Strong evolutionary constraints against amino acid changes in the P2 subdomain of sapovirus GI.1 capsid protein VP1.

Sapovirus (SaV) is a nonenveloped RNA virus that causes acute gastroenteritis in humans. Although SaV is a clinically important pathogen in children, an effective vaccine is currently unavailable. The capsid protein VP1 of SaVs forms the outer shell of the virion and is highly diverse, as often seen in the virion-surface proteins of RNA viruses, creating an obstacle for vaccine development. We here report a unique phenomenon pertaining to the variation of SaV VP1. Phylogenetic and information entropy analyses using full-length VP1 sequences from a public database consistently showed that the amino acid sequences of the VP1 protein have been highly conserved over more than 40 years in the major epidemic genotype GI.1 but not in GI.2. Structural modeling showed that even the VP1 P2 subdomain, which is arranged on the outermost shell of the virion and presumably exposed to anti-SaV antibodies, remained highly homogeneous in GI.1 but not in GI.2. These results suggest strong evolutionary constraints against amino acid changes in the P2 subdomain of the SaV GI.1 capsid and illustrate a hitherto unappreciated mechanism, i.e., preservation of the VP1 P2 subdomain, involved in SaV survival. Our findings could have important implications for the development of an anti-SaV vaccine.

Comprehensive full genome analysis of norovirus strains from eastern India, 2017-2021.

BACKGROUND: Worldwide, noroviruses are the leading cause of acute gastroenteritis (AGE) in people of all age groups. In India, norovirus rates between 1.4 to 44.4% have been reported. Only a very few complete norovirus genome sequences from India have been reported. OBJECTIVE: To perform full genome sequencing of noroviruses circulating in India during 2017-2021, identify circulating genotypes, assess evolution including detection of recombination events. METHODOLOGY: Forty-five archived norovirus-positive samples collected between October 2017 to July 2021 from patients with AGE from two hospitals in Kolkata, India were processed for full genome sequencing. Phylogenetic analysis, recombination breakpoint analysis and comprehensive mutation analysis were also performed. RESULTS: Full genome analysis of norovirus sequences revealed that strains belonging to genogroup (G)I were genotyped as GI.3[P13]. Among the different norovirus capsid-polymerase combinations, GII.3[P16], GII.4 Sydney[P16], GII.4 Sydney[P31], GII.13[P16], GII.16[P16] and GII.17 were identified. Phylogenetic analysis confirmed phylogenetic relatedness with previously reported norovirus strains and all viruses were analyzed by Simplot. GII[P16] viruses with multiple residue mutations within the non-structural region were detected among circulating GII.4 and GII.3 strains. Comprehensive mutation analysis and selection pressure analysis of GII[P16] viruses showed positive as well as negative selection sites. A GII.17 strain (NICED-BCH-11889) had an untypeable polymerase type, closely related to GII[P38]. CONCLUSION: This study highlights the circulation of diverse norovirus strains in eastern India. These findings are important for understanding norovirus epidemiology in India and may have implications for future vaccine development.

Prevalence and Characterization of Gastroenteritis Viruses among Hospitalized Children during a Pilot Rotavirus Vaccine Introduction in Vietnam.

Rotavirus (RV), norovirus (NoV), sapovirus (SaV), and human astrovirus (HAstV) are the most common viral causes of gastroenteritis in children worldwide. From 2016 to 2021, we conducted a cross-sectional descriptive study to determine the prevalence of these viruses in hospitalized children under five years old in Nam Dinh and Thua Thien Hue provinces in Vietnam during the pilot introduction of the RV vaccine, Rotavin-M1 (POLYVAC, Hanoi, Vietnam). We randomly selected 2317/6718 (34%) acute diarrheal samples from children <5 years of age enrolled at seven sentinel hospitals from December 2016 to May 2021; this period included one year surveillance pre-vaccination from December 2016 to November 2017. An ELISA kit (Premier Rotaclone®, Meridian Bioscience, Inc., Cincinnati, OH, USA) was used to detect RV, and two multiplex real-time RT-PCR assays were used for the detection of NoV, SaV and HAstV. The prevalence of RV (single infection) was reduced from 41.6% to 22.7% (p < 0.0001) between pre- and post-vaccination periods, while the single NoV infection prevalence more than doubled from 8.8% to 21.8% (p < 0.0001). The SaV and HAstV prevalences slightly increased from 1.9% to 3.4% (p = 0.03) and 2.1% to 3.3% (p = 0.09), respectively, during the same period. Viral co-infections decreased from 7.2% to 6.0% (p = 0.24), mainly due to a reduction in RV infection. Among the genotypeable samples, NoV GII.4, SaV GI.1, and HAstV-1 were the dominant types, representing 57.3%, 32.1%, and 55.0% among the individual viral groups, respectively. As the prevalence of RV decreases following the national RV vaccine introduction in Vietnam, other viral pathogens account for a larger proportion of the remaining diarrhea burden and require continuing close monitoring.

