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.

Genetic and Antigenic Characterization of Avian Avulavirus Type 6 (AAvV-6) Circulating in Canadian Wild Birds (2005-2017).

We describe for the first time the genetic and antigenic characterization of 18 avian avulavirus type-6 viruses (AAvV-6) that were isolated from wild waterfowl in the Americas over the span of 12 years. Only one of the AAvV-6 viruses isolated failed to hemagglutinate chicken red blood cells. We were able to obtain full genome sequences of 16 and 2 fusion gene sequences from the remaining 2 isolates. This is more than double the number of full genome sequences available at the NCBI database. These AAvV-6 viruses phylogenetically grouped into the 2 existing AAvV-6 genotype subgroups indicating the existence of an intercontinental epidemiological link with other AAvV-6 viruses isolated from migratory waterfowl from different Eurasian countries. Antigenic maps made using HI assay data for these isolates showed that the two genetic groups were also antigenically distinct. An isolate representing each genotype was inoculated in specific pathogen free (SPF) chickens, however, no clinical symptoms were observed. A duplex fusion gene based real-time assay for the detection and genotyping of AAvV-6 to genotype 1 and 2 was developed. Using the developed assay, the viral shedding pattern in the infected chickens was examined. The chickens infected with both genotypes were able to shed the virus orally for about a week, however, no significant cloacal shedding was detected in chickens of both groups. Chickens in both groups developed detectable levels of anti-hemagglutinin antibodies 7 days after infection.

In Vitro and In Vivo Characterization of a Pigeon Paramyxovirus Type 1 Isolated from Domestic Pigeons in Victoria, Australia 2011.

Significant mortalities of racing pigeons occurred in Australia in late 2011 associated with a pigeon paramyxovirus serotype 1 (PPMV-1) infection. The causative agent, designated APMV-1/pigeon/Australia/3/2011 (P/Aus/3/11), was isolated from diagnostic specimens in specific pathogen free (SPF) embryonated eggs and was identified by a Newcastle Disease virus (NDV)-specific RT-PCR and haemagglutination inhibition (HI) test using reference polyclonal antiserum specific for NDV. The P/Aus/3/11 strain was further classified as PPMV-1 using the HI test and monoclonal antibody 617/161 by HI and phylogenetic analysis of the fusion gene sequence. The isolate P/Aus/3/11 had a slow haemagglutin-elution rate and was inactivated within 45 min at 56 °C. Cross HI tests generated an R value of 0.25, indicating a significant antigenic difference between P/Aus/3/11 and NDV V4 isolates. The mean death time (MDT) of SPF eggs infected with the P/Aus/3/11 isolate was 89.2 hr, characteristic of a mesogenic pathotype, consistent with other PPMV-1 strains. The plaque size of the P/Aus/3/11 isolate on chicken embryo fibroblast (CEF) cells was smaller than those of mesogenic and velogenic NDV reference strains, indicating a lower virulence phenotype in vitro and challenge of six-week-old SPF chickens did not induce clinical signs. However, sequence analysis of the fusion protein cleavage site demonstrated an 112RRQKRF117 motif, which is typical of a velogenic NDV pathotype. Phylogenetic analysis indicated that the P/Aus/3/11 isolate belongs to a distinct subgenotype within class II genotype VI of avian paramyxovirus type 1. This is the first time this genotype has been detected in Australia causing disease in domestic pigeons and is the first time since 2002 that an NDV with potential for virulence has been detected in Australia.

Characterization of ten paramyxovirus type 1 viruses isolated from pigeons in China during 1996-2019.

Pigeon paramyxovirus type 1 (PPMV-1) is an antigenic variant of avian paramyxovirus type 1, which mainly infects pigeons. Here, we characterized ten PPMV-1 viruses isolated from pigeons in China during 1996-2019. Phylogenetic analysis of available complete genomes, F and HN genes of PPMV-1 from China showed that multiple PPMV-1 genotypes (I, II, VI, and VII) exist in pigeons in China. Ten PPMV-1 viruses isolated in this study belonged to genotypes VI.1.2.2.2, VI.2.1.1.2.1, VI.2.1.1.2.2 and VII respectively. Genotype VI is predominant in pigeons. VI.2.1.1.2.2 contains most recently isolated PPMV-1 viruses, suggesting that VI.2.1.1.2.2 is a prevalent genotype in pigeons in China. In vitro and in vivo studies showed that four representative viruses from genotypes VI.2.1.1.2.1 (TA14), VI.2.1.1.2.2 (SD19), VI.1.2.2.2 (SD16), and VII (JN08) could replicate efficiently in chicken embryo fibroblasts, while the replication titer of JN08 (VII) virus was significantly lower than that of VI gene viruses in pigeon embryo fibroblasts. The TA14 (VI.2.1.1.2.1) and SD19 (VI.2.1.1.2.2) viruses caused 20 % and 30 % mortality in pigeons, respectively. No birds infected with SD16 (VI.1.2.2.2) died during the study period. JN08 (VII) virus did not cause obvious clinical signs in infected pigeons. All data indicated that VI.2.1.1.2.2 is the prevalent genotype circulating in China and poses a major threat to pigeons, suggesting that a matched vaccine is necessary to control the disease.

