RNA sequencing of avian paramyxovirus (Paramyxoviridae, Avulavirinae) isolates from wild mallards in Belgium, 2021: complete genomes and coinfections.

We used untargeted RNA sequencing to characterize three Avulavirinae isolates from pooled samples obtained from wild mallards in Belgium in 2021. The complete genome sequences of two avian Orthoavulavirus-1 (AOAV-1) strains and one avian Paraavulavirus-4 (APMV-4) strain were determined confirming hemagglutination inhibition testing of the virus isolates. In addition, the applied sequencing strategy identified an avian influenza virus (AIV) coinfection in all three virus isolates, confirming weak-positive AIV realtime RT-PCR results from the original sample material. In one AOAV-1 isolate, partial sequences covering all genome segments of an AIV of subtype H11N9 could be de novo assembled from the sequencing data. Besides an AIV coinfection, RNA metagenomic data from the APMV-4 isolate also showed evidence of Alpharetrovirus and Megrivirus coinfection. In total, two AOAV-1 of Class II, genotype I.2 and one APMV-4 complete genome sequences were assembled and compared to publicly available sequences, highlighting the importance of surveillance for poultry pathogens in wild birds. Beyond the insights from full genome characterization of virus isolates, untargeted RNA sequencing strategies provide additional insights in the RNA virome of clinical samples as well as their derived virus isolates that are particularly useful when targeting wild avifauna reservoirs of poultry pathogens.

The genetic and biological characterization of the first avian paramyxovirus serotype 14 isolated from chicken in China.

In October 2020, an avian paramyxovirus serotype 14 (APMV-14)-designated chicken/Fujian/2160/2020 (FJ2160) was isolated from tracheal and cloacal swab sample of chicken collected from live bird market in Fujian province in China during the active surveillance program. The complete genome of FJ2160 comprised 15,444 nucleotides (nt) complying with the paramyxovirus "rule of six" and encoded six non-overlapping structural proteins in the order of 3'-NP-P-M-F-HN-L-'5. The complete genome sequence analysis showed that FJ2160 had the highest identity (90.0%) with the APMV-14 isolated from Japan, while the nucleotide sequence identities of FJ2160 and other APMVs ranged from 42.4 to 51.1%. The F protein cleavage site was TREGR↓L, which resembled a lentogenic strain of APMV-1. Phylogenetic analysis revealed that the FJ2160 closest relative was APMV-14. The intracerebral pathogenicity index (ICPI) tests indicated that the virus was lentogenic. This is the first report of APMV-14 in China. These results provide evidence that APMV-14 could infect chickens and reveal the genetic characteristics and biological properties of the virus, which can help to better understand this new emerging APMV-14.

Does Avian Coronavirus Co-Circulate with Avian Paramyxovirus and Avian Influenza Virus in Wild Ducks in Siberia?

Avian coronaviruses (ACoV) have been shown to be highly prevalent in wild bird populations. More work on avian coronavirus detection and diversity estimation is needed for the breeding territories of migrating birds, where the high diversity and high prevalence of Orthomyxoviridae and Paramyxoviridae have already been shown in wild birds. In order to detect ACoV RNA, we conducted PCR diagnostics of cloacal swab samples from birds, which we monitored during avian influenza A virus surveillance activities. Samples from two distant Asian regions of Russia (Sakhalin region and Novosibirsk region) were tested. Amplified fragments of the RNA-dependent RNA-polymerase (RdRp) of positive samples were partially sequenced to determine the species of Coronaviridae represented. The study revealed a high presence of ACoV among wild birds in Russia. Moreover, there was a high presence of birds co-infected with avian coronavirus, avian influenza virus, and avian paramyxovirus. We found one case of triple co-infection in a Northern Pintail (Anas acuta). Phylogenetic analysis revealed the circulation of a Gammacoronavirus species. A Deltacoronavirus species was not detected, which supports the data regarding the low prevalence of deltacoronaviruses among surveyed bird species.

Discovery of Avian Paramyxoviruses APMV-1 and APMV-6 in Shorebirds and Waterfowl in Southern Ukraine.

