Porcine rotavirus C in pigs with gastroenteritis on Thai swine farms, 2011–2016

Swine are economically important food animals, but highly contagious porcine epidemic diarrhea virus (PEDV) and rotavirus can afflict pig herds and contribute significantly to piglet morbidity and mortality. While there have been studies on rotavirus group A (RVA) in Thailand, reports of rotavirus group C (RVC) are limited. Here, we aimed to identify the prevalence of RVC circulating on Thai commercial swine farms. We analyzed 769 feces and intestine mucosal contents of pigs affected with diarrhea between 2011 and 2016 using RT-PCR specific for the PEDV spike (S), rotavirus glycoprotein (G) VP7, and protease-sensitive protein (P) VP4 genes. We found that 6.6% (51/769) of samples tested positive for RVC, of which 11 samples were co-infected with RVA and four samples were co-infected with PEDV. Three samples tested positive for all three viruses. Phylogenetic analysis of the VP7 gene showed that the most frequent RVC genotype was G1, which grouped with the prototypic RVC Cowden strain. While G6 and G9 were also common, G3 was relatively rare. Analysis of the VP4 gene revealed that the most common P type was P[5], followed by P[4], P[7], and P[1]. In all, there were six G/P combinations (G6P[5], G1P[1], G1P[4], G1P[5], G9P[4], and G9P[7]), of which G6P[5] was the most predominant.

Molecular characterization of porcine rotavirus C in pigs with gastroenteritis in Thailand, 2011 – 2016

Swine are economically important food animals, but highly contagious enteric viruses can affect entire swine herds and contribute significantly to piglet morbidity and mortality. The most frequent viruses associated with pig gastroenteritis have been reported as porcine epidemic diarrhea virus (PEDV) and rotavirus. Rotavirus is an important cause of diarrhea in piglets and pigs worldwide, and group A and C types are those that pig herds are mostly affected by. In Thailand, studies on rotavirus group A (RVA) have been reported continuously, whereas information on group C is still limited. In this study, we aimed to identify rotavirus group C (RVC) from the feces and intestinal contents of pigs affected with diarrhea. Seven hundred and sixty-nine samples were collected from swine herds located in difference provinces throughout Thailand. The specimens were tested using virus-specific RT-PCR to detect the gene encoding RVC capsid protein VP7 and VP4. Sequencing analyses showed that 6.6% (51/769) of samples were positive for RVC, one third of which tested as single positive for RVC (34/51). Co-infections with the most frequent enteric viruses, RVA and PEDV were also analyzed. Co-infections of RVA/RVC accounted for 21.6% (11/51) of samples and of PEDV/RVC for 7.8% (4/51) of samples, while three samples (5.9%) tested positive for all three viruses. Infections were not associated with seasonality, since the virus was detected throughout the year. RVC was detected in pigs up to 8 weeks old. Analysis of the partial VP7 gene sequences was suggestive that the predominant genotype was G1, which was closely related to the prototype Cowden strain. Due to P[5] was the most prevalent of VP4 genotype. This study demonstrated the low prevalence of RVC in Thailand, a virus not previously documented in this country.

Molecular characterization of porcine rotavirus C in pigs with gastroenteritis in Thailand, 2011 – 2016

Swine are economically important food animals, but highly contagious enteric viruses can affect entire swine herds and contribute significantly to piglet morbidity and mortality. The most frequent viruses associated with pig gastroenteritis have been reported as porcine epidemic diarrhea virus (PEDV) and rotavirus. Rotavirus is an important cause of diarrhea in piglets and pigs worldwide, and group A and C types are those that pig herds are mostly affected by. In Thailand, studies on rotavirus group A (RVA) have been reported continuously, whereas information on group C is still limited. In this study, we aimed to identify rotavirus group C (RVC) from the feces and intestinal contents of pigs affected with diarrhea. Seven hundred and sixty-nine samples were collected from swine herds located in difference provinces throughout Thailand. The specimens were tested using virus-specific RT-PCR to detect the gene encoding RVC capsid protein VP7 and VP4. Sequencing analyses showed that 6.6% (51/769) of samples were positive for RVC, one third of which tested as single positive for RVC (34/51). Co-infections with the most frequent enteric viruses, RVA and PEDV were also analyzed. Co-infections of RVA/RVC accounted for 21.6% (11/51) of samples and of PEDV/RVC for 7.8% (4/51) of samples, while three samples (5.9%) tested positive for all three viruses. Infections were not associated with seasonality, since the virus was detected throughout the year. RVC was detected in pigs up to 8 weeks old. Analysis of the partial VP7 gene sequences was suggestive that the predominant genotype was G1, which was closely related to the prototype Cowden strain. Due to P[5] was the most prevalent of VP4 genotype. This study demonstrated the low prevalence of RVC in Thailand, a virus not previously documented in this country.

