Genomic Evolution and Selective Pressure Analysis of a Novel Porcine Sapovirus in Shanghai, China.

Porcine sapovirus (PoSaV) is one of the most significant pathogens causing piglet diarrhea, and one with limited genetic characterization. In this study, the prevalence, infection pattern, and genetic evolution of porcine sapovirus were elucidated in detail. The positive rate of PoSaV was 10.1% (20/198), with dual, triple, and quadruple infections of 45%, 40%, and 5%, respectively. To further explore the viral composition in the PoSaV-positive diarrhea feces, metagenomic sequencing was carried out. The results confirmed that RNA viruses accounted for a higher proportion (55.47%), including the two primary viruses of PoSaV (21.78%) and porcine astrovirus (PAstV) (24.54%) in the tested diarrhea feces samples. Afterward, a full-length sequence of the PoSaV isolate was amplified and named SHCM/Mega2023, and also given the identifier of GenBank No. PP388958. Phylogenetic analysis identified the prevalent PoSaV strain SHCM/Mega2023 in the GIII genogroup, involving a recombinant event with MK962338 and KT922089, with the breakpoint at 2969-5132 nucleotides (nt). The time tree revealed that the GIII genogroup exhibits the widest divergence time span, indicating a high likelihood of viral recombination. Moreover, SHCM/Mega2023 had three nucleotide "RPL" insertions at the 151-153 nt site in the VP2 gene, compared to the other GIII strains. Further selective pressure calculations demonstrate that the whole genome of the SHCM/Mega2023 strain was under purifying selection (dN/dS < 1), with seven positively selected sites in the VP1 protein, which might be related to antigenicity. In conclusion, this study presents a novel genomic evolution of PoSaV, offering valuable insights into antigenicity and for vaccine research.

Porcine Sapovirus in Northern Vietnam: Genetic Detection and Characterization Reveals Co-Circulation of Multiple Genotypes.

Porcine sapovirus (PoSaV) has been reported in many countries over the world, which may cause gastroenteritis symptoms in pigs with all ages. There has been no report on PoSaV infection in Vietnam up to now. In this study, a total of 102 samples were collected from piglets, fattening pigs, and sows with diarrhea in several cities and provinces in northern Vietnam. The PoSaV genome was examined using polymerase chain reaction (PCR). Sequencing of the partial RNA-dependent RNA polymerase (RdRp) gene sequences (324 bp) was performed. Of the 102 tested samples, 10 (9.8%) and 7/20 (35%) were detected as positive for the PoSaV RdRp gene using the PCR method at the individual and farm levels, respectively. Genetic analysis of the partial RdRp gene region of about 324 bp indicated that the nucleotide identity of the current 10 Vietnamese viral strains ranged from 61.39% to 100%. Among the 10 strains obtained, 8 belonged to genotype III and the remaining 2 strains were clustered in genotype VIII. The Vietnamese genotype III viruses formed two sub-clusters. The Vietnamese PoSaV strains were closely related to PoSaVs reported in South Korea, Venezuela, and the Netherlands. This research was the first to describe PoSaV infection in northern Vietnam during 2022-2023.

High Genetic Diversity of Porcine Sapovirus From Diarrheic Piglets in Yunnan Province, China.

As one of the most important enteric viruses, sapovirus (SaV) can infect humans and a variety of animals. Until now, 19 SaV genogroups have been identified, among which 4 from human (GI, GII, GIV, and GV) and 8 from swine (GIII, GV-GXI). Porcine sapovirus (PoSaV) GIII has been prevalent in China; however, the status of PoSaV infection in Yunnan province remains unknown. In this study, 202 fecal samples were collected from piglets associated with outbreaks of acute diarrhea in Yunnan between January and May 2020. PoSaV detection revealed that the total PoSaV infection rate in Yunnan was 35.2%, with 21 PoSaV strains determined and phylogenetically analyzed. The phylogenetic tree analyses demonstrated that twenty PoSaV strains belonged to GIII and fell into five genotypes, whereas one PoSaV strain (YNQB) belonged to GV. Sequence alignments revealed deletions in VP2 region in 10 of the 20 GIII strains, as well as deletions and insertions in VP1 region of the GV strain (YNQB). Furthermore, genomic recombination analyses showed that two GIII strains (YNAN and YNJD) were recombinants, closely related to reference sequences MK965898 and LC215880, MK965898 and FJ387164, respectively. In summary, PoSaV-GIII strains were identified in Yunnan in 2020, and for the first time, a PoSaV-GV strain was identified from China, whereas the comprehensive analyses illustrated high genetic diversity of Yunnan PoSaV strains. This study may shed new light on the current PoSaV infections in Yunnan and pave the way toward further control of the PoSaV infections in China.

