruses of genotype VII had a high genetic diversity as compared to isolates from genotypes VI and XIII and, therefore, have more potential to evolve over a period of time. Taken together, the current study provides an insight into the genetic diversity and evolutionary dynamics of AAvV 1 strains circulating in Pakistan. Such findings are expected to facilitate better intervention strategies for the prevention and control of ND in disease-endemic countries across the globe particularly Pakistan.

Genetic characterization of one duck-origin paramyxovirus type 4 strain in China.

Avian paramyxovirus type 4 (APMV-4) has been frequently reported from wildfowl and waterfowl in recent year. However, few studies have reported on the molecular characteristics and regional transmission of APMV-4, knowledge of which is important for understanding the genetic diversity and epidemiology of avian paramyxovirus. Herein, we report the isolation of one APMV-4 strain, designated as QY17, from the duck in eastern China. The determined complete genome of the isolate with six gene segments 3'-N-P-M-F-HN-L-5' was 15,054 nt in length. Genetic analysis of the whole-fusion gene of this isolate showed that QY17 was derived from a Eurasian lineage. Further phylogenetic analysis showed that the duck-origin strain QY17 had a highly genetic relationship with representative APMV-4 strains from wildfowl in neighbouring regions. These genetic results suggested that APMV-4 viral exchange may occur in wildfowl and poultry via wild bird migration.

[Molecular-genetic characterization of Avian avulavirus 20 strains isolated from wild birds.]

INTRODUCTION: Previously unknown paramyxovirus strains were isolated from wild birds in 2013-2014 in Kazakhstan and subsequently identified as representatives of the novel Avian avulavirus 20 species. The aims and tasks were molecular genetic characterization of novel avulaviruses and investigation of their phylogenetic relationships. MATERIAL AND METHODS: Embryonated chicken eggs were inoculated with cloacal and tracheal swabs from wild birds with subsequent virus isolation. The complete nucleotide sequences of viral genomes were obtained by massive parallel sequencing with subsequent bioinformatics processing. RESULTS: By initial infection of chicken embryos with samples from 179 wild birds belonging to the Anatidae, Laridae, Scolopacidae and Charadriidae families, 19 hemagglutinating agents were isolated, and five of them were identified as representatives of new viral species. The study of their sequenced genomes revealed their similarity in size, but there was a significant genetic variability within the species. 2,640 nucleotide substitutions were identified and 273 of them were nonsynonymous, influencing the protein structure of viruses. It was shown that isolates Avian avulavirus 20/black-headed gull/Balkhash/5844/2013 and Avian avulavirus 20 /great black-headed gull/Atyrau/5541/2013 were 86% and 95% respectively identical to the previously described reference strain, indicating a significant evolutionary divergence within species. DISCUSSION: The authors suggest the existence of two independent lineages - the Caspian, represented by the reference strain Aktau/5976 and Atyrau/5541, as well as the second, geographically significantly distant Balkhash lineage. CONCLUSION: The study confirms the role of the birds of the Laridae family as the main reservoir of Avian avulavirus 20 in the avifauna that plays a key role in maintaining viruses of the genus Avulavirus in the biosphere and is a potential natural source for the emergence of new viral variants. Continuous surveillance of them in the wild is one of the most important tasks in ensuring the safety of the poultry industry.

Development of an avian avulavirus 1 (AAvV-1) L-gene real-time RT-PCR assay using minor groove binding probes for application as a routine diagnostic tool.

Newcastle disease is a devastating disease of poultry caused by Newcastle disease virus (NDV), a virulent form of avian avulavirus 1 (AAvV-1). A rapid, sensitive and specific means for the detection of NDV is fundamental for the control of this notifiable transboundary virus. Although several real-time RT-PCR assays exist for the detection of AAvV-1, diagnostic sensitivity and specificities can be sub-optimal. In this study, we describe a modification to an existing AAvV-1 l-gene RT-PCR screening assay, where the original probe set was replaced with minor groove binding (MGB) probes, to create the MGB l-gene assay. The diagnostic sensitivity and specificity of this assay was evaluated against a broad panel of both Class I and Class II AAvV-1 viruses of diverse and representative lineages/genotypes in both clinical samples and amplified viruses, and compared with a number of previously published real-time RT-PCR screening assays for AAvV-1. The MGB l-gene assay outperformed all other assays in this assessment, with enhanced sensitivity and specificity, detecting isolates from a broad range of virus lineages/genotypes (including contemporaneously-circulating strains). The assay has also proved its value for screening original clinical samples for the presence of AAvV-1, thus providing an improved screening assay for routine detection of this notifiable disease agent.

