Epidemiology of Astrovirus, Norovirus and Sapovirus in Greek pig farms indicates high prevalence of Mamastrovirus suggesting the potential need for systematic surveillance

Backround Astrovirus, Norovirus and Sapovirus exhibit a wide distribution in swine pig herds worldwide. However, the association of porcine Astrovirus (PAstV), porcine Norovirus (PoNoV) and porcine Sapovirus (PoSaV) with disease in pigs remains uncertain. In this study, we investigated the prevalence of PAstV, PoNoV and PoSaV in Greek pig farms using both conventional RT-PCR and SYBR-Green Real-time RT-PCR in an effort to compare the sensitivity of the two methods. We examined 1400 stool samples of asymptomatic pigs originating from 28 swine farms throughout Greece in pools of five. Results PAstV was detected in all 28 swine farms examined, with an overall prevalence of 267/280 positive pools (95.4%). Porcine Caliciviruses prevalence was found at 36 and 57 out of the 280 examined samples, by the conventional and SYBR-Green Real time RT-PCR, respectively. Sequencing and phylogenetic analysis of the positive samples revealed that the detected PAstV sequences are clustered within PAstV1, 3 and 4 lineages, with PAstV3 being the predominant haplotype (91.2%). Interestingly, sequencing of the Calicivirus positive samples demonstrated the presence of non-target viruses, i.e. Sapovirus, Kobuvirus and Sapelovirus sequences and one sequence highly similar to bat Astrovirus, while no Norovirus sequence was detected. Conclusions The high prevalence of PAstV in Greek pig farms poses a necessity for further investigation of the pathogenicity of this virus and its inclusion in surveillance programs in case that it proves to be important. To our knowledge, this is the first epidemiological study of these viruses in pig farms in Greece.

Dynamics of the Enteric Virome in a Swine Herd Affected by Non-PCV2/PRRSV Postweaning Wasting Syndrome

A commercial pig farm with no history of porcine circovirus 2 (PCV2) or porcine reproductive and respiratory syndrome virus (PRRSV) repeatedly reported a significant reduction in body weight gain and wasting symptoms in approximately 20–30% of the pigs in the period between three and six weeks after weaning. As standard clinical interventions failed to tackle symptomatology, viral metagenomics were used to describe and monitor the enteric virome at birth, 3 weeks, 4 weeks, 6 weeks, and 9 weeks of age. The latter four sampling points were 7 days, 3 weeks, and 6 weeks post weaning, respectively. Fourteen distinct enteric viruses were identified within the herd, which all have previously been linked to enteric diseases. Here we show that wasting is associated with alterations in the enteric virome of the pigs, characterized by: (1) the presence of enterovirus G at 3 weeks of age, followed by a higher prevalence of the virus in wasting pigs at 6 weeks after weaning; (2) rotaviruses at 3 weeks of age; and (3) porcine sapovirus one week after weaning. However, the data do not provide a causal link between specific viral infections and the postweaning clinical problems on the farm. Together, our results offer evidence that disturbances in the enteric virome at the preweaning stage and early after weaning have a determining role in the development of intestinal barrier dysfunctions and nutrient uptake in the postweaning growth phase. Moreover, we show that the enteric viral load sharply increases in the week after weaning in both healthy and wasting pigs. This study is also the first to report the dynamics and co-infection of porcine rotavirus species and porcine astrovirus genetic lineages during the first 9 weeks of the life of domestic pigs.

Molecular Detection and Characterization of Porcine Epidemic Diarrhea Virus and Porcine Aichivirus C Coinfection in México

Swine enteric viral infections are responsible for substantial economic losses in the pork industry worldwide. Porcine epidemic diarrhea (PEDV) is one of the main causative agents of diarrhea in lactating pigs, and reports of PEDV coinfection with other enteric viruses highlight the importance of viral interactions for disease presentation and outcomes. Using next-generation sequencing (NGS) and sequence analyses from samples taken from piglets with acute diarrhea, we explored the possible interactions between PEDV and other less reported pathogens. PEDV coinfection with porcine kobuvirus (PKV) was detected in 36.4% (27/74) of samples. Full genomes from porcine coronavirus and kobuvirus were obtained, as was a partial porcine sapovirus genome (PSaV). The phylogenetic results show the clustering of these strains corresponding to the geographical relationship. To our knowledge, this is the first full genome and isolation report for porcine kobuvirus in México, as well as the first phylogenetic analysis for porcine sapovirus in the country. The NGS approach provides a better perspective of circulating viruses and other pathogens in affected production units.