Emerging RNA virus infections are a growing concern among domestic poultry industries due to the severe impact they can have on flock health and economic livelihoods. Avian paramyxoviruses (APMV; avulaviruses, AaV) are pathogenic, negative-sense RNA viruses that cause serious infections in the respiratory and central nervous systems. APMV was detected in multiple avian species during the 2017 wild bird migration season in Ukraine and studied using PCR, virus isolation, and sequencing. Of 4090 wild bird samples collected, mostly from southern Ukraine, eleven isolates were grown in ovo and identified for APMV serotype by hemagglutinin inhibition test as: APMV-1, APMV-4, APMV-6, and APMV-7. To build One Health's capacity to characterize APMV virulence and analyze the potential risks of spillover to immunologically naïve populations, we sequenced virus genomes in veterinary research labs in Ukraine using a nanopore (MinION) platform. RNA was extracted and amplified using a multiplex tiling primer approach to specifically capture full-length APMV-1 (n = 5) and APMV-6 (n = 2) genomes at high read depth. All APMV-1 and APMV-6 fusion (F) proteins possessed a monobasic cleavage site, suggesting these APMVs were likely low virulence, annually circulating strains. Utilization of this low-cost method will identify gaps in viral evolution and circulation in this understudied but important critical region for Eurasia.

Intranasal Immunization with a Recombinant Avian Paramyxovirus Serotypes 2 Vector-Based Vaccine Induces Protection against H9N2 Avian Influenza in Chicken.

Commercial inactivated vaccines against H9N2 avian influenza (AI) have been developed in China since 1990s and show excellent immunogenicity with strong HI antibodies. However, currently approved vaccines cannot meet the clinical demand for a live-vectored vaccine. Newcastle disease virus (NDV) vectored vaccines have shown effective protection in chickens against H9N2 virus. However, preexisting NDV antibodies may affect protective efficacy of the vaccine in the field. Here, we explored avian paramyxovirus serotype 2 (APMV-2) as a vector for developing an H9N2 vaccine via intranasal delivery. APMV-2 belongs to the same genus as NDV, distantly related to NDV in the phylogenetic tree, based on the sequences of Fusion (F) and hemagglutinin-neuraminidase (HN) gene, and has low cross-reactivity with anti-NDV antisera. We incorporated hemagglutinin (HA) of H9N2 into the junction of P and M gene in the APMV-2 genome by being flanked with the gene start, gene end, and UTR of each gene of APMV-2-T4 to generate seven recombinant APMV-2 viruses rAPMV-2/HAs, rAPMV-2-NPUTR-HA, rAPMV-2-PUTR-HA, rAPMV-2-FUTR-HA, rAPMV-2-HNUTR-HA, rAPMV-2-LUTR-HA, and rAPMV-2-MUTR-HA, expressing HA. The rAPMV-2/HAs displayed similar pathogenicity compared with the parental APMV-2-T4 virus and expressed HA protein in infected CEF cells. The NP-UTR facilitated the expression and secretion of HA protein in cells infected with rAPMV-2-NPUTR-HA. Animal studies demonstrated that immunization with rAPMV-2-NPUTR-HA elicited effective H9N2-specific antibody (6.14 ± 1.2 log2) responses and conferred complete immune protection to prevent viral shedding in the oropharyngeal and cloacal swabs from chickens challenged with H9N2 virus. This study suggests that our recombinant APMV-2 virus is safe and immunogenic and can be a useful tool in the combat of H9N2 outbreaks in chicken.

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.

Detection of Velogenic Avian Paramyxoviruses in Rock Doves in New York City, New York.

Avian paramyxovirus 1 (APMV-1), also known as Newcastle disease virus (NDV), causes severe and economically important disease in poultry around the globe. Although a limited amount of APMV-1 strains in urban areas have been characterized, the role of the urban wild bird population as an APMV-1 reservoir is unclear. Because urban birds may have an important role for long-term circulation of the virus, fecal and swab samples were collected by community scientists from wild birds in New York City (NYC), New York, United States. These samples were screened for APMV-1 and genotypically characterized by sequencing of the complete genome. A total of 885 samples were collected from NYC parks and from a local wildlife rehabilitation clinic from October 2020 through June 2021, and 255 samples obtained from 197 birds have been processed to date. Eight birds (4.1%) screened positive for the APMV-1 nucleoprotein gene by conventional reverse transcription PCR (RT-PCR), and two live viruses were isolated via egg culture. A multibasic F protein cleavage sequence, 112R R K K R F117, an indicator of highly pathogenic velogenic APMV-1 strains, was present in the two samples fully sequenced by next generation sequencing. Phylogenetic analysis of the F gene coding sequence classified both isolates into genotype VI, a diverse and predominant genotype responsible for APMV-1 outbreaks in pigeon and dove species worldwide. IMPORTANCE Here we describe the first large-scale effort to screen for APMV-1 in New York City's wild bird population as part of the New York City Virus Hunters program, a community science initiative. We characterized two isolates of APMV-1, with phylogenetic analyses suggesting diversity in established and circulating strains of pigeon paramyxoviruses. Our isolates are also domestic reference strains for future APMV-1 vaccine developments. Future surveillance in this region may contribute to our understanding of APMV-1's evolution and genetic diversity, as well as inform poultry husbandry and vaccination practices in New York State.