Biological and phylogenetic characterization of a novel hemagglutination-negative avian avulavirus 6 isolated from wild waterfowl in China.

Up to now only nine whole genome sequences of avian avulavirus 6 (AAvV-6) had been documented in the world since the first discovery of AAvV-6 (AAvV-6/duck/HongKong/18/199/77) at a domestic duck in 1977 from Hong Kong of China. Very limited information is known about the regularities of transmission, genetic and biological characteristics of AAvV-6 because of the lower isolation rate and mild losses for poultry industry. To better further explore the relationships among above factors, an AAvV-6 epidemiological surveillance of domestic poultry and wild birds in six provinces of China suspected of sites of inter-species transmission and being intercontinental flyways during the year 2013-2017 was conducted. Therefore, 9,872 faecal samples from wild birds and 1,642 cloacal and tracheal swab samples from clinically healthy poultry of live bird market (LBM) were collected respectively. However, only one novel hemagglutination-negative AAvV-6 isolate (AAvV-6/mallard/Hubei/2015) was isolated from a fresh faecal sample obtained from mallard at a wetland of Hubei province. Sequencing and phylogenetic analyses of this AAvV-6 isolate (AAvV-6/mallard/Hubei/2015) indicated that this isolate grouping to genotype I were epidemiological intercontinentally linked with viruses from the wild birds in Europe and America. Meanwhile, at least two genotypes (I and II) are existed within serotype AAvV-6. In additional, this novel hemagglutination-negative AAvV-6 isolate in chicken embryos restored its hemagglutination when pre-treated with trypsin. These findings, together with data from other AAvV-6, suggest potential epidemiological intercontinental spreads among AAvV-6 transmission by wild migratory birds, and reveal potential threats to wild birds and domestic poultry worldwide.

Molecular characterization of two novel sub-sublineages of pigeon paramyxovirus type 1 in China.

Pigeon paramyxovirus type 1 (PPMV-1) infection is enzootic in pigeon flocks and poses a potential risk to the poultry industry in China. To gain insight into the biological characteristics and transmission routes of circulating PPMV-1 in pigeons, 13 PPMV-1 isolates from domestic pigeons isolated during 2011-2015 in Guangxi province, China, were characterized using a pathogenicity assessment and phylogenetic analysis. All PPMV-1 isolates were mesogenic or lentogenic strains and had a mean death time (MDT) in 9-day-old SPF chicken embryos and a intracerebral pathogenicity index (ICPI) values of 54-154 h and 0.00-0.90, respectively. Analysis of the F and HN gene sequences of the PPMV-1 isolates and the Newcastle Disease (ND) vaccine strain La Sota, revealed that the nucleotide sequence similarity of the F and HN genes were all < 85% between the PPMV-1 isolates and La Sota, significantly lower than those > 98% among the PPMV-1 isolates. The amino acids sequence of the F protein at the cleavage site of the 13 PPMV-1 isolates was 112RRQKR↓F117, characteristic of virulent Newcastle disease virus (NDV). All 13 isolates were classified as sublineage 4b by phylogenetic analysis and evolutionary distances, based on the F gene sequences. It was also found that the 13 isolates were divided into two novel sub-groups of sublineage 4b, sub-sublineages 4biig and 4biih. Since these two novel sub-sublineages had two different geographic sources, we speculated that they represent two different transmission routes of PPMV-1 in China. Phylogenetic analysis of these isolates will help to elucidate the sources of the transmission and evolution of PPMV-1 and may help to control PPMV-1 infection in the pigeon industry in China.

Genetic Diversity of Avian Paramyxovirus Type 6 Isolated from Wild Ducks in the Republic of Korea.

Eleven avian paramyxovirus type 6 (APMV-6) isolates from Eurasian Wigeon ( n=5; Anas penelope), Mallards ( n=2; Anas platyrhynchos), and unknown species of wild ducks ( n=4) from Korea were analyzed based on the nucleotide (nt) and deduced amino acid sequences of the fusion (F) gene. Fecal samples were collected in 2010-14. Genotypes were assigned based on phylogenetic analyses. Our results revealed that APMV-6 could be classified into at least two distinct genotypes, G1 and G2. The open reading frame (ORF) of the G1 genotype was 1,668 nt in length, and the putative F0 cleavage site sequence was 113PAPEPRL119. The G2 genotype viruses included five isolates from Eurasian wigeons and four isolates from unknown waterfowl species, together with two reference APMV-6 strains from the Red-necked Stint ( Calidris ruficollis) from Japan and an unknown duck from Italy. There was an N-truncated ORF (1,638 nt), due to an N-terminal truncation of 30 nt in the signal peptide region of the F gene, and the putative F0 cleavage site sequence was 103SIREPRL109. The genetic diversity and ecology of APMV-6 are discussed.

