High genetic diversity of hepatitis E virus in swine in São Paulo State, Brazil

Hepatitis E virus (HEV), a member of the Orthohepevirus A species, can infect mammals and humans and may be classified into genotypes and subtypes (Smith et al., 2020). In South America, genotype 1 (HEV-1) is restricted to humans, and transmission occurs via the fecal– oral route. Zoonotic genotype 3 (HEV-3) is related to humans, pigs, wild boar, sewage, rivers, drinking water plants, and slaughterhouse effluent (Pisano et al., 2018).

Both serological and molecular detection of HEV-3 infection in domestic pigs have been demonstrated in several Brazilian regions and South America, and the subtypes are 3a-f and h (Campos et al., 2018;Pisano et al., 2018;Silva et al., 2018;Oliveira-Filho et al., 2019), but there are only two records of HEV-3 in pigs in São Paulo state (Paiva et al., 2007;Bertolini, 2017).
Therefore, due to scarce information concerning this viral infection, the aim of this work was to assess the occurrence of HEV in samples from a biological bank of swine feces from the state of São Paulo.
Fecal samples from 89 swine collected between 2008 and 2009 from the metropolitan region of Campinas were analyzed using nested reverse transcriptase-polymerase chain reaction (RT-nPCR) and Sanger sequencing. This study was carried out following the recommendations in the *Corresponding author: marcosbryan@usp.br Submitted: October 3, 2020. Accepted: May 24, 2021. Brazilian National Guidelines on Animal Experimentation. The protocol was approved by the Universidade Santo Amaro Ethics Committee on Animal Use (32/2013).
Arq. Bras. Med. Vet. Zootec., v.73, n.5, p.1237-1242, 2021 The first amplification was carried out in a final volume of 25μL, comprising 2.5µL of cDNA 1 X Promega Go Taq® Green Master Mix (Promega, Madison, USA), 10 pmol of each primer and DEPC water q.s.p. Conditions for the first amplification were initial denaturation at 94°C for 4 min, followed by 40 cycles (94°C/15 s denaturation, 52°C/30 s annealing, and 72°C/1 min extension) and a final extension at 72°C for 5 min. The second amplification was performed using 1μL of the amplicon and the same reagents, concentrations, and amplification conditions from the first amplification, except for the extension time (72 °C/30 s). The ORF-1 (287 bp) and ORF-2 (348 bp) fragments were run on a 1.5% agarose gel, stained with SYBR Safe® (Thermo Fisher Scientific, Carlsbad, USA), and visualized under UV light.
The resulting sequences were genotyped by phylogenetic reconstruction using reference sequences (Silva et al., 2018;Smith et al., 2020) and GenBank sequences. GenBank accession numbers, genotypes, and countries of origin are related in the phylogenetic tree. Phylogenetic trees were generated by the neighbor-joining method with the Kimura 2-parameter method (ORF-1) or the maximum composite likelihood method (ORF-2) for evolutionary distances, complete deletion and 1,000 bootstrap replicates with Mega 7.0.21 (Kumar et al., 2016). Avian HEV was used as an outgroup. The sequence identity matrix was generated with BioEdit software version 7.0.8.0 (http://www.mbio.ncsu.edu/bioedit/bioedit.html).
Seven out of 89 stool samples were positive for the ORF-1 region, three collected in 2008 and four in 2009. Of the seven, three showed positive amplification for ORF-2, which suggests differences in sensitivity between protocols or gene diversity (Silva et al., 2018). A possible explanation is that there are differences in the anchoring of the primers due to possible mutations in the Brazilian sequences, which is reflected in the lower proportion of detection.
Phylogenetic analysis of both partial ORF-1 and ORF-2 sequences ( fig. 1 and 2) showed that all seven samples belonged to HEV-3 zoonotic genotypes (Pisano et al., 2018;Oliveira-Filho et al., 2019). The finding of HEV-3 in the metropolitan region of Campinas increased the viral dispersion previously described in the southeastern region (Paiva et al., 2007;Bertolini 2017) and may explain the presence of HEV seropositivity in human populations in 2000 (Gonçales et al., 2000). The sequences were deposited in GenBank under accession numbers MF981263-MF981272. Figure 1 shows the genetic diversity of HEV samples from the state of São Paulo. The presence of subgenotypes 3b (MF981263, MF981264, and MF981265), 3h (MF981266 and MF981267), and 3j (MF981268) can be observed by phylogenetic partial ORF-1 analysis.
The sequences MF981266 and MF981267 obtained in this study clustered with Brazilian sequences (KU888662-KU888663) from pork bioproducts at Rio Grande do Sul (Heldt et al., 2016), with 98.3% to 98.9% nucleotide identity, and clustered with subtype HEV-3h, showing 87% nucleotide identity with the reference subtype JQ013794 (Smith et al., 2020) from humans. This clade forms a sister group with swine isolates from Rio Grande do Sul that have similarities with human samples from Uruguay. This subtype was first reported in backyard pigs in Rio Grande do Sul (Silva et al., 2018). It is noteworthy that both MF981266 and MF981267 were identified in 2008, suggesting the presence of this genotype for at least one decade in the state of São Paulo.
The other single sequence (EF491206) from São Paulo (Paiva et al., 2007) clustered with newly described subtype HEV-3i (FJ998008) reference sequences (Smith et al., 2020), which have not been previously described in Brazil. Figure 2. The tree was inferred using the Neighbor-Joining method using the Maximum Composite Likelihood parameter model. It was constructed from 260 nucleotide sequences of a partial ORF-2. The analysis involved 27 nucleotide sequences. Phylogenetic analyses were conducted in MEGA 7.0.21 with 1,000 bootstrap replicates. Bootstrap values of >70 was shown. Avian HEV was used as an outgroup. The sequences from this study are indicated by a black form.
Considering the genotypic variety found in São Paulo state in samples collected in the late 2000s, molecular epidemiology studies are necessary in humans and animals to understand the dispersion of these genotypes and the current seropositivity found in humans. The present data demonstrated the presence of HEV subtypes not previously described in Brazil, indicated elevated genetic diversity in the state of São Paulo and reinforced the zoonotic potential of HEV-3.

CONTRIBUTION
AC and MBH were responsible for supervision. AC, PEB, MBH were responsible for the planning of the experiments, interpretation and discussion of the data and drafted the manuscript. AC, AMVP, CSM, SATM, AOS were responsible for the execution of the experiments and interpretation of the data. AMMGC and FAB were responsible for discussion of the data and manuscript draf. All authors reviewed the results and approved the final version of the manuscript. Council for Scientific and Technological Development) for the research productivity fellowships (PQ). RMB thank CNPq for PIBIC fellowship.

FUNDING
This work was supported by the National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq) and Coordination of Superior Level Staff Improvement (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES), Financial Code 001.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are openly available in GenBank at https://www.ncbi.nlm.nih.gov/genbank/, reference number MF981263-MF981272.