Porcine Sapovirus-Induced Tight Junction Dissociation via Activation of RhoA/ROCK/MLC Signaling Pathway

Tight junctions (TJs) are a major barrier and also an important portal of entry for different pathogens. Porcine sapovirus (PSaV) induces early disruption of the TJ integrity of polarized LLC-PK cells, allowing it to bind to the buried occludin co-receptors hidden beneath the TJs on the basolateral surface. However, the signaling pathways involved in the PSaV-induced TJ dissociation are not yet known. Here, we found that the RhoA/ROCK/MLC signaling pathway was activated in polarized LLC-PK cells during the early infection of PSaV Cowden strain in the presence of bile acid. Specific inhibitors of RhoA, ROCK, and MLC restored PSaV-induced reduction of transepithelial resistance, increase of paracellular flux, intracellular translocation of occludin, and lateral membrane lipid diffusion. Moreover, each inhibitor significantly reduced PSaV replication, as evidenced by a reduction in viral protein synthesis, genome copy number, and progeny viruses. The PKC/MLCK and RhoA/ROCK/MYPT signaling pathways, known to dissociate TJs, were not activated during early PSaV infection. Among the above signaling pathways, the RhoA/ROCK/MLC signaling pathway was only activated by PSaV in the absence of bile acid, and specific inhibitors of this signaling pathway restored early TJ dissociation. Our findings demonstrate that PSaV binding to cell surface receptors activates the RhoA/ROCK/MLC signaling pathway, which in turn disrupts TJ integrity via the contraction of the actomyosin ring. Our study contributes to understanding how PSaV enters the cells and will aid in developing efficient and affordable therapies against PSaV and other calicivirus infections. IMPORTANCE Porcine sapovirus (PSaV), one of the most important enteric pathogens, is known to disrupt tight junction (TJ) integrity to expose its buried co-receptor occludin in polarized LLC-PK cells. However, the cellular signaling pathways that facilitate TJ dissociation are not yet completely understood. Here, we demonstrate that early infection of PSaV in polarized LLC-PK cells in either the presence or absence of bile acids activates the RhoA/ROCK/MLC signaling pathway, whose inhibitors reverse the early PSaV infection-induced early dissociation of TJs and reduce PSaV replication. However, early PSaV infection did not activate the PKC/MLCK and RhoA/ROCK/MYPT signaling pathways, which are also known to dissociate TJs. This study provides a better understanding of the mechanism involved in early PSaV infection-induced disruption of TJs, which is important for controlling or preventing PSaV and other calicivirus infections.

Detection and genetic characterization of porcine sapovirus from pigs with diarrhea

Porcine Sapovirus (SaV) was first identified by electron microscopy in the United States in 1980 and has since been reported from both asymptomatic and diarrheic pigs usually in mixed infection with other enteric pathogens. SaV as the sole etiological agent of diarrhea 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 diarrhea in pigs. A highly sensitive and specific rRT-PCR was established to detect porcine SaV GIII. Examination of 184 fecal samples from the outbreak farm showed that pigs with clinical diarrhea had significantly lower Ct values (15.9 ± 0.59) compared to clinically unaffected pigs (35.8 ± 0.71). Further survey of 336 fecal samples from different states in the United States demonstrated that samples from pigs with clinical diarrhea had a comparable positive rate (45.3%) with those from non-clinical pigs (43.1%). However, the SaV-positive pigs with clinical diarrhea had significantly higher viral loads (Ct = 26.0 ± 0.5) than those 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 intra-genogroup recombination occurred within the field isolates of SaV GIII. These results suggest that porcine SaV GIII plays an important etiologic role in swine enteritis and diarrhea 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.

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.

Early Porcine Sapovirus Infection Disrupts Tight Junctions and Uses Occludin as a Coreceptor