Sero-detection of antibodies to Avian metaavulavirus 2 in peri-domestic birds, Nigeria.

Avian metaavulavirus 2 (AMAV-2) previously known as the avian paramyxovirus-2 causes mild to severe respiratory disease, reduced hatchability and infertility of eggs, including increase in white-shelled eggs in chickens and Turkey breeders. When exacerbated by secondary pathogens and environmental stresses, infection is more severe leading to significant economic losses. This study was conducted to determine, if any, the presence of antibodies to Avian metaavulavirus 2 (AMAV-2) in peri-domestic birds in Bauchi State, Nigeria. In all, one hundred sera samples from pigeons (n = 10) and doves (n = 90 were collected in Bauchi, Nigeria. Based on hemagglutination-inhibition (HI) test, overall seroprevalence of 27.0% (27/100) was recorded. In pigeon, the seroprevalence was 80.0% while 21.1% was recorded for dove with HI antibody titers ranging from 3log2 to 8log2. There was statistical significance obtained between dove and pigeon sera tested (p < .05). Until now and to the best of our knowledge, there are no reports on AMAV-2 in poultry or wild birds in Nigeria. This study, thus, provides preliminary information on AMAV-2 seroprevalence in Nigerian peri-domestic birds. The need to conduct further studies in other avian species and wild birds in Nigeria is highlighted.

Genetic diversity of avian paramyxoviruses isolated from wild birds and domestic poultry in Taiwan between 2009 and 2020.

Avian paramyxoviruses (APMVs) belonging to the subfamily Avulavirinae within the family Paramyxoviridae. APMVs consist of twenty-two known species and are constantly isolated from a wide variety of avian species around the world. In this study, the APMV isolates obtained from wild birds and domestic poultry during 2009-2020 in Taiwan were genetically characterized by phylogenetic analysis of their complete fusion protein gene or full-length genome. As a result, 57 APMV isolates belonging to seven different species were obtained during this period and subsequently identified as APMV-1 (n=17), APMV-2 (n=1), APMV-4 (n=25), APMV-6 (n=8), APMV-12 (n=2), APMV-21 (n=2) and APMV-22 (n=2). Sanger sequencing was performed to provide 22 full-length genome sequences and 35 complete fusion protein gene sequences for the APMV isolates. Phylogenetic analysis showed that the recovered viruses were closely related to Eurasian strains, except five class I APMV-1 and four APMV-4 isolates were related to North America strains. Our findings provided more evidence for the intercontinental transmission of APMVs between Eurasia and North America by wild birds. In addition, according to the criteria of the classification system based on complete fusion protein gene sequences, three novel genotypes within APMV-2, APMV-12, and APMV-22 were identified. Together, this investigation provided a broader perspective on the genetic diversity, evolution, and distribution of APMVs in multiple avian host species sampled in Taiwan.

Novel avian orthoavulavirus 13 in wild migratory waterfowl: biological and genetic considerations.

Avian orthoavulavirus 13 (AOAV-13), formerly known as Avian paramyxovirus 13 (APMV-13), is found scatteredly in wild birds around the world. Although four complete genome sequences of AOAV-13 had been identified since the first discovery in Japan in 2003, the information available on the genetic variation and biological characteristics of AOAV-13 is still limited. In the present study, we isolated six AOAV-13 strains from fecal samples of wild migratory waterfowls during annual (2014-2018) viral surveillance of wild bird populations from wetland and domestic poultry of live bird markets (LBMs) in China. The phylogenetic analyses based on the HN and F genes showed that they had very close relationship and the molecular clock estimations showed a low evolutionary rate of AOAV-13. However, Bean goose/Hubei/V97-1/2015 is 1953 nt in size (ORF, 1, 776 nt), which is a unique size and longer than other reported AOAV-13 strains. Additionally, four repeats of conserved sequences "AAAAAT" was presented in the 5'-end trailer region of Swan goose/Hubei/VI49-1/2016, which is unprecedented in the AOAV-13. These findings highlight the importance of continuous monitoring the specific species of APMVs.

Avian Paramyxoviruses as Vectors for Vaccine Development.

Avian paramyxoviruses (APMVs) have gained a great attention to be developed as vaccine vectors against human and veterinary pathogens. Avirulent APMVs are highly safe to be used as vaccine vectors for avian and non-avian species. APMV vectored vaccines induce robust cellular and humoral immune responses in a broad range of hosts. APMV vectors can be a good platform by facilitating rapid generation of vaccines against emerging pathogens. In this chapter, we discuss application of reverse genetics of APMVs for vaccine development, design of APMV vectored vaccines, cloning of protective antigen(s) into a vector, recovery of vectored vaccines and characterization of generated vaccine viruses.

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.

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.