Complete Genome Sequencing, Molecular Epidemiological, and Pathogenicity Analysis of Pigeon Paramyxoviruses Type 1 Isolated in Guangxi, China during 2012-2018.

Newcastle disease is an important poultry disease that also affects Columbiform birds. The viruses adapted to pigeons and doves are referred to as pigeon paramyxoviruses 1 (PPMV-1). PPMV-1 are frequently isolated from pigeons worldwide and have the potential to cause disease in chickens. The complete genomes of 18 PPMV-1 isolated in China during 2012-2018 were sequenced by next-generation sequencing (NGS). Comprehensive phylogenetic analyses showed that five of the viruses belong to sub-genotype VI1.2.1.1.2.1 and 13 isolates belong to sub-genotype VI.2.1.1.2.2. The results demonstrate that these sub-genotypes have been predominant in China during the last decade. The viruses of these sub-genotypes have been independently maintained and continuously evolved for over 20 years, and differ significantly from those causing outbreaks worldwide during the 1980s to 2010s. The viral reservoir remains unknown and possibilities of the viruses being maintained in both pigeon farms and wild bird populations are viable. In vivo characterization of the isolates' pathogenicity estimated mean death times between 62 and 114 hours and intracerebral pathogenicity indices between 0.00 and 0.63. Cross-reactivity testing showed minor antigenic differences between the studied viruses and the genotype II LaSota vaccine. These data will facilitate PPMV-1 epidemiology studies, vaccine development, and control of Newcastle disease in pigeons and poultry.

Experimental pigeon paramyxovirus-1 infection in chicken: evaluation of infectivity, clinical and pathological manifestations and diagnostic methods.

Several pigeon paramyxovirus-1 (PPMV-1) outbreaks in feral pigeons were described recently in Switzerland. The potential of PPMV-1 to induce the notifiable Newcastle disease in chickens is discussed controversially. Therefore, in order to study epidemiologically relevant parameters such as the kinetics of PPMV-1 replication and shedding as well as seroconversion after infection, chickens were infected experimentally with a Swiss PPMV-1 isolate. This generated also defined sample material for the comparison of diagnostic tests. The infectivity of the Swiss PPMV-1 isolate for chickens was demonstrated successfully by virus shedding after experimental inoculation. Our data suggest that long-lasting shedding for up to 60 days can occur in chickens infected with PPMV-1. The isolate used here was of low pathogenicity for chickens. Different quantitative reverse transcription PCR assays were evaluated with a set of Swiss PPMV-1 isolates, and various samples from experimentally infected chickens were analysed with respect to their suitability for viral RNA detection. At 14 days post-infection, virus genome was detected mainly in spleen, caecal tonsils, heart, cloacal swabs, liver, proventriculus, duodenum and kidney tissue samples. Overall, the level of virus replication was low. Not all assays used routinely in diagnostics were capable of detecting viral genome from the isolates tested. Possible explanations are the genetic divergence of PPMV-1 and the low level of viral RNA in the samples. In contrast, two methods that are not used routinely proved more suitable for virus-genome detection. Importantly, the collection of material from various different organs is recommended, in addition to the kidney and brain analysed routinely. In conclusion, this study shows that there is a need to reconsider the type of samples and the protocols used for the detection of PPMV-1 RNA in chickens.

Novel avian metaavulavirus isolated from birds of the family Columbidae in Taiwan.