Molecular characterization and genetic diversity of avian paramyxovirus type 4 isolated in South Korea from 2013 to 2017.

In recent years, avian paramyxovirus type 4 (APMV-4) frequently isolated from wild and domestic bird populations particularly waterfowls worldwide. However, molecular characteristics and genetic diversity of APMV-4 are uncertain, owing to the limited availability of sequence information. A total of 11 APMV-4 strains from 9850 fecal, swab, and environmental samples were isolated during the surveillance program in wintering seasons of 2013-2017 in South Korea. We performed genetic characterization and phylogenetic analysis to investigate the genetic diversity and relatedness between isolates from the region. We report high APMV-4 genetic diversity (multiple genotypes and sub-genotypes) among wild bird and poultry populations in Korea and that the potential virus exchange occurs between neighboring countries via wild bird migration. Furthermore, our study results suggest the possibility of transcontinental transmission of APMV-4 between Asia and Europe.

Molecular Characterization of Avian Paramyxovirus Types 4 and 8 Isolated from Wild Migratory Waterfowl in Mongolia.

Avian paramyxoviruses (APMVs) constitute some of the most globally prevalent avian viruses and are frequently isolated from wild migratory bird species. Using 1,907 fresh fecal samples collected during the 2012 avian influenza surveillance program, we identified two serotypes of APMV: APMV-4 ( n=10) and APMV-8 ( n=1). Sequences for these isolates phylogenetically clustered with Asian APMV-4 and APMV-8 recently isolated from wild birds in Korea, Japan, China, and Kazakhstan. Analysis by DNA barcoding indicated that the Mongolian APMV-4 and APMV-8 strains were isolated from Anseriformes species including Mallards ( Anas platyrhynchos) and Whooper Swans ( Cygnus cygnus). The close genetic relatedness to Asian isolates, and to similar host species, suggested that wild bird species in the Anatidae family might play an important role as a natural reservoir in the spread of APMV-4 and APMV-8. However, we did not find conclusive evidence to support this hypothesis owing to the limited number of strains that could be isolated. Enhanced surveillance of poultry and wild bird populations in Asia is therefore crucial for the understanding of global AMPV transmission, ecology, evolution, and epidemiology.

Next-generation sequencing of five new avian paramyxoviruses 8 isolates from Kazakhstan indicates a low genetic evolution rate over four decades.

Five avian paramyxoviruses of serotype 8 (APMV-8) were isolated during a study monitoring wild birds in Kazakhstan in 2013 and each was further characterized. The viruses were isolated from three White-fronted geese (Anser albifrons), one Whooper swan (Cygnus cygnus), and one Little stint (Calidris minuta). Before our study, only two complete APMV-8 sequences had been reported worldwide since their discovery in the USA and Japan in the 1970s. We report the complete genome sequences of the newly detected viruses and analyze the genetic evolution of the APMV-8 viruses over four decades.

Complete genome sequence of a novel avian paramyxovirus isolated from wild birds in South Korea.

A novel avian paramyxovirus (APMV), Cheonsu1510, was isolated from wild bird feces in South Korea and serologically and genetically characterized. In hemagglutination inhibition tests, antiserum against Cheonsu1510 showed low reactivity with other APMVs and vice versa. The complete genome of Cheonsu1510 comprised 15,408 nucleotides, contained six open reading frames (3'-N-P-M-F-HN-L-5'), and showed low sequence identity to other APMVs (< 63%) and a unique genomic composition. Phylogenetic analysis revealed that Cheonsu1510 was related to but distinct from APMV-1, -9, and -15. These results suggest that Cheonsu1510 represents a new APMV serotype, APMV-17.

Phylogenomics and Infectious Potential of Avian Avulaviruses Species-Type 1 Isolated from Healthy Green-Winged Teal (Anas carolinensis) from a Wetland Sanctuary of Indus River.

