Surveillance and genetic diversity analysis of avian astrovirus in China

Avian astroviruses (AAstVs) have caused major problem for poultry breeding industries in China in recent years, and the goose gout caused by goose astrovirus has produced particularly great economic losses. To better understand the prevalence and genetic diversity of AAstVs in China, 1210 poultry samples collected from eight provinces were tested with reverse transcription-polymerase chain reaction (RT-PCR) to detect AAstV infections in different poultry populations. Then, Open reading frames 2 (ORF2) was amplified by specific primers, and the genetic evolution was analyzed. Our surveillance data demonstrate the diversity of AAstVs in China insofar as we detected 17 AAstVs, including seven chicken astroviruses (CAstVs), five avian nephritis viruses (ANVs), two goose astroviruses (GoAstVs), two duck astrovirus (DAstVs), and one new AAstV belonging to Avastrovirus Group 3. The positive rate of AAstV infection was 1.40%. Host analysis showed that CAstVs and ANVs were isolated from chickens, DAstVs and GoAstVs were isolated from ducks. Host-species-specific AAstVs infections were also identified in numerous samples collected at each stage of production. This study provides further evidence to better understand the epidemiology of AAstVs in different species of poultry in China.

In recent years, AAstV infections have been shown to be associated with many diseases [9] that pose a threat to the poultry breading industries. These include poultry enteritis mortality syndrome, runting-stunting syndrome in broilers, kidney and visceral gout in broilers, and fatal hepatitis in ducklings [13,14]. A previous study reported that the antibody positive rate for CAstV in chickens in China is 60.68% [15]. Astroviruses infections are becoming more common in China, including those of ANV, CAstV, DAstV, and GoAstV [12,[16][17][18]. Some astrovirus isolates have caused fatal visceral gout in domestic goslings [19]. These studies suggest that more attention must be paid to astrovirus infections in poultry.
In the present study, we undertook an epidemiological investigation in 2020 with the routine surveillance of AAstVs using universal RT-PCR, to better understand the prevalence and distribution of AAstVs in China. Our results demonstrate the genetic diversity of AAstVs in China, and extend the epidemiological information available on these viruses.

Ethics statement
This study was conducted according to the Animal Welfare Guidelines of the World Organization for Animal Health [20] and was approved by the Animal Welfare Committee of the China Animal Health and Epidemiology Center (CAHEC, Qingdao, Shandong).

Sample collection
A total of 1210 swab and tissue samples were collected from 20 regions in eight provinces of China in 2020 (the sample background information was shown in Table 1), mainly for routine surveillance of avian diseases, such as avian influenza virus (AIVs) and Newcastle disease virus (NDVs) infection, among others. The swab and tissue samples were collected by taking smears from both the cloacal and oropharyngeal tracts of the poultry, and were stored in 1.5 mL of phosphate-buffered saline (pH 7.2) containing 10% glycerol. The RNA was extracted with the

Detection of AAstVs with conserved RT-PCR assay
Conserved RT-PCR and sequencing were used to analyze the genetic characteristics of the AAstVs circulating in poultry. In brief, the RNAs described above were amplified with the Takara One Step RT-PCR Kit (Takara), using primers to conserved sites in the AAstV genomes: 5'-GAYTGGACNMGNTWYGAYGGNACNATNCC-3' (forward) and 5'-YTTNAC CCACATNCCRAA-3' (reverse) [21]. The assay amplified a 430-bp region of the viral RdRp, which has been shown to detect all astroviruses circulating in avian species, including chickens, ducks, and geese. The RT-PCR assay was performed in a 25μL reaction system, which was incubated at 50˚C for 30 min and then denatured at 94˚C for 5 min, followed by 45 cycles of 94˚C for 1 min, 45˚C for 1 min and 72˚C for 40 s. The PCR products were purified by DNA extraction kit (Sangon, Shanghai, China), and sequenced directly with the ABI 3730xl DNA Analyzer. Then, the primers in the Table 2 are used to amplify ORF2 (Amplification primers are shown in Table 2). RT-PCR detection was performed in a 25μL reaction system, which was incubation at 50˚C for 30 min and then denatured at 94˚C for 5 min, followed by 45 cycles of 94˚C for 1 min, 45˚C for 1 min and 72˚C for 1 min 30 s. The PCR products were purified and sequenced, and the sequence obtained by sequencing is used to construct a phylogenetic tree.

Phylogenetic analysis
The ORF2 sequences were aligned, and the substitutions and phylogenetic relationships were calculated with the software package MEGA 6.0 [22,23]. The phylogenetic relationships of the sequences were calculated with the maximum likelihood method, which is assumed to best describe the substitution pattern. Gaps were handled by partial deletion, and bootstrap values were calculated from 1000 replicates.

Surveillance of Astrovirus circulating in poultry
Seventeen samples tested positive for AAstV with RT-PCR and the PCR products were sequenced. The accession numbers deposited in GenBank for the sequences of the 17 Astroviruses are MZ097383-MZ097399 (Table 3).