Genetic characterization of porcine sapoviruses identified from pigs during a diarrhoea outbreak in Iowa, 2019.

Porcine sapovirus (SaV) was first identified by electron microscopy in the United States in 1980 and has since been reported from both asymptomatic and diarrhoeic pigs usually in mixed infection with other enteric pathogens. SaV as the sole aetiological agent of diarrhoea in naturally infected pigs has not previously been reported in the United States. Here, we used four independent lines of evidence including metagenomics analysis, real-time RT-PCR (rRT-PCR), histopathology, and in situ hybridization to confirm porcine SaV genogroup III (GIII) as the sole cause of enteritis and diarrhoea in pigs. A highly sensitive and specific rRT-PCR was established to detect porcine SaV GIII. Examination of 184 faecal samples from an outbreak of diarrhoea on a pig farm showed that pigs with clinical diarrhoea had significantly lower Ct values (15.9 ± 0.59) compared to clinically unaffected pigs (35.8 ± 0.71). Further survey of 336 faecal samples from different states in the United States demonstrated that samples from pigs with clinical diarrhoea had a comparable positive rate (45.3%) with those from asymptomatic pigs (43.1%). However, the SaV-positive pigs with clinical diarrhoea had significantly higher viral loads (Ct  = 26.0 ± 0.5) than the SAV-positive but clinically healthy pigs (Ct  = 33.2 ± 0.9). Phylogenetic analysis of 20 field SaVs revealed that all belonged to SaV GIII and recombination analysis indicated that intragenogroup recombination had occurred within the field isolates of SaV GIII. These results suggest that porcine SaV GIII plays an important aetiologic role in swine enteritis and diarrhoea and rRT-PCR is a reliable method to detect porcine SaV. Our findings provide significant insights to better understand the epidemiology and pathogenicity of porcine SaV infection.

Genetically divergent porcine sapovirus identified in pigs, United States.

Porcine sapoviruses (SaVs) are genetically diverse and widely distributed in pig-producing countries. Eight genogroups of porcine SaV have been identified, and genogroup III is the predominant type. Most of the eight genogroups of porcine SaV are circulating in the United States. In the present study, we report detection of porcine SaVs in pigs at different ages with clinical diarrhoea using next-generation sequencing and genetic characterization. All seven cases have porcine SaV GIII strains detected and one pooled case was found to have a porcine SaV GVI strain IA27912-B-2018. Sequence analysis showed that seven GIII isolates were genetically divergent and formed four different lineages on the trees of complete genome, RdRP, VP1 and VP2. In addition, these seven GIII isolates had three different deletion/insertion patterns in an identified variable region close to the 3' end of VP2. The GVI strain IA27912-B-2018 was closely related to strains previously detected in the United States and Japan. A 3-nt deletion in VP1 region of GVI IA27912-B-2018 was identified. Our study showed that genetically divergent SaVs of different genogroups are co-circulating in pigs in the United States. Future studies comparing the virulence of these different genogroups in pigs are needed to better understand this virus and to determine if surveillance and vaccine development are needed to monitor and control porcine SaVs.

One-step triplex reverse-transcription PCR detection of porcine epidemic diarrhea virus, porcine sapelovirus, and porcine sapovirus.

