Coinfection and Genetic Characterization of Porcine Astrovirus in Diarrheic Piglets in China From 2015 to 2018

Porcine astrovirus (PAstV) is broadly distributed globally and exists as at least five distinct genotypes. PAstV, which was recently identified as an important pathogen of diarrhea in piglets, is widely distributed in China. However, few studies have investigated the coinfection and genetic characterization of PAstV in diarrheic piglets in China. In this study, 89 PAstV-positive samples were identified in 543 diarrhea samples in China from 2015 to 2018, of which 75.28% (67/89) were coinfected with three to five different porcine pathogens, while none were positive for PAstV only. Among the 543 diarrhea samples, statistical analysis showed that PAstV-induced diarrhea was potentially associated with coinfection of PEV (p < 0.01) and GARV (p < 0.01). Phylogenetic analysis showed that the 27 identified PAstV strains belong to three different genotypes and that PAstV-2 (81.48%, 22/27) was predominant in diarrheic piglets in China, followed by PAstV-4 (11.11%, 3/27) and PAasV-5 (7.41%, 2/27). Sequence analysis revealed that the 27 RdRp genes identified in this study had nucleotide homologies of 53.8–99.5%. In addition, the RdRp gene of PAstV-4 strain JL/MHK/2018/0115 harbored a unique insert of three nucleotides (GAA) as compared with other known PAstV-4 strains. Furthermore, the genotypes of PAstV varied among different geographical locations, although PAstV-2 was the most widely distributed in China. These data demonstrate that PAstV coinfection with other porcine pathogens was common and there was genetic diversity of PAstV in diarrheic piglets in China.

Diarrhea of piglets has long been a problem afflicting the global pig industry. Coinfections with more than one porcine pathogen are common and often more clinically severe (7,8). A previous surveillance study conducted by our group found a high percentage of coinfection among diarrheic piglets (9). PAstV, which has been identified as an important agent of diarrhea (10), and frequently presents as a coinfection with other porcine pathogens (3,5,6,9,11). However, data regarding coinfections with PAstV in diarrheic piglets in China are limited. Therefore, coinfections with PAstV and other porcine pathogens should be monitored in China.
To date, five genotypes of PAstV (PAstV-1 to PAstV-5) with different prevalences have been identified worldwide. Although all five PAstV genotypes have been reported in Europe, the most common is reportedly PAstV-4 (4), while PAstV-2 and PAstV-4 are the most common throughout Asia (5,6,12). However, information available on the genetic characterization of PAstV in China is fairly limited (2,11). Therefore, it is necessary to investigate the genetic diversity and evolution of PAstV currently in China.
In this study, 89 PAstV-positive diarrhea samples were collected to investigate the prevalence of PAstV coinfection with 12 other porcine pathogens. The obtained RdRp genes were genetically characterized in order to provide insights into the epidemiology of PAstV circulating among diarrheic piglets in China.
Sequencing and Analysis of the RdRp Gene of PAstV RNA extraction and cDNA synthesis were performed as previously described by Wang et al. (13). The PAstV RdRp gene was amplified using the nested RT-PCR method described by Chu et al. (14) and then cloned into the vector pMD18-T (TaKaRa Biotechnology Co., Ltd, Dalian, China) in accordance with the manufacturer's protocol. Three positive clones of each amplicon were subjected to Sanger sequencing. Sequence analysis was conducted using the EditSeq tool included with the Lasergene DNASTAR TM 5.06 software package (DNASTAR Inc., Madison, WI, USA). Multiple-sequence alignments were performed using the multiple-sequence alignment tool included with the DNAMAN 6.0 software package (Lynnon BioSoft, Pointe-Claire, QB, Canada).

Phylogenetic Analysis
Sequences of the PAstV RdRp gene retrieved from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) were used for sequence alignments and phylogenetic analyses. Multiplesequence alignments were generated using the ClustalX alignment program included with the MEGA 6.06 software package (15). A phylogenetic tree was constructed from the aligned nucleotide sequences using the p-distance model and 1000 bootstrap replicates and annotated with Interactive Tree Of Life (iTOL) software (http://itol.embl.de/) (16).