Distribution of Human Sapovirus Strain Genotypes over the Last Four Decades in Japan: a Global Perspective.

Sapovirus (SaV) infections are a public health problem because they cause acute gastroenteritis in humans of all ages, both sporadically and as outbreaks. However, only a limited amount of SaV sequence information, especially whole-genome sequences for all the SaV genotypes, is publicly available. Therefore, in this study, we determined the full/near-full-length genomic sequences of 138 SaVs from the 2001 to 2015 seasons in 13 prefectures across Japan. The genogroup GI was predominant (67%, n = 92), followed by genogroups GII (18%, n = 25), GIV (9%, n = 12), and GV (6%, n = 9). Within the GI genogroup, four different genotypes were identified: GI.1 (n = 44), GI.2 (n = 40), GI.3 (n = 7), and GI.5 (n = 1). We then compared these Japanese SaV sequences with 3,119 publicly available human SaV sequences collected from 49 countries over the last 46 years. The results indicated that GI.1, and GI.2 have been the predominant genotypes in Japan, as well as in other countries, over at least four decades. The 138 newly determined Japanese SaV sequences together with the currently available SaV sequences, could facilitate a better understanding of the evolutionary patterns of SaV genotypes.

Myeloid-associated differentiation marker is an essential host factor for human parechovirus PeV-A3 entry.

Human parechovirus (PeV-A) is an RNA virus that belongs to the family Picornaviridae and it is currently classified into 19 genotypes. PeV-As usually cause mild illness in children and adults. Among the genotypes, PeV-A3 can cause severe diseases in neonates and young infants, resulting in neurological sequelae and death. In this study, we identify the human myeloid-associated differentiation marker (MYADM) as an essential host factor for the entry of six PeV-As (PeV-A1 to PeV-A6), including PeV-A3. The infection of six PeV-As (PeV-A1 to PeV-A6) to human cells is abolished by knocking out the expression of MYADM. Hamster BHK-21 cells are resistant to PeV-A infection, but the expression of human MYADM in BHK-21 confers PeV-A infection and viral production. Furthermore, VP0 capsid protein of PeV-A3 interacts with one extracellular domain of human MYADM on the cell membrane of BHK-21. The identification of MYADM as an essential entry factor for PeV-As infection is expected to advance our understanding of the pathogenesis of PeV-As.

Characterization of a Human Sapovirus Genotype GII.3 Strain Generated by a Reverse Genetics System: VP2 Is a Minor Structural Protein of the Virion.

We devised a reverse genetics system to generate an infectious human sapovirus (HuSaV) GII.3 virus. Capped/uncapped full-length RNAs derived from HuSaV GII.3 AK11 strain generated by in vitro transcription were used to transfect HuTu80 human duodenum carcinoma cells; infectious viruses were recovered from the capped RNA-transfected cells and passaged in the cells. Genome-wide analyses indicated no nucleotide sequence change in the virus genomes in the cell-culture supernatants recovered from the transfection or those from the subsequent infection. No virus growth was detected in the uncapped RNA-transfected cells, suggesting that the 5'-cap structure is essential for the virus' generation and replication. Two types of virus particles were purified from the cell-culture supernatant. The complete particles were 39.2-nm-dia., at 1.350 g/cm3 density; the empty particles were 42.2-nm-dia. at 1.286 g/cm3. Two proteins (58-kDa p58 and 17-kDa p17) were detected from the purified particles; their molecular weight were similar to those of VP1 (~60-kDa) and VP2 (~16-kDa) of AK11 strain deduced from their amino acids (aa) sequences. Protein p58 interacted with HuSaV GII.3-VP1-specific antiserum, suggesting that p58 is HuSaV VP1. A total of 94 (57%) aa of p17 were identified by mass spectrometry; the sequences were identical to those of VP2, indicating that the p17 is the VP2 of AK11. Our new method produced infectious HuSaVs and demonstrated that VP2 is the minor protein of the virion, suggested to be involved in the HuSaV assembly.