Swine diarrhea can be caused by multiple agents, including porcine epidemic diarrhea virus (PEDV), porcine sapelovirus (PSV), and porcine sapovirus (SaV). We designed a one-step triplex reverse-transcription PCR (RT-PCR) detection method including 3 pairs of primers that focused on the S1 gene of PEDV, a conserved gene of PSV, and the VP1 gene of SaV. The optimal concentrations of upstream and downstream primers in the triplex RT-PCR were 0.24 µM for PEDV, 0.15 µM for PSV, and 0.2 µM for SaV, and the optimal annealing temperature was 55.5°C. Triplex RT-PCR assessment of 402 piglet diarrhea samples was compared with conventional individual RT-PCR. Concordance rates in both tests for individual viruses were 100%, 97.6%, and 94.4% for PEDV, PSV, and SaV, respectively. PEDV, PSV, and SaV were detected in 57.2%, 10.4%, and 9.0% of the samples, respectively. The high sensitivity and specificity of this triplex RT-PCR-based detection method for PEDV, PSV, and SaV could allow rapid detection and analysis of mixed infections by these 3 viruses.

Molecular detection of enteric viruses and the genetic characterization of porcine astroviruses and sapoviruses in domestic pigs from Slovakian farms.

BACKGROUND: Surveillance and characterization of pig enteric viruses such as transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), rotavirus, astrovirus (PAstV), sapovirus (PSaV), kobuvirus and other agents is essential to evaluate the risks to animal health and determination of economic impacts on pig farming. This study reports the detection and genetic characterization of PAstV, PSaV in healthy and diarrheic domestic pigs and PEDV and TGEV in diarrheic pigs of different age groups. RESULTS: The presence of PAstV and PSaV was studied in 411 rectal swabs collected from healthy (n = 251) and diarrheic (n = 160) pigs of different age categories: suckling (n = 143), weaned (n = 147) and fattening (n = 121) animals on farms in Slovakia. The presence of TGEV and PEDV was investigated in the diarrheic pigs (n = 160). A high presence of PAstV infections was detected in both healthy (94.4%) and diarrheic (91.3%) pigs. PSaV was detected less often, but also equally in clinically healthy (8.4%) and diarrheic (10%) pigs. Neither TGEV nor PEDV was detected in any diarrheic sample. The phylogenetic analysis of a part of the RdRp region revealed the presence of all five lineages of PAstV in Slovakia (PAstV-1 - PAstV-5), with the most frequent lineages being PAstV-2 and PAstV-4. Analysis of partial capsid genome sequences of the PSaVs indicated that virus strains belonged to genogroup GIII. Most of the PSaV sequences from Slovakia clustered with sequences originating from neighbouring countries. CONCLUSIONS: Due to no significant difference between healthy and diarrheic pigs testing of the presence of PAstV and PSaV provides no diagnostic value. Genetic diversity of PAstV was very high as all five lineages were identified in pig farms in Slovakia. PSaV strains were genetically related to the strains circulating in Central European region.

Tissue Distribution and Visualization of Internalized Human Norovirus in Leafy Greens.