Avian paramyxoviruses (APMVs) consist of twenty known species and have been isolated from domestic and wild birds around the world. In 2009, the isolate APMV/dove/Taiwan/AHRI33/2009 was isolated from swabs of red turtle doves (Streptopelia tranquebarica) during active surveillance of avian influenza in resident birds in Taiwan, and it was initially identified as paramyxovirus based on electron microscopy. Hemagglutination inhibition assays indicated antigenic heterogeneity of AHRI33 with the known APMV-1, -2, -3, -4, -6, -8, and -9 species, only showing weak but measurable cross-reactivity with APMV-7. Pathogenicity ICPI test revealed that the virus was avirulent for chickens. The AHRI33 virus genome revealed a typical APMV structure consisting of six genes 3'-NP-P-M-F-HN-L-5', and the length of the genome was 16,914 nucleotides, the third longest among the members of the subfamily Avulavirinae. Estimates of the nucleotide sequence identities of the genome between each prototype of APMVs had shown AHRI33 to be more closely related to APMV-7 than to the others, with a sequence identity of 62.8%. Based on topology of the phylogenetic tree of RdRp genes and the branch length between the nearest node and the tip of the branch, AHRI33 met the criteria for designation as distinct species. Together, the data suggest that the isolate APMV/dove/Taiwan/AHRI33/2009 should be considered as the prototype strain of the new species Avian metaavulavirus 21 in the genus Metaavulavirus in the subfamily Avulavirinae.

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.

The Emergence of Avian Orthoavulavirus 13 in Wild Migratory Waterfowl in China Revealed the Existence of Diversified Trailer Region Sequences and HN Gene Lengths within this Serotype.

Avian orthoavulavirus 13 (AOAV-13), also named avian paramyxovirus 13 (APMV-13), has been found sporadically in wild birds around the world ever since the discovery of AOAV-13 (AOAV-13/wild goose/Shimane/67/2000) in a wild goose from Japan in 2000. However, there are no reports of AOAV-13 in China. In the present study, a novel AOAV-13 virus (AOAV-13/wild goose/China/Hubei/V93-1/2015), isolated from a wild migratory waterfowl in a wetland of Hubei province of China, during active surveillance from 2013 to 2018, was biologically and genetically characterized. Phylogenetic analyses demonstrated a very close genetic relationship among all AOAV-13 strains, as revealed by very few genetic variations. Moreover, pathogenicity tests indicated that the V93-1 strain is a low virulent virus for chickens. However, the genome of the V93-1 virus was found to be 16,158 nucleotides (nt) in length, which is 12 nt or 162 nt longer than the other AOAV-13 strains that have been reported to date. The length difference of 12 nt in strain V93-1 is due to the existence of three repeats of the conserved sequence, "AAAAAT", in the 5'-end trailer of the genome. Moreover, the HN gene of the V93-1 virus is 2070 nt in size, encoding 610 aa, which is the same size as the AOAV-13 strain from Japan, whereas that of two strains from Ukraine and Kazakhstan are 2080 nt in length, encoding 579 aa. We describe a novel AOAV-13 in migratory waterfowl in China, which suggests that diversified trailer region sequences and HN gene lengths exist within serotype AOAV-13, and highlight the need for its constant surveillance in poultry from live animal markets, and especially migratory birds.

Novel penguin Avian avulaviruses 17, 18 and 19 are widely distributed in the Antarctic Peninsula.

Three novel Avian avulavirus species were discovered and isolated during 2017 from Gentoo penguins (Pygoscelis papua) at Kopaitic island in the Northwestern region of the Antarctic Peninsula. The viruses were officially named as Avian avulavirus 17 (AAV17), Avian avulavirus 18 (AAV18) and Avian avulavirus 19 (AAV19), collectively referred to as penguin avulaviruses (PAVs). To determine whether these viruses are capable of infecting the three species of Pygoscelis spp. penguins (Gentoo, Adelie and Chinstrap) and assess its geographical distribution, serum samples were collected from seven locations across the Antarctic Peninsula and Southern Shetland Islands. The samples were tested by Hemagglutination inhibition assay using reference viruses for AAV17, AAV18 and AAV19. A total of 498 sera were tested, and 40 were positive for antibodies against AAV17, 20 for AAV18 and 45 for AAV19. Positive sera were obtained for the penguin's species for each virus; however, antibodies against AAV18 were not identified in Adelie penguins. Positive penguins were identified in all regions studied. Positive locations include Ardley Island and Cape Shirreff at Livingston Island (Southern Shetland Region); Anvers Island, Doumer Island and Paradise Bay in the Central Western region; and Avian Island at Southwestern region of the Antarctic Peninsula. The lowest occurrence was observed at the Southwestern region at Lagotellerie Island, where all samples were negative. On the other hand, Cape Shirreff and Paradise Bay showed the highest antibody titres. Field samples did not evidence cross-reactivity between viruses, and detection was significantly higher for AAV19 and lower for AAV18. This is the first serologic study on the prevalence of the novel Avian avulaviruses including different locations in the white continent. The results indicate that these novel viruses can infect the three Pygoscelis spp. penguins, which extend across large distances of the Antarctic Peninsula.