Phylogenetic and diversity analyses of Astrovirus circulating in poultry
A phylogenetic analysis revealed that the 17 astroviruses detected in poultry could be classified into five lineages, and corresponded to seven CAstVs, five ANVs, two GoAstVs, two DAstVs, and one new AAstV belonging to Avastrovirus Group 3 (Fig 2). 17 isolates were analyzed for homology with 43 strains downloaded from Genbank, and the results are shown in Table 4, and it is worth noting that the new AAstV isolate have high homology with the Hong Kong strain, which ranging from 92.6% to 92.8%. The number of samples and the AAstV-positive rate in each province are shown in Table 5, and the geographical distribution of the samples detected in this study is shown in Fig 1. The rate of AAstV-positive samples was 1.40%, and the positivity rates of CAstV, ANV, DAstV, GoAstV and AAstV 3 were 0.66%, 0.41%, 0.08%, 0.17% and 0.08%, respectively. These data suggested that CAstV and ANV circulate in chickens, DAstV and GoAstV circulate in ducks. Two GoAstV strains were found in ducks when the host were analyzed ( Table 6), whereas GoAstV was not detected in goose populations.  Of the 1210 samples tested, 526 were collected from retail markets, 553 were from wholesale markets, 15 were from slaughterhouse, and 116 were from poultry farms, as shown in Table 7.
Host-species-specific AAstV infections were identified in numerous samples collected at all the four types of poultry sites. Poultry farms had the highest positive rate, followed by the retail markets.

Discussion
DAstV was the first-discovered AAstV, in 1965, when it caused a case of viral hepatitis [1]. The virus was confirmed as DAstV-2 until 1985 [24]. In 2011-2012, an outbreak of gout was reported in broilers in India. After the isolation and identification of the virus, Bulbule confirmed that a new type of CAstV was one of the pathogens responsible for gout disease in chicken flocks in India [13]. In China, gout disease was first reported in 1-week-old goslings in Anhui province in 2015, after which the disease spread to most provinces in China in 2017, with a high incidence rate of 80%-90% and a mortality rate of 20%-70% [19]. Duck viral hepatitis is recognized as an acute, highly contagious, and fatal disease, for which there is no specific treatment. ANV and CAstV are associated with growth problems, kidney lesions and enteritis in young chickens, and CAstV infection has been shown to be prevalent in broilers. In addition to these three groups of DAstVs found primarily in domestic ducks, other AAstVs have also been identified in domestic ducks, including ANV, TAstV-1, TAstV-2 and CAstV [10,25,26]. The occurrence of different AAstVs in domestic ducks raises concern about the role of domestic ducks as reservoirs for diverse astroviruses. Previous studies have detected ANV in dead-in-shell goslings, suggests that attention must be paid to astrovirus infections in domestic geese [26].
AAstV are widely spread throughout the world and have become increasingly harmful to the poultry industry. However, their epidemiology and pathogenic mechanism are not yet clear. In this study, we conducted a surveillance of AAstVs with a conserved RT-PCR assay, which detected several different AAstVs, including CAstV, ANV, DAstV and GoAstV. Our results suggested that a diversity of astroviruses is present in poultry, including CAstVs, ANVs, DAstVs, GoAstVs, and novel AAstV.
Our results also suggested that AAstVs are present in many provinces of China, and that CAstV and ANV are circulating in chickens and DAstV and GoAstV in ducks. Interestingly, the two GoAstV strains were isolated from ducks in an analysis of their hosts, which suggests that CAstV, ANV and DAstV were all prevalent in their dominant hosts in 2020, whereas GoAstV was not detected in geese. This finding warrants further research.
In this study, 17 of AAstV were detected in 5 provinces of China, and nearly half of the strains were isolated from Jiangsu Province, This is consistent with the results of serological testing by Xue et al, which suggested that the epidemic of AAstVs in this areas was very serious [15]. Two CAstVs and two GoAstVs were detected in samples collected from a symptomatic poultry in Shandong province. This is consistent with the previous detection and identification of AAstVs in Shandong Province [16]. A novel AAstV was detected in Guangdong Province, which was classified as Avastroviruses Group 3, but its pathogenicity in poultry is still unclear and requires further research. The results of this study suggest that other new astroviruses are yet to be found.
Of the 1210 samples tested, host-species-specific AAstV infections were identified in samples collected from many different poultry populations. Therefore, each stage of the production process, from breeding to marketing to slaughter (in poultry farms, live poultry markets, and slaughterhouses) may play important roles in the circulation of AAstVs in poultry, as they do in the circulation of AIVs.
To our knowledge, this study is the first to report a systematic phylogenetic analysis of AAstV strains in China. The data presented extend the epidemiological information available on AAstVs in China. However, the continuous surveillance of avastrovirus should be increased to better understand the diversity, distribution, cross-species transmission and clinical significance of AAstVs in nature.
Supporting information S1 Table. Relevant data for geographic distribution maps. A total of samples collected from 8 provinces were tested (column ID = "NameEN"). The number of positives detected in each province ranges from 0 to 8 (column ID = "Total"), The positive rate varies from 0-10% (column ID = "Rate").