Lettuce has been implicated in human norovirus (HuNoV) outbreaks. The virus is stable on the leaf surface for at least 2 weeks; however, the dynamics of virus internalization have not been fully investigated. The purpose of this study was to assess the internalization and distribution of HuNoV and two surrogate viruses, porcine sapovirus (SaV) and Tulane virus (TV), in lettuce and spinach. Viral inoculations through the roots of seedlings and the petiole of leaves from mature plants were performed, and the viruses were tracked on days 1 and 6 post-root inoculation and at 16 h and 72 h post-petiole inoculation. Confocal microscopy was used to visualize root-internalized HuNoV. In both lettuce and spinach, (i) HuNoV was internalized into the roots and leaves at similar RNA titers, whereas surrogate viruses were more restricted to the roots, (ii) all three viruses were stable inside the roots and leaves for at least 6 days, and (iii) HuNoV disseminated similarly inside the central veins and leaf lamina, whereas surrogate viruses were more restricted to the central veins. Infectious TV, but not SaV, was detectable in all tissues, suggesting that TV has greater stability than SaV. HuNoV was visualized inside the roots' vascular bundle and the leaf mesophyll of both plants. In conclusion, using surrogate viruses may underestimate the level of HuNoV internalization into edible leaves. The internalization of HuNoV through roots and cut leaves and the dissemination into various spinach and lettuce tissues raise concerns of internal contamination through irrigation and/or wash water.IMPORTANCE Human noroviruses are the leading cause of foodborne outbreaks, with lettuce being implicated in the majority of outbreaks. The virus causes acute gastroenteritis in all age groups, with more severe symptoms in children, the elderly, and immunocompromised patients, contributing to over 200,000 deaths worldwide annually. The majority of deaths due to HuNoV occur in the developing world, where limited sanitation exists along with poor wastewater treatment facilities, resulting in the contamination of water resources that are often used for irrigation. Our study confirms the ability of lettuce and spinach to internalize HuNoV from contaminated water through the roots into the edible leaves. Since these leafy greens are consumed with minimal processing that targets only surface pathogens, the internalized HuNoV presents an added risk to consumers. Thus, preventive measures should be in place to limit the contamination of irrigation water. In addition, better processing technologies are needed to inactivate internalized viral pathogens.

Short communication: Immune responses in sows induced by porcine sapovirus virus-like particles reduce viral shedding in suckled piglets.

Porcine sapovirus (PoSaV) is a potential threat to public health owing to its capacity for reassortment with human sapovirus strains. However, there is still no vaccine available for the prevention and control of this infectious disease. In this study, we developed PoSaV virus-like particles (VLPs) using a baculovirus expression system. Immunization with PoSaV VLPs induced high titers of serum antibody specific for VP1 in sows. The results of our challenge study demonstrated that maternally-derived antibodies (MDA) induced by VLP immunization dramatically reduced viral shedding of PoSaV in the feces of next generation piglets. Therefore, the results of this study indicate that the immune responses of sows elicited by PoSaV VLPs can inhibit in vivo viral replication in their offspring and represent a promising strategy for developing vaccines against PoSaV.

Genomic organization and recombination analysis of a porcine sapovirus identified from a piglet with diarrhea in China.

BACKGROUND: Sapovirus (SaV), a member of the family Caliciviridae, is an etiologic agent of gastroenteritis in humans and pigs. To date, both intra- and inter-genogroup recombinant strains have been reported in many countries except for China. Here, we report an intra-genogroup recombination of porcine SaV identified from a piglet with diarrhea of China. METHODS: A fecal sample from a 15-day-old piglet with diarrhea was collected from Shanghai, China. Common agents of gastroenteritis including porcine circovirus type 2, porcine rotavirus, porcine transmissible gastroenteritis virus, porcine SaV, porcine norovirus, and porcine epidemic diarrhea virus were detected by RT-PCR or PCR method. The complete genome of porcine SaV was then determined by RT-PCR method. Phylogenetic analyses based on the structural region and nonstructural (NS) region were carried out to group this SaV strain, and it was divided into different genotypes based on these two regions. Recombination analysis based on the genomic sequence was further performed to confirm this recombinant event and locate the breakpoint. RESULTS: All of the agents showed negative results except for SaV. Analysis of the complete genome sequence showed that this strain was 7387 nt long with two ORFs and belonged to SaV GIII. Phylogenetic analyses of the structural region (complete VP1 nucleotide sequences) grouped this strain into GIII-3, whereas of the nonstructural region (RdRp nucleotide sequences) grouped this strain into GIII-2. Recombination analysis based on the genomic sequence confirmed this recombinant event and identified two parental strains that were JJ259 (KT922089, GIII-2) and CH430 (KF204570, GIII-3). The breakpoint located at position 5139 nt of the genome (RdRp-capsid junction region). Etiologic analysis showed the fecal sample was negative with the common agents of gastroenteritis, except for porcine SaV, which suggested that this recombinant strain might lead to this piglet diarrhea. CONCLUSIONS: P2 strain was an intra-genogroup recombinant porcine SaV. To the best of our knowledge, this study would be the first report that intra-genogroup recombination of porcine SaV infection was identified in pig herd in China.