Given the importance of Avian avulaviruses (AAvVs) in commercial poultry, continuous monitoring and surveillance in natural reservoirs (waterfowls) is imperative. Here, we report full genomic and biologic characterization of two virulent AAvVs isolated from apparently asymptomatic green-winged teal (Anas carolinensis). Genetic characterization (genome length, coding potential, and presence of typical cleave motif [112RRQKR| F117]) and biologic assessment (HA, log 29; mean death time, 49.2-50 hr; 10-6.51 50% egg infective dose [EID50]/0.1 mL; and 1.5 intracerebral pathogenicity index [ICPI] value) revealed virulence of both isolates. Phylogenetic analysis of the complete genome and hypervariable region of the fusion (F) gene revealed clustering of both isolates within class II strains in close association with domestic poultry-origin AAvVs representing genotype VII and subgenotype VIIi. The inferred residue analysis of F and hemagglutinin-neuraminidase genes showed a number of substitutions in critical domains compared with reference strains of each genotype (I-XVIII). The isolates showed a high nucleotide resemblance (99%) with strain isolated previously from backyard poultry; however, they also showed a variable similarity (16.1% to 19.3%) with the most commonly used vaccine strains, Mukteswar (EF201805) and LaSota (AF077761). In accordance with pathogenicity assessment and horizontal transmission, the clinical and histopathologic observations in experimental chickens indicated the velogenic viscerotropic nature of AAvV 1 isolates. Taken together, this study confirms the evolutionary nature of AAvVs and their potential role in disease occurrence, necessitating continuous surveillance of migratory/aquatic fowls to better elucidate infection epidemiology and potential impacts on commercial poultry.

Análisis filogenético y potencial infeccioso de avulavirus aviares de tipo 1 aislados de cercetas americanas (Anas carolinensis) de un santuario en los humedales del río Indo Dada la importancia de los avulavirus aviares en la avicultura, es imperativo tanto el monitoreo como la vigilancia continuos en los reservorios naturales (aves acuáticas). En este artículo se describe la caracterización genética completa y las características biológicas de dos avulavirus aviares virulentos aislados de cercetas americanas (Anas carolinensis) aparentemente asintomáticas. La caracterización genética (longitud del genoma, potencial de codificación y presencia del motivo típico de disociación [112RRQKR| F117]) y la evaluación biológica (ensayo de hemaglutinación [HA], log 29; tiempo promedio de mortalidad, 49.2­50 horas; 10­6.51 dosis infectantes50% [EID50] /0.1mL y el índice de patogenicidad intracerebral [ICPI] de 1.5, revelaron la virulencia de ambos aislamientos. El análisis filogenético del genoma completo y la región hipervariable del gene de fusión (F) revelaron la agrupación de ambos aislamientos con cepas de la clase II en estrecha asociación con los avulavirus de origen avícola que representan el genotipo VII y el subgenotipo VIIi. El análisis de residuos deducidos de los genes F y de la hemaglutininaneuraminidasa mostró varias sustituciones en los dominios críticos en comparación con las cepas de referencia de cada genotipo (IXVIII). Los aislamientos mostraron una gran semejanza en la secuencia de nucléotidos (99%) con una cepa aislada previamente de aves de traspatio; sin embargo, también mostraron similitudes variables (de 16.1% a 19.3%) con las cepas de vacunas más utilizadas, Mukteswar (EF201805) y LaSota (AF077761). De acuerdo con la evaluación de patogenicidad y la transmisión horizontal, las observaciones clínicas e histopatológicas en los pollos experimentales indicaron la naturaleza velogénica viscerotrópica de estos aislamientos de avulavirus del tipo 1. En conjunto, este estudio confirma la naturaleza evolutiva de los avulavirus aviares y su posible papel en la aparición de enfermedades, lo que requiere una vigilancia continua de las aves migratorias acuáticas para dilucidar mejor la epidemiología de la infección y el posible impacto en las aves comerciales.

Dispersal and Transmission of Avian Paramyxovirus Serotype 4 among Wild Birds and Domestic Poultry.

Avian paramyxovirus serotype 4 (APMV-4) is found sporadically in wild birds worldwide, and it is an economically important poultry pathogen. Despite the existence of several published strains, very little is known about the distribution, host species, and transmission of APMV-4 strains. To better understand the relationships among these factors, we conducted an APMV-4 surveillance of wild birds and domestic poultry in six provinces of China suspected of being intercontinental flyways and sites of interspecies transmission. APMV-4 surveillance was conducted in 9,160 wild birds representing seven species, and 1,461 domestic poultry in live bird markets (LMBs) from December 2013 to June 2016. The rate of APMV-4 isolation was 0.10% (11/10,621), and viruses were isolated from swan geese, bean geese, cormorants, mallards, and chickens. Sequencing and phylogenetic analyses of the 11 isolated viruses indicated that all the isolates belonging to genotype I were epidemiologically connected with wild bird-origin viruses from the Ukraine and Italy. Moreover, chicken-origin APMV-4 strains isolated from the LBMs were highly similar to wild bird-origin viruses from nearby lakes with free-living wild birds. In additional, a hemagglutination-negative APMV-4 virus was identified. These findings, together with recent APMV-4 studies, suggest potential virus interspecies transmission between wild birds and domestic poultry, and reveal possible epidemiological intercontinental connections between APMV-4 transmission by wild birds.