Atomic Structure of the Human Sapovirus Capsid Reveals a Unique Capsid Protein Conformation in Caliciviruses.

Sapovirus (SaV) is a member of the Caliciviridae family, which causes acute gastroenteritis in humans and animals. Human sapoviruses (HuSaVs) are genetically and antigenically diverse, but the lack of a viral replication system and structural information has hampered the development of vaccines and therapeutics. Here, we successfully produced a self-assembled virus-like particle (VLP) from the HuSaV GI.6 VP1 protein, and the first atomic structure was determined using single-particle cryo-electron microscopy (cryo-EM) at a 2.9-Å resolution. The atomic model of the VP1 protein revealed a unique capsid protein conformation in caliciviruses. All N-terminal arms in the A, B, and C subunits interacted with adjacent shell domains after extending through their subunits. The roof of the arched VP1 dimer was formed between the P2 subdomains by the interconnected ß strands and loops, and its buried surface was minimized compared to those of other caliciviruses. Four hypervariable regions that are potentially involved in the antigenic diversity of SaV formed extensive clusters on top of the P domain. Potential receptor binding regions implied by tissue culture mutants of porcine SaV were also located near these hypervariable clusters. Conserved sequence motifs of the VP1 protein, "PPG" and "GWS," may stabilize the inner capsid shell and the outer protruding domain, respectively. These findings will provide the structural basis for the medical treatment of HuSaV infections and facilitate the development of vaccines, antivirals, and diagnostic systems. IMPORTANCE SaV and norovirus, belonging to the Caliciviridae family, are common causes of acute gastroenteritis in humans and animals. SaV and norovirus infections are public health problems in all age groups, which occur explosively and sporadically worldwide. HuSaV is genetically and antigenically diverse and is currently classified into 4 genogroups consisting of 18 genotypes based on the sequence similarity of the VP1 proteins. Despite these detailed genetic analyses, the lack of structural information on viral capsids has become a problem for the development of vaccines or antiviral drugs. The 2.9-Å atomic model of the HuSaV GI.6 VLP presented here not only revealed the location of the amino acid residues involved in immune responses and potential receptor binding sites but also provided essential information for the design of stable constructs needed for the development of vaccines and antivirals.

A Human Intestinal Cell Line Suitable for the Propagation of Human Parechovirus Type 1 to 6 with a Clear Cytopathic Effect.

Human parechoviruses (HPeVs) are being increasingly recognized as pathogens that cause mild-to-life-threatening diseases in children and adults. Recently, nucleic acid detection has become the mainstream method for pathogen detection. However, virus isolation is important for virus detection and further virologic characterization studies, and securing human pathogenic virus bioresources. We recently explored conventional cell lines suitable for human sapovirus isolation and accidentally identified a human duodenal cell line, HuTu80, that supported efficient growth of human parechovirus type 3 (HPeV-3) with clear cytopathic effects (CPE). Subsequently, we confirmed that all representative prototype HPeV type 1-6 strains were propagated efficiently in HuTu80 cells with clear CPE within 4 days. Another human ileocecal cell line, HCT-8 (HRT-18), also supports HPeV propagation except for HPeV-3. Titer values in HuTu80 and HCT-8 reached approximately 6.83-8.83 and 6.50-8.17 log10 50% tissue culture infectious dose/50 µL, respectively, when inoculated with multiplicity of infection of 0.0025. Previously reported cell lines likely support HPeV types 1-6 with different efficiency, especially for HPeV-3. In summary, HuTu80 can be used as an additional cell line for HPeV isolation, propagation with a clear CPE to produce a high titer value and for the virus neutralization assays.

Caliciviruses induce mRNA of tumor necrosis factor α via their protease activity.

Calicivirus infection in patients and animals is associated with the production of multiple inflammatory cytokines, including tumor necrosis factor α (TNF-α). Here we studied the feline calicivirus (FCV) non-structural proteins and found that the FCV protease was a key factor for TNF-α gene expression in cultured cells. The expression of the TNF-α gene in cells expressing FCV, human norovirus, and rabbit hemorrhagic disease virus protease was compared, revealing that the induction of TNF-α could be a common phenomenon during the infection by the viruses in the Caliciviridae. The level of TNF-α mRNA in the cells expressing mutant proteases that lacked the active site was measured. These data indicate that the protease activity is crucial for TNF-α expression. These findings provide new insight into the induction of inflammation during calicivirus infection.