Coinfection of PAstV With Multiple Porcine Pathogens in Diarrheic Piglets
As reported in our previous study, the PAstV-positive rate in diarrheic piglets was 16.39% (89/543), indicating wide distribution in China (9). In the present study, PAstV coinfection with 12 other porcine pathogens in diarrheic piglets was investigated. Of the 89 PAstV-positive diarrhea samples, the rate of PAstV coinfection with 12 other porcine pathogens ranged from 7.87% (7/89) to 85.39% (76/89) ( Figure 1A). The average number of viruses detected in each sample was 4.12, while 75.28% (67/89) of samples had three to five different viruses, 3.37% (3/89) had seven to eight different viruses, and none were positive for PAstV only (Figure 1B). Coinfections of PAstV with other porcine pathogens, such as rotavirus, PEDV, TGEV, porcine circovirus-2 (PCV2), and porcine hemagglutinating encephalomyelitis virus (PHEV), have been reported previously (3,5,6,11). In the present study, the rate of PKV coinfection in PAstV-positive samples was relatively high (85.39%, 76/89), and there was evidence that u shed more PKV than did healthy individuals (18), indicating that PKV may have a potential role in PAstV-induced diarrhea in piglets. PEDV is the major cause of viral diarrheal disease in swine in China (9). Although there was a high prevalence of PEDV in piglets infected with PAstV in our previous study, statistical analysis indicated that PEDVinduced diarrhea was not associated with PAstV coinfection (p > 0.05) (9). The pathogenicity of PEV is typically mild in pigs (19), and GARV is among the most common pathogens of diarrhea in piglets (20). In this study, the positivity rates of PEV (69.66%, 62/89) and GARV (35.96%, 32/89) were relatively high in PAstVpositive samples, suggesting a highly significant association of coinfection with PEV (p < 0.01) and GARV (p < 0.01) in PAstV-positive diarrhea samples of piglets (Table 1). However, since evidence of coinfections of PAstV with PEV and GARV causing diarrhea in piglets is somewhat limited, further studies are warranted.

Genotype Distribution of PAstV in China
The results of the present study showed that PAstV-2 was circulating in ten different provinces, covering five regions of China (Figure 3), suggesting that PAstV-2 is the most widely distributed strain in China, which is supported by recent studies conducted in the provinces of Hebei, Hunan, Sichuan, and Guangxi (11,21,22,25). Previous studies have reported the existence of multiple PAstV genotypes in China. For example, PAstV-1 was identified in Shanghai as well as the provinces of Hunan and Guangxi (22,25), while PAstV-3 was identified in Guangxi province only (22), PAstV-4 has been reported in the provinces of Tianjin, Hunan, and Guangxi (22,24,25), and PAstV-5 has been detected in the provinces of Hebei, Sichuan, Hunan, and Guangxi (11,22,(24)(25)(26). In the present study, three PAstV-4 strains were identified in the provinces of Jilin, Shanxi, and Jiangxi, and two PAstV-5 strains were identified in Shandong and Jiangxi provinces, respectively. These results suggest the presence of various genotypes of PAstV in different regions of China. Moreover, five PAstV strains identified in Jiangxi province exhibited three distinct genotypes (PAstV-2, PAstV-4, and PAstV-5). Previous studies reported the presence of two or more genotypes of PAstV in the same province of China, such as four different genotypes of PAstV circulating in Hunan province (21,25), while all five genotypes of PAstV were identified in Guangxi province (22), indicating a remarkable diversity of genotypes of PAstV cocirculating among pig farms in China.
In conclusion, results from the present study provide evidence that coinfection of PAstV with multiple porcine pathogens is common in diarrheic piglets in China, and PAstV-induced diarrhea is potentially associated with PEV and GARV coinfection. Phylogenetic analysis revealed that PAstV-2 was predominant in diarrheic piglets in China and multiple genotypes of PAstV were co-circulating in China from 2015 to 2018. In addition, one PAstV-4 strain was shown to harbor a unique insert within the RdRp gene. These results increase our current understanding of the coinfection and genetic characterization of PAstV in diarrheic piglets in China and provide valuable information for further studies of PAstV.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

ETHICS STATEMENT
This animal study was reviewed and approved by Animal Experiments Committee of the Heilongjiang Bayi Agricultural University (registration protocol 201501003). Written informed consent was obtained from the owners for the participation of their animals in this study.

AUTHOR CONTRIBUTIONS
DS conceived the study. MS, SQ, DY, DG, and BY analyzed the data. MS and SQ wrote the manuscript. All authors contributed to the article and approved the submitted version.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets. 2020.00462/full#supplementary-material Table S1 | The background information of 89 PAstV-positive samples.