Serological and molecular investigation of porcine sapovirus infection in piglets in Xinjiang, China.

Porcine sapovirus (PoSaV) is one of the important pathogens that cause acute gastroenteritis in piglets. A survey on the infection and epidemic status of PoSaV in Xinjiang Province, Northwest China, was conducted in this study. We applied indirect viral protein 1 (VP1)-ELISA method to detect specific antibodies in 1218 serum samples of 3-month-old piglets collected from eight regions in Xinjiang during 2013-2014 and also detected PoSaV in 146 diarrhea stools of piglets in these eight regions using RT-PCR technology. The results showed that the PoSaV-serological positive rates in piglets in eight different regions in Xinjiang were between 32.82 and 47.06% with a mean rate of 37.68%. The average positive rate of PCR in stools of piglets was 3.42%. Sequencing and comparative analysis of five PCR-amplified DNA fragments revealed that four epidemic strains of PoSaV (swine/XJ-KO1, swine/XJ-AK2, swine/XJ-KS1, and swine/XJ-SHZ1) shared high nucleotide and amino acid identities with Cowden strain, while strain swine/XJ-AK1 shared higher high identities with Po/OH-JJ681/2000/US isolate. Phylogenetic clustering further verified that the epidemic strains of PoSaVs, i.e., swine/XJ-KO1, swine/XJ-AK2, swine/XJ-KS1, and swine/XJ-SHZ1, belong to genogroup (GIII) while swine/XJ-AK1 belongs to GVI. This survey confirmed for the first time that PoSaV infection was common in piglets in Xinjiang, China, and that the epidemic strains exist at least in both GIII and GVI clusters. This study provided the useful epidemiological data for scientific control and prevention of this disease.

Solution structure of the porcine sapovirus VPg core reveals a stable three-helical bundle with a conserved surface patch.

Viral protein genome-linked (VPg) proteins play a critical role in the life cycle of vertebrate and plant positive-sense RNA viruses by acting as a protein primer for genome replication and as a protein cap for translation initiation. Here we report the solution structure of the porcine sapovirus VPg core (VPg(C)) determined by multi-dimensional NMR spectroscopy. The structure of VPg(C) is composed of three α-helices stabilized by several conserved hydrophobic residues that form a helical bundle core similar to that of feline calicivirus VPg. The putative nucleotide acceptor Tyr956 within the first helix of the core is completely exposed to solvent accessible surface to facilitate nucleotidylation by viral RNA polymerase. Comparison of VPg structures suggests that the surface for nucleotidylation site is highly conserved among the Caliciviridae family, whereas the backbone core structures are different. These structural features suggest that caliciviruses share common mechanisms of VPg-dependent viral replication and translation.

Emerging and re-emerging swine viruses.

In the past two decades or so, a number of viruses have emerged in the global swine population. Some, such as porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2), cause economically important diseases in pigs, whereas others such as porcine torque teno virus (TTV), now known as Torque teno sus virus (TTSuV), porcine bocavirus (PBoV) and related novel parvoviruses, porcine kobuvirus, porcine toroviruses (PToV) and porcine lymphotropic herpesviruses (PLHV), are mostly subclinical in swine herds. Although some emerging swine viruses such as swine hepatitis E virus (swine HEV), porcine endogenous retrovirus (PERV) and porcine sapovirus (porcine SaV) may have a limited clinical implication in swine health, they do pose a potential public health concern in humans due to zoonotic (swine HEV) or potential zoonotic (porcine SaV) and xenozoonotic (PERV, PLHV) risks. Other emerging viruses such as Nipah virus, Bungowannah virus and Menangle virus not only cause diseases in pigs but some also pose important zoonotic threat to humans. This article focuses on emerging and re-emerging swine viruses that have a limited or uncertain clinical and economic impact on pig health. The transmission, epidemiology and pathogenic potential of these viruses are discussed. In addition, the two economically important emerging viruses, PRRSV and PCV2, are also briefly discussed to identify important knowledge gaps.