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

Abstract

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.

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

“…During the years from 2013 to 2018, both cloacal and oropharyngeal swab samples from clinically healthy domestic poultry from the live bird market ( n = 3974) and fresh fecal samples from migratory birds in wetlands ( n = 14,909) were collected from six provinces of China, including two provinces in Central China (Hubei and Hunan), one in East China (Anhui), one in Northeast China (Jilin), one in Northwest China (Qinghai) and one in North China (Neimenggu), as part of both the avian influenza virus (AIV) and NDV surveillance program [18,19,20,21]. The detailed information on sample collection, transportation, and handing as described in our previous study [18,19].…”

“…RNA extraction, cNDA synthesis, and seminested PCR and sanger sequencing for L gene of paramyxoviruses were performed according to the methods described in our previous study [20]. To explore the genetic characteristics of this V93-1 isolate, a complete viral genome sequence was determined by next-generation sequencing (NGS) [24], based on the random sequencing of total RNA using the de novo assembly method.…”

“…To explore the genetic characteristics of this V93-1 isolate, a complete viral genome sequence was determined by next-generation sequencing (NGS) [24], based on the random sequencing of total RNA using the de novo assembly method. Briefly, 2 mL of allantoic fluids containing V93-1 virus was used for RNA isolation using the TriPure Isolation Reagent (Roche, Shanghai, China), and then reverse transcription reactions were conducted using the protocols described in a previous study [20]. The sequencing library (using 1.5 ng purified cDNA) with an insert size of 200 bp was prepared by end-repairing, dA-tailing, adaptor ligation, and PCR amplification.…”

“…The pathogenicity test of the V93-1 virus, including the mean death time (MDT), the intra-cerebral pathogenicity index (ICPI), and intravenous pathogenicity index (IVPI), were determined according to the protocols described in the Office International des Epizooties (OIE) manual for ND [22]. Virus infection of MDCK, HD 11 and DF-1 cells with the presence or absence of TPCK-trypsin were performed according to the methods as previously described [20].…”

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

“…Of the eighteen AAvV-6 viruses isolated in this study, complete genome sequences were obtained for fifteen viruses. The genome for strain AAVV-6 mallard/QC/610/2005 was incomplete, missing the first 14 The fusion gene cleavage site sequence for prototype genotype AAvV-6 viruses (PEPR/L) was identical in the two Canadian genotype 1 isolates. The Canadian genotype 2 isolates also shared identical cleavage sites (REPR/L) and also matched previously reported genotype 2 viruses (Table 3).…”