The predicted stem-loop structure in the 3'-end of the human norovirus antigenomic sequence is required for its genomic RNA synthesis by its RdRp.

The norovirus genome consists of a single positive-stranded RNA. The mechanism by which this single-stranded RNA genome is replicated is not well understood. To reveal the mechanism underlying the initiation of the norovirus genomic RNA synthesis by its RNA-dependent RNA polymerase (RdRp), we used an in vitro assay to detect the complementary RNA synthesis activity. Results showed that the purified recombinant RdRp was able to synthesize the complementary positive-sense RNA from a 100-nt template corresponding to the 3'-end of the viral antisense genome sequence, but that the RdRp could not synthesize the antisense genomic RNA from the template corresponding to the 5'-end of the positive-sense genome sequence. We also predicted that the 31 nt region at the 3'-end of the RNA antisense template forms a stem-loop structure. Deletion of this sequence resulted in the loss of complementary RNA synthesis by the RdRp, and connection of the 31 nt to the 3'-end of the inactive positive-sense RNA template resulted in the gain of complementary RNA synthesis by the RdRp. Similarly, an electrophoretic mobility shift assay further revealed that the RdRp bound to the antisense RNA specifically, but was dependent on the 31 nt at the 3'-end. Therefore, based on this observation and further deletion and mutation analyses, we concluded that the predicted stem-loop structure in the 31 nt end and the region close to the antisense viral genomic stem sequences are both important for initiating the positive-sense human norovirus genomic RNA synthesis by its RdRp.

Intertypic reassortment of mammalian orthoreovirus identified in wastewater in Japan.

Mammalian orthoreovirus (MRV), a non-enveloped virus with a ten-segmented double-stranded RNA genome, infects virtually all mammals, including humans. Human infection with MRV seems to be common in early childhood, but is rarely symptomatic. Despite the ubiquitous presence of MRV in mammals as well as in environmental waters, the molecular characterisation of the MRV genome remains to be fully elucidated. In this study, two novel strains, MRV-2 THK0325 and MRV-1 THK0617, were unintentionally isolated from wastewater in Japan via an environmental surveillance of enteric viruses. Homology and phylogenetic analysis demonstrated that all the segments of THK0325 were closely related to the MRV-2 Osaka strains, which were recently proposed to have existed for at least two decades in Japan. Most of the segments in THK0617 also showed a close relationship with the MRV-2 Osaka strains, but the M2, S1, and S3 segments belong to another MRV cluster. According to the S1 sequence, the determinant of serotype THK0617 was classified as MRV-1, and both the M2 and S3 segments were closely related to MRV-1 and -3 from the tree shrew in China. These results suggest that the MRV-2 Osaka-like strain spread widely throughout Japan, accompanied by intertypic reassortment occurring in East Asia.

Isolation and characterization of mammalian orthoreovirus type 3 from a fecal sample from a wild boar in Japan.

Mammalian orthoreoviruses (MRVs) have been identified in various mammalian species, including humans, bats, and pigs. However, isolation and complete genome sequences of MRVs from wild boars have not yet been reported. In this study, we isolated, sequenced, and analyzed an MRV from a free-living wild boar in Japan using the porcine-sapelovirus-resistant cell line N1380. Complete and empty virus particles were obtained from the N1380 cell culture supernatants, and complete genome sequences were obtained from complete virus particles. Sequence analysis revealed that the isolated MRV, named TY-14, could be classified as MRV3 and had a close genetic relationship to an MRV2 isolate from a lion in a Japanese zoo (L2, L3, and M3 genes) and a human MRV2 isolate from Japan (S2 gene). Phylogenetic analysis showed that TY-14 clustered only with bat MRVs in the M1 phylogenetic tree but formed a cluster with several animal MRVs in the M2 and S3 phylogenetic trees and branched independently in the L1, S1, and S4 phylogenetic trees, suggesting a genetic relationship to viruses of unknown origin. Recombination events were identified in the M2 gene. These results suggest that TY-14 was generated by reassortment and recombination events involving MRVs circulating in Japan, viruses from bats, and other viruses of unknown origin.

