








key: cord-326596-8ux1q9xw
authors: Chen, Yanyu; Ding, Zhuang; Liu, Xinxin; Chen, Jianjun; Li, Junjiao; Fei, Yidong; Liu, Zhe; Stoeger, Tobias; Bi, Yuhai; Yin, Renfu
title: Biological and phylogenetic characterization of a novel hemagglutination‐negative avian avulavirus 6 isolated from wild waterfowl in China
date: 2018-09-08
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.13005
sha: 
doc_id: 326596
cord_uid: 8ux1q9xw

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.

Over the last 40 years, many viruses from the Paramyxoviridae family isolated from not only human or animal but also in birds have been newly identified (Kolakofsky & Roux, 1987; Samal, 2011) .

Paramyxoviruses are enveloped, non-segmented, pleomorphic RNA viruses containing a single stranded, negative-sense genome. Avian paramyxoviruses that have been isolated from birds; however, due to changes in taxonomy is now referred to as avian avulavirus (AAvV) (Amarasinghe et al., 2017 ). There are 13 described AAvV *Equal contributors. serotypes (AAvV-1 to -13) based on neuraminidase inhibition tests and hemagglutination inhibition (HI), and eight another putative serotypes have been recently isolated (AAvV-14 to -21) (Jeong et al., 2017; Lee et al., 2017; Neira et al., 2017; Thampaisarn et al., 2017; Thomazelli et al., 2017; Yamamoto, Ito, & Ito, 2016) . While very limited information is known about the biological and molecular characteristics of AAvV-2 to -21, extensive study has been mainly conducted on AAvV-1 (Newcastle disease virus, NDV) (Cardenas-Garcia et al., 2015; Umali, Ito, Katoh, & Ito, 2014) .

Newcastle disease (ND), caused by the virulent AAvV-1, a wellcharacterized AAvV serotype, is a highly contagious devastating viral disease to the domestic poultry worldwide because of its high mortality and heavy losses for economy (Saif & Barnes, 2008) .

Other serotypes AAvV, such as AAVV-2, -3 and -7, are also known to cause reproductive and respiratory diseases in turkeys and chickens, sometimes resulting in death of the infected birds (Samuel, Subbiah, Shive, Collins, & Samal, 2011; Warke, Stallknecht, Williams, Pritchard, & Mundt, 2008) . Meanwhile, some serotypes AAvV strains display their specific host restriction, such as AAvV-5 cause diarrhoea and high mortality in budgerigars but not in chickens and ducks (Briand, Henry, Massin, & Jestin, 2012) . However, was first identified at a domestic duck in 1977 from Hong Kong (duck/Hong Kong/18/199/77) and then was found to cause drop in egg production and mild respiratory disease in turkeys, but was avirulent in chickens (Chang et al., 2001; Tian et al., 2012; Xiao et al., 2010) . But recent serosurveillance of commercial chickens in the USA showed the likely prevalence of all serotypes AAvV including AAvV-6, excepted with AAvV-5 (Warke, Appleby, & Mundt, 2008) .

The genome size of AAvV range from 14.9 to 17.4 kb that is transcribed into at least six genes, which separately encode for up to nine different proteins (Saif & Barnes, 2008) . However, AAvV-6 has an RNA genome consists of seven genes in the order of 3′-NP (56-1,626)-P(1,634-3,119)-M(3,122-4,526)-F(4,586-6,420 or 4,586-6,416)-SH(6,470-7,061 or 6,464-7,037)-HN(7,072-9,102 or 7,066-9,096)-L(9, 166-16,182 or 9,160-16,176 )-5′ in length with 16,230 or 16,236 nucleotides (Xiao et al., 2010; Yamamoto, Ito, Tomioka, & Ito, 2015) . Six major proteins are encoded, including the nucleocapsid protein (NP, 128-1,525, 1,398 nt), phosphoprotein (P, 1,687-2,979, 1,293 nt), matrix protein (M, 3,235-4,335, 1,101 nt), fusion protein (F, 4, 265, 1, 668 nt or 4, 265, 1, 638 nt), 7, 963 or 7, 957, 1, 842 nt) and large polymerase protein (L, 9,278-16,003 or 9,272-15,997, 6,726 nt) . In addition, the small hydrophobic protein (SH, 6, 970 or 6, 964, 429 nt) that AAvV-6 has, is not found in the other serotypes (Sobolev et al., 2016; ).