ABSTRACT The genus Sapovirus belongs to the family Caliciviridae, and its members are common causative agents of severe acute gastroenteritis in both humans and animals. Some caliciviruses are known to use either terminal sialic acids or histo-blood group antigens as attachment factors and/or cell surface proteins, such as CD300lf, CD300ld, and junctional adhesion molecule 1 of tight junctions (TJs), as receptors. However, the roles of TJs and their proteins in sapovirus entry have not been examined. In this study, we found that porcine sapovirus (PSaV) significantly decreased transepithelial electrical resistance and increased paracellular permeability early in infection of LLC-PK cells, suggesting that PSaV dissociates TJs of cells. This led to the interaction between PSaV particles and occludin, which traveled in a complex into late endosomes via Rab5- and Rab7-dependent trafficking. Inhibition of occludin using small interfering RNA (siRNA), a specific antibody, or a dominant-negative mutant significantly blocked the entry of PSaV. Transient expression of occludin in nonpermissive Chinese hamster ovary (CHO) cells conferred susceptibility to PSaV, but only for a limited time. Although claudin-1, another TJ protein, neither directly interacted nor was internalized with PSaV particles, it facilitated PSaV entry and replication in the LLC-PK cells. We conclude that PSaV particles enter LLC-PK cells by binding to occludin as a coreceptor in PSaV-dissociated TJs. PSaV and occludin then form a complex that moves to late endosomes via Rab5- and Rab7-dependent trafficking. In addition, claudin-1 in the TJs opened by PSaV infection facilitates PSaV entry and infection as an entry factor. IMPORTANCE Sapoviruses (SaVs) cause severe acute gastroenteritis in humans and animals. Although they replicate in intestinal epithelial cells, which are tightly sealed by apical-junctional complexes, such as tight junctions (TJs), the mechanisms by which SaVs hijack TJs and their proteins for successful entry and infection remain largely unknown. Here, we demonstrate that porcine SaVs (PSaVs) induce early dissociation of TJs, allowing them to bind to the TJ protein occludin as a functional coreceptor. PSaVs then travel in a complex with occludin into late endosomes through Rab5- and Rab7-dependent trafficking. Claudin-1, another TJ protein, does not directly interact with PSaV but facilitates the entry of PSaV into cells as an entry factor. This work contributes to our understanding of the entry of SaV and other caliciviruses into cells and may aid in the development of efficient and affordable drugs to treat SaV infections.

Phosphatidylinositol 3-Kinase/Akt and MEK/ERK Signaling Pathways Facilitate Sapovirus Trafficking and Late Endosomal Acidification for Viral Uncoating in LLC-PK Cells

ABSTRACTSapovirus, an important cause of acute gastroenteritis in humans and animals, travels from the early to the late endosomes and requires late endosomal acidification for viral uncoating. However, the signaling pathways responsible for these viral entry processes remain unknown. Here we demonstrate the receptor-mediated early activation of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein extracellular signal-regulated kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways involved in sapovirus entry processes. Both signaling pathways were activated during the early stage of porcine sapovirus (PSaV) infection. However, depletion of the cell surface carbohydrate receptors by pretreatment with sodium periodate or neuraminidase reduced the PSaV-induced early activation of these signaling pathways, indicating that PSaV binding to the cell surface carbohydrate receptors triggered these cascades. Addition of bile acid, known to be essential for PSaV escape from late endosomes, was also found to exert a stiffening effect to stimulate both pathways. Inhibition of these signaling pathways by use of inhibitors specific for PI3K or MEK or small interfering RNAs (siRNAs) against PI3K or MEK resulted in entrapment of PSaV particles in early endosomes and prevented their trafficking to late endosomes. Moreover, phosphorylated PI3K and ERK coimmunoprecipitated subunit E of the V-ATPase proton pump that is important for endosomal acidification. Based on our data, we conclude that receptor binding of PSaV activates both PI3K/Akt and MEK/ERK signaling pathways, which in turn promote PSaV trafficking from early to late endosomes and acidification of late endosomes for PSaV uncoating. These signaling cascades may provide a target for potent therapeutics against infections by PSaV and other caliciviruses.IMPORTANCESapoviruses cause acute gastroenteritis in both humans and animals. However, the host signaling pathway(s) that facilitates host cell entry by sapoviruses remains largely unknown. Here we demonstrate that porcine sapovirus (PSaV) activates both PI3K/Akt and MEK/ERK cascades at an early stage of infection. Removal of cell surface receptors decreased PSaV-induced early activation of both cascades. Moreover, blocking of PI3K/Akt and MEK/ERK cascades entrapped PSaV particles in early endosomes and prevented their trafficking to the late endosomes. PSaV-induced early activation of PI3K and ERK molecules further mediated V-ATPase-dependent late endosomal acidification for PSaV uncoating. This work unravels a new mechanism by which receptor-mediated early activation of both cascades may facilitate PSaV trafficking from early to late endosomes and late endosomal acidification for PSaV uncoating, which in turn can be a new target for treatment of sapovirus infection.