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 Protective Efficacies of Novel Avian Paramyxovirus-Vectored Vaccines against Virulent Infectious Bronchitis Virus in Chickens.

Viral vectored vaccines are desirable alternatives for conventional infectious bronchitis virus (IBV) vaccines. We have recently shown that a recombinant Newcastle disease virus (rNDV) strain LaSota expressing the spike (S) protein of IBV strain Mass-41 (rLaSota/IBV-S) was a promising vaccine candidate for IBV. Here we evaluated a novel chimeric rNDV/avian paramyxovirus serotype 2 (rNDV/APMV-2) as a vaccine vector against IBV. The rNDV/APMV-2 vector was chosen because it is much safer than the rNDV strain LaSota vector, particularly for young chicks and chicken embryos. In order to determine the effectiveness of this vector, a recombinant rNDV/APMV-2 expressing the S protein of IBV strain Mass-41 (rNDV/APMV-2/IBV-S) was constructed. The protective efficacy of this vector vaccine was compared to that of the rNDV vector vaccine. In one study, groups of one-day-old specific-pathogenic-free (SPF) chickens were immunized with rLaSota/IBV-S and rNDV/APMV-2/IBV-S and challenged four weeks later with the homologous highly virulent IBV strain Mass-41. In another study, groups of broiler chickens were single (at day one or three weeks of age) or prime-boost (prime at day one and boost at three weeks of age) immunized with rLaSota/IBV-S and/or rNDV-APMV-2/IBV-S. At weeks six of age, chickens were challenged with a highly virulent IBV strain Mass-41. Our challenge study showed that novel rNDV/APMV-2/IBV-S provided similar protection as rLaSota/IBV-S in SPF chickens. However, compared to prime-boost immunization of chickens with chimeric rNDV/APMV-2, rLaSota/IBV-S and/or a live IBV vaccine, single immunization of chickens with rLaSota/IBV-S, or live IBV vaccine provided better protection against IBV. In conclusion, we have developed the novel rNDV/APMV-2 vector expressing S protein of IBV that can be a safer vaccine against IB in chickens. Our results also suggest a single immunization with a LaSota vectored IBV vaccine candidate provides better protection than prime-boost immunization regimens.

Molecular evolution and genetic variations of V and W proteins derived by RNA editing in Avian Paramyxoviruses.

The newly assigned subfamily Avulavirinae in the family Paramyxoviridae includes avian paramyxoviruses (APMVs) isolated from a wide variety of avian species across the globe. Till date, 21 species of APMVs are reported and their complete genome sequences are available in GenBank. The APMV genome comprises of a single stranded, negative sense, non-segmented RNA comprising six transcriptional units (except APMV-6 with seven units) each coding for a structural protein. Additionally, by co-transcriptional RNA editing of phosphoprotein (P) gene, two mRNAs coding for accessory viral proteins, V and W, are generated along with unedited P mRNA. However, in APMV-11, the unedited mRNA codes for V protein while +2 edited mRNA translates to P protein, similar to members of subfamily Rubulavirinae in the same family. Such RNA editing in paramyxoviruses enables maximizing the coding capacity of their smaller genome. The three proteins of P gene: P, V and W, share identical N terminal but varied C terminal sequences that contribute to their unique functions. Here, we analyzed the P gene editing site, V and W sequences of all 21 APMV species known so far (55 viruses) by using bioinformatics and report their genetic variations and molecular evolution. The variations observed in the sequence and hexamer phase positions of the P gene editing sites is likely to influence the levels and relative proportions of P, V and W proteins' expressions which could explain the differences in the pathogenicity of APMVs. The V protein sequences of APMVs had conserved motifs similar to V proteins of other paramyxoviruses including the seven cysteine residues involved in MDA5 interference, STAT1 degradation and interferon antagonism. Conversely, W protein sequences of APMVs were distinct. High sequence homology was observed in both V and W proteins between strains of the same species than between species except in APMV-3 which was the most divergent APMV species. The estimates of synonymous and non-synonymous substitution rates suggested negative selection pressure on the V and W proteins within species indicating their low evolution rate. The molecular clock analysis revealed higher conservation of V protein sequence compared to W protein indicating the important role played by V protein in viral replication, pathogenesis and immune evasion. However, we speculate the genetic diversity of W proteins could impact the degree of pathogenesis, variable interferon antagonistic activity and the wide host range exhibited by APMV species. Phylogenetically, V proteins of APMVs clustered into three groups similar to the recent classification of APMVs into three new genera while no such pattern could be deciphered in the analysis of W proteins except that strains of same species grouped together. This is the first comprehensive study describing in detail the genetic variations and the molecular evolution of P gene edited, accessory viral proteins of Avian paramyxoviruses.

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.