Sapoviruses detected from acute gastroenteritis outbreaks and hospitalized children in Taiwan.

BACKGROUND/PURPOSE: Sapoviruses (SaVs) become important pathogens causing both sporadic and outbreaks of acute gastroenteritis (AGE) after rotavirus vaccination era worldwide. SaVs were included in AGE screening items when norovirus and rotavirus were negative in Taiwan CDC since 2008. However, no complete SaV genome sequence of any genotype detected in Taiwan was determined. This study aimed to investigate SaVs infection and complete genome sequences detected in Taiwan. METHODS: This prospective survey, SaVs samples with untyped or weak PCR result were selected for testing the new design qRT-PCR assay from AGE hospitalized children during 2008-2011, 2016-2017 and AGE outbreak in 2012-2014. Those were genetically characterized using long RT-PCR with different primer combinations as well as primer independent deep sequencing and with 5' RACE and 3' terminal region targeting RT-PCR. RESULTS: Overall, 14 SaV-AGE hospitalized children and 4 SaV-AGE outbreaks were enrolled in this study. In addition to the AGE symptoms, 6 children also showed URI symptoms (cough, pharyngitis, rhinorrhea and nasal congestion). The detected 19 SaVs were classified as eight genotypes (GI.1, GI.2, GI.3, GII.2, GII.3, GII.5, GII.8, and GIV.1) and the complete genome sequence of representative strain for each genotype were determined except GI.3. The GII.3 was the most major genotype following GI.1 and GIV.1. CONCLUSION: Our result confirmed that SaV is one of the pathogens detected from Taiwanese AGE patients. Multiple SaV genotype strains would associate with AGE as similar to those detected in different countries/areas. The whole genome of SaV strains detected including rarely reported GII.8 was firstly determined.

Human sapovirus propagation in human cell lines supplemented with bile acids.

Human sapoviruses (HuSaVs) cause acute gastroenteritis similar to human noroviruses. Although HuSaVs were discovered four decades ago, no HuSaV has been grown in vitro, which has significantly impeded the understanding of viral biology and the development of antiviral strategies. In this study, we identified two susceptible human cell lines, that originated from testis and duodenum, that support HuSaV replication and found that replication requires bile acids. HuSaVs replicated more efficiently in the duodenum cell line, and viral RNA levels increased up to ∼6 log10-fold. We also detected double-stranded RNA, viral nonstructural and structural proteins in the cell cultures, and intact HuSaV particles. We confirmed the infectivity of progeny viruses released into the cell culture supernatants by passaging. These results indicate the successful growth of HuSaVs in vitro. Additionally, we determined the minimum infectious dose and tested the sensitivities of HuSaV GI.1 and GII.3 to heat and ultraviolet treatments. This system is inexpensive, scalable, and reproducible in different laboratories, and can be used to investigate mechanisms of HuSaV replication and to evaluate antivirals and/or disinfection methods for HuSaVs.

Multiple genotypes of enterovirus G carrying a papain-like cysteine protease (PL-CP) sequence circulating on two pig farms in Japan: first identification of enterovirus G10 carrying a PL-CP sequence.

Two and three genotypes of enterovirus G (EV-G) carrying a papain-like cysteine protease (PL-CP) sequence were detected on two pig farms and classified into genotypes G1 and G10, and G1, G8, and G17, respectively, based on VP1 sequences. A G10 EV-G virus bearing a PL-CP sequence was detected for the first time. Phylogenetic analysis of the P2 and P3 regions grouped the viruses by farm with high sequence similarity. Furthermore, clear recombination break points were detected in the 2A region, suggesting that PL-CP EV-G-containing strains gained sequence diversity through recombination events among the multiple circulating EV-G genotypes on the farms.

Polymerase chain reaction primer sets for the detection of genetically diverse human sapoviruses.

Sapoviruses are increasingly being recognized as pathogens associated with gastroenteritis in humans. Human sapoviruses are currently assigned to 18 genotypes (GI.1-7, GII.1-8, GIV.1, and GV.1-2) based on the sequence of the region encoding the major structural protein. In this study, we evaluated 11 polymerase chain reaction (PCR) assays using published and newly designed/modified primers and showed that four PCR assays with different primer combinations amplified all of the tested human sapovirus genotypes using either synthetic DNA or cDNA prepared from human sapovirus-positive fecal specimens. These assays can be used as improved broadly reactive screening tests or as tools for molecular characterization of human sapoviruses.