Mechanism of Cell Culture Adaptation of an Enteric Calicivirus, the Porcine Sapovirus Cowden Strain

ABSTRACTThe porcine sapovirus (SaV) (PoSaV) Cowden strain is one of only a few culturable enteric caliciviruses. Compared to the wild-type (WT) PoSaV Cowden strain, tissue culture-adapted (TC) PoSaV has two conserved amino acid substitutions in the RNA-dependent RNA polymerase (RdRp) and six in the capsid protein (VP1). By using the reverse-genetics system, we identified that 4 amino acid substitutions in VP1 (residues 178, 289, 324, and 328), but not the substitutions in the RdRp region, were critical for the cell culture adaptation of the PoSaV Cowden strain. The other two substitutions in VP1 (residues 291 and 295) reduced virus replicationin vitro. Three-dimensional (3D) structural analysis of VP1 showed that residue 178 was located near the dimer-dimer interface, which may affect VP1 assembly and oligomerization; residues 289, 291, 324, and 328 were located at protruding subdomain 2 (P2) of VP1, which may influence virus binding to cellular receptors; and residue 295 was located at the interface of two monomeric VP1 proteins, which may influence VP1 dimerization. Although reversion of the mutation at residue 291 or 295 from that of the TC strain to that of the WT reduced virus replicationin vitro, it enhanced virus replicationin vivo, and the revertants induced higher-level serum and mucosal antibody responses than those induced by the TC PoSaV Cowden strain. Our findings reveal the molecular basis for PoSaV adaptation to cell culture. These findings may provide new, critical information for the cell culture adaptation of other PoSaV strains and human SaVs or noroviruses.IMPORTANCEThe tissue culture-adapted porcine sapovirus Cowden strain is one of only a few culturable enteric caliciviruses. We discovered that 4 amino acid substitutions in VP1 (residues 178, 289, 324, and 328) were critical for its adaptation to LLC-PK cells. Two substitutions in VP1 (residues 291 and 295) reduced virus replicationin vitrobut enhanced virus replication and induced higher-level serum and mucosal antibody responses in gnotobiotic pigs than those induced by the tissue culture-adapted strain. Structural modeling analysis of VP1 suggested that residue 178 may affect VP1 assembly and oligomerization; residues 289, 291, 324, and 328 may influence virus binding to cellular receptors; and residue 295 may influence VP1 dimerization. Our findings will provide new information for the cell culture adaptation of other sapoviruses and possibly noroviruses.

Detection of nongroup A rotaviruses in faecal samples of pigs in the Czech Republic

Besides group A rotaviruses, group B and C rotaviruses have been detected as the cause of diarrheal diseases in pigs. Of a set of 329 faecal samples from pigs, 16 samples were selected in which rotavirus was detected by electron microscopy and at the same time group A rotavirus was excluded by ELISA method. Rotaviruses were assayed using specific primers for detection of group B and C rotaviruses, and RT-PCR and semi-nested PCR methods. In one sample, no rotavirus of group B or C was detected; in the remaining 15 samples rotavirus group C was detected, in two samples together with group B rotavirus. Sequencing of the obtained PCR products and comparison with corresponding gene sequences revealed 80% nucleotide sequence identity between group B rotaviruses and available sequences of porcine isolates. A nucleotide sequence identity of 92% was obtained in group C rotaviruses as compared with the Cowden strain.

Detection and phylogenetic analysis of porcine enteric calicivirus, genetically related to the Cowden strain of sapovirus genogroup III, in Brazilian swine herds

Sapovirus of the Caliciviridae family is an important agent of acute gastroenteritis in children and piglets. The Sapovirus genus is divided into seven genogroups (G), and strains from the GIII, GVI and GVII are associated with infections in swine. Despite the high prevalence in some countries, there are no studies related to the presence of porcine enteric sapovirus infections in piglets in Brazil. In the present study, 18 fecal specimens from piglets up to 28 days were examined to determine the presence of sapovirus genome by RT-PCR assay, using primers designed to amplify a 331 bp segment of the RNA polymerase gene. In 44.4% (8/18) of fecal samples, an amplified DNA fragment was obtained. One of these fragments was sequenced and submitted to molecular and phylogenetic analysis. This analysis revealed high similarity, with nucleotides (87%) and amino acids (97.8%), to the Cowden strain, the GIII prototype of porcine enteric calicivirus. This is the first description of sapovirus in Brazilian swine herds.

Characterization of Group C Rotaviruses Associated with Diarrhea Outbreaks in Feeder Pigs

Feces and serum specimens were collected from three farms in Michigan on which ∼50-lb (8- to 9-week-old) pigs experienced diarrhea just after placement into all-in-all-out finishing barns. The clinical signs (profuse watery diarrhea lasting about 2 weeks and no vomiting) were similar on all farms, and the morbidity rate was high (ranging from 60 to 80%) but without mortality. Eleven diarrheic fecal samples from the farms were tested for group A and C rotaviruses by immune electron microscopy (IEM) and various assays. IEM indicated that the fecal samples reacted only with antiserum against group C rotaviruses, and polyacrylamide gel electrophoresis indicated that the samples had characteristic genomic electropherotypes for group C rotavirus. Group C rotavirus was detected by cell culture immunofluorescence (CCIF) tests in nine fecal samples, but no group A rotavirus was detected by enzyme-linked immunosorbent assay or CCIF. By reverse transcription (RT)-PCR, all 11 fecal samples were positive for group C rotaviruses, with only 2 samples positive for group A rotaviruses. However, a second amplification of RT-PCR products using nested primers detected group A rotaviruses in all samples. Analysis of nucleotide and deduced amino acid sequences of the RT-PCR product (partial-length VP7) of the group C rotavirus showed 87.2 to 91% nucleotide identity and 92.6 to 95.9% amino acid identity among two strong samples from the different farms and the Cowden strain of porcine group C rotavirus. All nine convalescent-phase serum samples tested had neutralizing antibodies to the Cowden strain, and the majority of them had neutralizing antibody against group A rotaviruses (OSU or/and Gottfried strains) by fluorescent focus neutralization tests. Although group C rotaviruses have been reported as a cause of sporadic diarrhea in suckling or weanling pigs, to our knowledge, this is the first report of epidemic diarrhea outbreaks associated with group C rotavirus in older pigs.