The few reports on the incidence of AAvV-6 in commercial and domestic poultry from different parts of the world have shown a notable presence of several of this virus (Chang et al., 2001; . Despite this, knowledge about the regularities of transmission, genetic and biological characteristics of AAvV-6 viruses in commercial poultry and wild birds in the China recent years remains limited. Therefore, in this study, an AAvV-6 surveillance of domestic poultry and wild birds in six provinces of China suspected of sites of inter-species transmission and being intercontinental flyways from December 2013 to June 2017 was conducted.

All experimental protocols (Approval ID: 20130113-1, approval date:

15th Jan 2013) used in this work were reviewed and approved by the Experimental Animal Council of Jilin University, China. 

Presence and identification of AAvV-6 in each individual collected specimen was performed through allantoic cavities inoculation of 9 to 10-day-old specific-pathogen-free (SPF) chicken embryos (Merial, Beijing, China) (Kim, King, Suarez, Wong, & Afonso, 2007; Yin et al., 2017) . The presence of the AAvV-6 in allantoic fluid was identified by RT-PCR and sequencing for paramyxoviruses (Tong, Chern, Li, Pallansch, & Anderson, 2008) .

The chicken fibroblast cell line DF-1 and the chicken bone marrow macrophages cell line HD11 were grown in DMEM containing 10% foetal bovine serum (FBS) (Gibco, Life Technologies) and complete DMEM/F12 containing 10% FBS respectively. Cells were planted into a 24-well cell culture plate at a viable cell density (determined by Trypan blue exclusion, Sigma, Shanghai, China) of 3 × 10 5 cells per well at 37°C under 5% CO 2 for 8 hr. Cells then were washed three times with phosphate buffered saline and supernatant was changed into fresh medium supplemented with 100 μg/ml streptomycin and 100 U/ml penicillin without FBS.

Thereafter, cells were absorbed with virus at 100 μl allantoic fluid containing the Hubei isolate for 1 hr in the presence or absence of TPCK-trypsin (Sigma, Shanghai, China) and fresh medium was added into the well and then incubated with 72 hr post infection (hpi). Subsequent to infection, virus titre in the supernatants was measured using a micro-hemagglutination assay (HA) method (Zhang et al., 2018) .

Viral RNA was isolated from allantoic fluid using the AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit (Axygen, Shanghai, China) according to the manufacturer's instructions. Following extraction, cDNA synthesis was performed by using GoScript ™ Reverse Transcription System (Promega, Shanghai, China) following the manufacturer's instructions using random primer. Then samples were measured by seminested PCR for L gene of paramyxoviruses using 2×EasyTaq PCR kit (TransGen Biotech, Beijing, China) (Tong et al., 2008) . The first amplification in the seminested PCR assay consists of 10 μl 2×EasyTaq PCR supermix, 2 μl cDNA, 10 μM PAR-F1 primer, 10 μM PAR-R primer and H 2 O to achieve a final volume of 20 μl. The cycling reactions consisted of a cycle of 94°C for 2 min followed by 40 cycles of 94°C for 15 s, 48-50°C for 30 s and 72°C for 30 s. For the second amplification in the seminested PCR assay, we used 2 μl aliquot from the first PCR reaction, 10 μl 2×EasyTaq PCR supermix,10 μM PAR-F2 primer, 10 μM PAR-R primer and H 2 O to achieve a final volume of 20 μl.

The cycling conditions consisted of an initial denaturation at 94°C for 2 min followed by 40 cycles of 94°C for 15 s, 48-50°C for 30 s and 72°C for 30 s. After that, the PAR-F2 and PAR-R primers were used for PCR amplicons sequencing (Sangon Biotech, Shanghai, China).

