Astroviruses (AstVs) are emerging pathogens, belonging to members of the family Astroviridae. These viruses are divided into two genera: Mamastrovirus and Avastrovirus, which infect mammals and birds, respectively (1). AstV infections cause a wide range of clinical signs from gastroenteric (e.g., human, turkey, sheep, and pig) to neurologic (e.g., human, mink, cattle, sheep, and pig) disease (2–10). They are non-enveloped small viruses (30 nm) with a positive-sense single-stranded RNA genome of 6.4–7.9 kb (11). The genome contains three open reading frames (ORFs): the ORF1a and ORF1b, encoding non-structural proteins, and the ORF2, which encodes the capsid (11, 12).

In humans, AstV is the third most common cause of viral diarrhea in young children worldwide, with high seroprevalences as 94% in children of 6–9 years old (13, 14). Despite this high prevalence, due to the lack of cell culture systems and animal models, AstV are among the least studied enteric RNA viruses (12). However, advances in sequencing technologies have increased the availability of genome sequences and the identification of new strains. Typically, human AstV (HAstV) infections cause acute self-limiting mild diarrhea (12). Nevertheless, immunocompromised patients occasionally exhibit systemic spread, resulting in neurologic disease (15). In addition, a zoonotic potential of AstV is suspected but remains unclear (16–18). Genetic and evolutionary studies support the idea that both cross-species transmission and recombination events among AstV of human, porcine, and other species origin, may have occurred (11, 16).

Porcine astrovirus (PoAstV) has high genetic diversity, it is worldwide distributed, and it is commonly detected and shed by healthy and diarrheic swine (19). Five lineages of porcine AstV (PoAstV1–5) have been described by Laurin, Dastor (20), but only PoAstV-1 has been officially classified under the species Mamastrovirus 3 (21). In the US, pigs are commonly (13.9%) co-infected with multiple astrovirus strains (19). High prevalence and co-infections may create appropriate conditions for viral recombination and the potential emergence of viral variants that pose a higher risk of clinical disease. Recently, PoAstV has been linked to extraintestinal infections suggesting more complex pathogenesis and serious outcomes than previously thought (3, 4, 22).

In Chile, a recent study reported human AstV infections (14%) as a predominant cause of viral gastroenteritis in rural zones, in addition to norovirus (15%) and rotavirus (14%) (23). However, animal AstV has not been described in Chile. Considering the high prevalence and worldwide distribution of the AstV and PoAstV, the suspected zoonotic potential, and the lack of information regarding these viruses in a regional context, this study aims to determine the presence and genetic diversity of PoAstV circulating in Chilean intensive pig farms. These samples were taken in the context of other viral disease surveillance programs.

All samples were successfully sequenced by Illumina. The most consistent virus family found in the samples was Astroviridae. Additionally, reads that belong to other families, such as Caliciviridae, Parvoviridae, and Reoviridae, were found at lower rates and were not considered for further analysis as they are beyond the aim of this study. Porcine astrovirus reads were observed in all samples/farms included, confirming its ubiquity in Chilean swine intensive production. Overall results of PoAstV identified 229,496 reads for PoAstV-5, 33,917 for PoAstV-4 and 31,751 for PoAstV-2. Only 10 reads were classified as PoAstV3, however, the limited reads do not provide sufficient evidence to confirm the presence of this lineage.

PoAstV-4 was identified in 16 out of 17 farms, PoAstV-2 in 15 and PoAstV-5 in 13 farms (Supplementary Table 1). These results are in agreement with estimations made in the US and some European and Asian countries, where the most prevalent lineage is PoAstV-4, mainly followed by PoAstV-2 (19, 29–33). Contrary to the situation in China, where the most widely distributed strain is presumably PoAstV-2 (34).

We identified at least two different PoAstV lineages co-circulating in 15 out of 17 farms (Supplementary Table 1). Thus, the Chilean swine exhibits conditions for PoAstV recombination events. The circulation of multiple PoAstV strains in the same farm has been reported previously in China, Denmark, Slovakia, Thailand, and the USA (31, 35–37). Co-infection of different lineages in the same individual has also been reported (19, 38).

Eleven complete or near-to-complete genomes of PoAstV were obtained, which were used for the phylogenetic analysis (Table 1). The genomes were obtained from both fecal and oral fluids samples and were recovered from 8 different farms. Figure 1 shows the geographic distribution of the sequences obtained (Figure 1). Interestingly, from farm two, it was possible to obtain the genome of PoAst-2, 4, and 5.

The phylogenetic analysis grouped the Chilean strains into 5 (Figure 2 and Supplementary Figure 1). The seven PoAstV-5 strains are grouped into one monophyletic cluster (97.4% pairwise identity), closely related to strains detected in the USA. Interestingly, the PoAstV-5 were obtained from seven different farms distributed in five geographic regions. The three PoAstV-2 genomes were detected from three different farms in different regions and grouped into two separate sub-clusters. The PoAstV-2/Swine/CHI/FB036/2017 (Metropolitan region) and PoAstV-2/Swine/CHI /CF2673/2017 (Maule region) genomes formed one sub-cluster, while PoAstV-2 /Swine/CHI/FB0148/2017 (Araucania region) grouped with sequences from USA and Japan. Finally, the two PoAstV-4 from two different farms and regions (Metropolitan and Valparaiso region) were phylogenetically distant also related to Japanese strains. The phylogeny demonstrates a genetic relationship between Chilean PoAstV-5 strains, suggesting the same origin for those strains. On the contrary, PoAstV-2 and PoAstV-4 results indicate more diversity even with fewer sequences (For details on genetic distances see Supplementary Tables 2, 3).

Due to the limited number of sequences obtained in this study and the scarce of sequences in GenBank database, conclusions about the origin of the viral strains cannot be elucidated. Indeed, most of the available sequences in GenBank database are from the USA, Japan, and China. Other limitations of the phylogenetic analysis are the incomplete genome coverage in several samples (Table 1) and the potential errors derived from sequencing methods, as these may alter the phylogenetic tree estimation.

This is the first report characterizing the PoAstV sequences circulating in Chile.

This result represents, in turn, the first PoAstV genomes from swine in Latin America. PoAstV studies in Latin America are very scarce, and only two have been published. One study identified PoAstV in healthy pigs from a farm in Brazil (39), and another study conducted in Colombia, which obtained partial PoAstV sequences from diarrheic piglets and humans (40). To date, most of the PoAstV sequences available in GenBank database were obtained in the Northern hemisphere.

Our results support the detection of PoAstV in the Chilean swine population, similar to other observations worldwide. Further studies are needed to understand the relevance of PoAstV to swine health and the evolution and spread of PoAstV locally and globally.

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.

The animal study was reviewed and approved by Institutional Animal Care and Use Committees of the Universidad de Chile, protocol number 02–2016. Written informed consent was obtained from the owners for the participation of their animals in this study.

NA, GR-T, and VN: study design and conceptualization. SM and VN: funding and resources. CF and JM: samples collection and processing. CF, JM, and SM: performed the assays. CF, NA, CV, BB, and VN: data analysis. CF, NA, GR-T, and VN: wrote the paper. All authors critically evaluated the paper. All authors contributed to the article and approved the submitted version.

Animal Virology Laboratory, Faculty of Veterinary and Animal Sciences, Universidad de Chile and Programa Fondecyt 11170877 and 1211517.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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