A comparative phylogenomic analysis of avian avulavirus 1 isolated from non-avian hosts: conquering new frontiers of zoonotic potential among species.

A number of avian avulavirus 1 (AAvV 1) isolates have been reported from avian and non-avian hosts worldwide with varying clinical consequences. In this regard, robust surveillance coupled with advanced diagnostics, genomic analysis, and disease modelling has provided insight into the molecular epidemiology and evolution of this virus. The genomic and evolutionary characteristics of AAvV 1 isolates originating from avian hosts have been well studied, but those originating from non-avian hosts have not. Here, we report a comparative genomic and evolutionary analysis of so-far reported AAvV 1 isolates originating from hosts other than avian species (humans, mink and swine). Phylogenetic analysis showed that AAvV 1 isolates clustered in five distinct genotypes (I, II, VI, VII and XIII). Further analysis revealed clustering of isolates into clades distant enough to be considered distinct subgenotypes, along with a few substitutions in several significant motifs. Although further investigation is needed, the clustering of AAvV 1 strains isolated from non-avian hosts into novel subgenotypes and the presence of substitutions in important structural and biological motifs suggest that this virus can adapt to novel hosts and therefore could have zoonotic potential.

Co-Circulation and Excretion Dynamics of Diverse Rubula- and Related Viruses in Egyptian Rousette Bats from South Africa.

The Egyptian rousette bat (Rousettus aegyptiacus) has previously been implicated as the natural host of a zoonotic rubulavirus; however, its association with rubulaviruses has been studied to a limited extent. Urine, spleen, and other organs collected from the R. aegyptiacus population within South Africa were tested with a hemi-nested RT-PCR assay targeting a partial polymerase gene region of viruses from the Avula- and Rubulavirus genera. Urine was collected over a 14-month period to study the temporal dynamics of viral excretion. Diverse rubulaviruses, including viruses related to human mumps and parainfluenza virus 2, were detected. Active excretion was identified during two peak periods coinciding with the host reproductive cycle. Analysis of additional organs indicated co-infection of individual bats with a number of different putative rubulaviruses, highlighting the limitations of using a single sample type when determining viral presence and diversity. Our findings suggest that R. aegyptiacus can harbor a range of Rubula- and related viruses, some of which are related to known human pathogens. The observed peaks in viral excretion represents potential periods of a higher risk of virus transmission and zoonotic disease spill-over.

[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.

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.

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.

Novel avian paramyxovirus isolated from gulls in Caspian seashore in Kazakhstan.

Three isolates APMV/gull/Kazakhstan/5976/2014, APMV/gull/Kazakhstan/ 5977/2014 and APMV/gull/Kazakhstan/5979/2014, were obtained from independent samples during annual surveillance for avian influenza and paramyxoviruses in wild birds from the Caspian Sea coast in Western Kazakhstan, and were initially identified as putative paramyxoviruses on the basis of electron microscopy. Hemagglutination Inhibition Assays with antisera to nine known APMV serotypes (APMV1-9) indicated no relation to any of them. Next generation sequencing of whole genome sequences indicated the three isolates were genetically identical, and had a nucleotide structure typical for all APMVs, consisting of six genes 3'-NP-P-M-F-HN-L-5'. Phylogenetic analyses, and assessment of amino acid identities, suggested the most closely related lineages to be APMV-2, 8, 10 and 15, but the novel isolate had less than 64% identity to them and all other known avian paramyxoviruses. This value was above levels considered to generally define other APMV serotypes. Estimates of the evolutionary divergence of the nucleotide sequences of the genomes of APMVs have shown that novel Kazakhstan APMV strain was closest to APMV-2, APMV-8, APMV-10 and APMV-15, with calculated distance values of 2.057, 2.058, 2.026 and 2.286 respectively, which is above values considered to differentiate other serotypes (observed minimum was 1.108 between APMV-1 and recently isolated APMV/UPO216/Korea). Together, the data suggest that isolate APMV/gull/Kazakhstan/5976/2014 and other two should be considered as the first representative of a novel APMV-20 group, and is the first time that avian paramyxoviruses have been found infecting members of the gull family, extending the known taxonomic host range.