The BLAST search identified the relatedness of the isolated viruses with other reported AAvV-6 strains and therefore this Hubei stain was designated as AAvV-6/mallard/Hubei/2015. After that this AAvV-6 in this study were amplified for the entire genome using 16 primer pairs (Table 1 ). The cycling reactions consisted of a cycle of 95°C for 3 min followed by 40 cycles of 95°C for 1 min, 45-57°C for 45 s and 72°C for 150 s. PCR amplicons sequencing was performed by Major-bio Company (Beijing, China).

The pathogenicity of the AAvV-6 isolate was determined by (a) 

HA and HI assay were carried out according to the OIE guidelines (Newcastle disease 2018). In HI tests, anti-sera against avian influenza virus (AIV) H1, H5 and H9 (Weike Biotechnology, Harbin, China) and NDV LaSota strain (Weike Biotechnology, Harbin, China), AAvV-4 (prepared by our lab) were used as references.

Nucleotide sequences of AAvV-6 in this study were aligned through Mega X software with the sequences of representative AAvV-6 strains retrieved from GenBank database (http://www.ncbi.nlm.nih.

gov/GenBank). The homology analysis was carried out using the maximum likelihood method through MegAlign (DNASTAR A tremendous amount of information about AAvV-1 is available on the characteristics and genetic relationships because of the severe disease it causes in poultries worldwide (Shittu, Joannis, Odaibo, & Olaleye, 2016; Zhang et al., 2015) . By comparison, the pathological phenomenon which AAvV-6 causes are relatively weak, just manifested in decreased egg production and mild respiratory disease in turkeys and was avirulent in chickens (Alexander, 2000; Saif & Barnes, 2008; Sobolev et al., 2016) . As a low virulence virus for chickens and low separation rate, the potential harm of the AAvV-6 is easily overlooked. However, in a recent study of the pathogenicity of two AAvV-6 variant isolates, AAvV-6/red-necked stint/Japan/8KS0813/ 2008 and AAvV-6/duck/Hong Kong/18/199/1977 , as representative isolate of genotype I and II respectively, could replicate in respiratory tissues of infected mice and induce respiratory disease, sometimes resulting in death of the infected mice . Further researches about the virulence and susceptibility of AAvV-6 should be include more isolates, since differences of viral propagation properties in same cells were observed between the two variant isolates, owing to the change of host from red-necked stint to duck and sites where the two variant isolates separated at such a distance to some extent (Bui et al., 2014) . Therefore, the identification and isolation of Hubei isolate is beneficial for the further understanding of HA-negative AAvV-6 in this study for the high sequence identity (99.1%-99.2%) with two Jilin isolates (AAvV-6/mallard/Jilin/190/2011 and AAvV-6/mallard/Jilin/127/2011) and the same cleavage site with other AAvV-6 isolates.

In conclusion, our current data indicate that AAvV-6 is distributed sporadically in wild migratory birds, not in domestic birds, in The number of base substitutions per site from averaging over all sequence pairs between groups are shown. Standard error estimate(s) are shown above the diagonal and were obtained by a bootstrap procedure (500 replicates). Analyses were conducted using the Maximum Composite Likelihood model. The rate variation among sites was modelled with a gamma distribution (shape parameter = 1). The analysis involved (a) 24 nucleotide sequences (I, n = 13; II, n = 11), (b) 13 nucleotide sequences (Ia, n = 8; Ib, n = 4) and 11 nucleotide sequences (IIa, n = 5; IIb, n = 6). Codon positions included were 1st+2nd+3rd+Non-coding. All positions containing gaps and missing data were eliminated. There were a total of 1,638 positions in the final dataset. Evolutionary analyses were conducted in MEGA X.

(20160414029GH), and three grants from the National Science Foundation of China (31402195, 31472195 and 31570026).

RY, XL, YC and ZD designed and performed the study, drafted the manuscript and analyzed the data. All authors collected clinical samples. RY, XL, ZD and YC carried out experiments.

Yin http://orcid.org/0000-0001-7431-2523

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