Genetic diversity of enterovirus G detected in faecal samples of wild boars in Japan: identification of novel genotypes carrying a papain-like cysteine protease sequence.

The genetic diversity of enterovirus G (EV-G) was investigated in the wild-boar population in Japan. EV-G-specific reverse transcription PCR demonstrated 30 (37.5 %) positives out of 80 faecal samples. Of these, viral protein 1 (VP1) fragments of 20 samples were classified into G1 (3 samples), G4 (1 sample), G6 (2 samples), G8 (4 samples), G11 (1 sample), G12 (7 samples), G14 (1 sample) and G17 (1 sample), among which 11 samples had a papain-like cysteine protease (PL-CP) sequence, believed to be the first discoveries in G1 (2 samples) or G17 (1 sample) wild-boar EV-Gs, and in G8 (2 samples) or G12 (6 samples) EV-Gs from any animals. Sequences of the non-structural protein regions were similar among EV-Gs possessing the PL-CP sequence (PL-CP EV-Gs) regardless of genotype or origin, suggesting the existence of a common ancestor for these strains. Interestingly, for the two G8 and two G12 samples, the genome sequences contained two versions, with or without the PL-CP sequence, together with the homologous 2C/PL-CP and PL-CP/3A junction sequences, which may explain how the recombination and deletion of the PL-CP sequences occured in the PL-CP EV-G genomes. These findings shed light on the genetic plasticity and evolution of EV-G.

Porcine sapoviruses: Pathogenesis, epidemiology, genetic diversity, and diagnosis.

The first porcine Sapovirus (SaV) Cowden strain was discovered in 1980. To date, eight genogroups (GIII, V-IX) and three genogroups (GIII, GV, and GVI) of porcine SaVs have been detected from domestic pigs worldwide and wild boars in Japan, respectively based on the capsid sequences. Although GIII Cowden strain replicated in the villous epithelial cells and caused intestinal lesions in the proximal small intestines (mainly in duodenal and less in jejunum), leading to mild to severe diarrhea, in the orally inoculated neonatal gnotobiotic pigs, the significance of porcine SaVs in different ages of pigs with diarrhea in the field is still undetermined. This is due to two reasons: 1) similar prevalence of porcine SaVs was detected in diarrheic and non-diarrheic pigs; and 2) co-infection of porcine SaVs with other enteric pathogens is common in pigs. Diagnosis of porcine SaV infection is mainly based on the detection of viral nucleic acids using reverse transcription (RT)-PCR and sequencing. Much is unknown about these genetically diverse viruses to understand their role in pig health and to evaluate whether vaccines are needed to prevent SaV infection.

Complete genome sequencing and genetic analysis of a Japanese porcine torovirus strain detected in swine feces.

We sequenced the complete genome of a porcine torovirus (PToV) strain from Japan for the first time. Whole-genome analysis revealed that this strain (Iba/2018) has a mosaic sequence composed of at least three genome backgrounds, related to US, Chinese and German PToV strains. Clear recombination breakpoints were detected in the M and HE coding regions. A similarity plot and structural analysis demonstrated that the HE coding region exhibits the highest diversity, and the most sequence variation was found in the lectin domain. PToVs were divided into two lineages in the HE region, whereas clear lineages were not found in other regions.

First identification of Sapoviruses in wild boar.

Sapoviruses (SaVs) are enteric viruses that have been detected in human and animals previously; however, SaVs have not been identified in wild boar yet. Using a metagenomics approach, we identified SaVs in fecal samples of free-living wild boars in Japan for the first time. Six of the 48 specimens identified belonged to one genogroup (G)III, one GV and four GVI SaV sequence reads. We successfully determined complete genome of GV and GVI SaV strains using the long reverse transcription PCR strategy and the 5' rapid amplification of cDNA end method. Phylogenetic tree analysis and pairwise distance calculation revealed that GV SaV detected from wild boar was related to recently assigned GV.5 strains from pig, while GVI SaV was assigned to a new genotype within GVI. Moreover, wild boar may act as a reservoir for transmission of SaVs to the pig population (and vice versa) because GIII, GV, and GVI SaVs were all detected in pigs previously.