Detection and genetic diversity of porcine group A rotaviruses in historic (2004) and recent (2011 and 2012) swine fecal samples in Ohio: predominance of the G9P[13] genotype in nursing piglets.

Epidemiological surveillance of porcine group A rotavirus (RVA) strains was conducted in five swine herds in Ohio using historical (2004) and recent (2011 to 2012) fecal samples. Of the 371 samples examined, 9.4% (35/371) were positive for RVA. The RVA detection rates increased from 5.9% in 2004 and 8.5% in 2011 to 13.8% in 2012. A total of 23 positive samples were analyzed for RVA G and P genotypes. The dominant G-P combination was G9P[13] found in 60.9% of positive samples. The other combinations were G9P[7] (8.7%), G4P[13] (8.7%), G11P[13] (4.3%), and G11P[7] (4.3%). Sequence analysis of partial VP7 genes of selected strains revealed that the G4 strains were closely related to one another (95%) and, to a lesser extent, to human (82 to 84%) and porcine (84 to 86%) G4 strains. The G11 strains detected shared identical VP7 gene sequences (100%) and were closely related to human (85 to 86%) and other porcine (83%) G11 strains. The G9 strains identified were closely related to one another and to human and other porcine strains (96 to 97%, 89 to 91%, and 89 to 91% nucleotide identities, respectively). The VP4 gene analysis revealed that P[7] strains were closely related to each other and to P[7] strains isolated from porcine, bovine, and panda samples (91 to 99%, 92 to 99% and 92 to 99%, respectively). The P[13] strains showed a higher diversity among themselves and with other porcine P[13] strains, ranging from 83% to 99% and from 82 to 97%, respectively. Our results demonstrate broad genetic heterogeneity of the RVA strains and suggest the possibility of genetic reassortment between different RVA genotypes within these farms.

Intestinal mucin-type O-glycans: the major players in the host-bacteria-rotavirus interactions.

Rotavirus (RV) causes severe diarrhea in young children and animals worldwide. Several glycans terminating in sialic acids (SAs) and histo-blood group antigens (HBGAs) on intestinal epithelial cell (IEC) surface have been recognized to act as attachment sites for RV. IECs are protected by the double layer of mucus of which O-glycans (including HBGAs and SAs) are a major organic component. Luminal mucins, as well as bacterial glycans, can act as decoy molecules removing RV particles from the gut. The composition of the intestinal mucus is regulated by complex O-glycan-specific interactions among the gut microbiota, RV and the host. In this review, we highlight O-glycan-mediated interactions within the intestinal lumen prior to RV attachment to IECs. A better understanding of the role of mucus is essential for the development of alternative therapeutic tools including the use of pre- and probiotics to control RV infection.

Glycan-mediated interactions between bacteria, rotavirus and the host cells provide an additional mechanism of antiviral defence.

Limited efficacy of rotavirus (RV) vaccines in children in developing countries and in animals remains a significant problem necessitating further search for additional approaches to control RV-associated gastroenteritis. During cell attachment and entry events, RV interacts with cell surface O-glycans including histo-blood group antigens (HBGAs). Besides modulation of the protective immunity against RV, several commensal and probiotic bacteria were shown to express HBGA-like substances suggesting that they may affect RV attachment and entry into the host cells. Moreover, some beneficial bacteria have been shown to possess the ability to bind host HBGAs via sugar specific proteins called lectins. However, limited research has been done to evaluate the effects of HBGA-expressing and/or HBGA-binding bacteria on RV infection. The aim of this study was to investigate the ability of selected commensal and probiotic bacteria to bind different RV strains via HBGAs and to block RV infection of IPEC-J2 cells. Our data indicated that Gram-negative probiotic Escherichia coli Nissle 1917 (E. coli Nissle 1917) and commensal Gram-positive (Streptococcus bovis and Bifidobacterium adolescentis) and Gram-negative (Bacteroides thetaiotaomicron, Clostridium clostridioforme and Escherichia coli G58 (E. coli G58) bacteria of swine origin expressed HBGAs which correlated with their ability to bind group A and C RVs. Additionally, Gram-positive E. coli 1917 and E. coli G58 demonstrated the ability to block RV attachment onto IPEC-J2 cells. Taken together, our results support the hypothesis that physical interactions between RVs and HBGA-expressing beneficial bacteria may limit RV replication.

Reduced rotavirus vaccine efficacy in protein malnourished human-faecal-microbiota-transplanted gnotobiotic pig model is in part attributed to the gut microbiota.

The low efficacy of human rotavirus (HRV) vaccines in low- and middle-income countries (LMIC) remains a major challenge for global health. Protein-calorie malnutrition (kwashiorkor) affects the gut microbiota and compromises immune development, leading to environmental enteropathy, vaccine failures, and increased susceptibility to enteric diseases in young children. Relationship between diet and reduced vaccine efficacy in developing countries is not well established; therefore, we investigated the interconnections between the host-microbiota-nutrition-HRV vaccine using HRV-vaccinated, human infant faecal microbiota (HIFM)-transplanted neonatal gnotobiotic pigs fed with a protein deficient or sufficient diet. The microbiota from faecal, intestinal (duodenum, ileum, jejunum, and colon), and systemic tissue (liver, spleen, and mesenteric lymph node [MLN]) samples was analysed before and after HRV challenge using MiSeq 16S rRNA sequencing. Overall, microbiota from deficient fed HIFM pigs displayed, compared to the sufficient group, significantly higher Shannon index, especially in the faeces and lower intestines; higher level of Proteus and Enterococcus, and lower level of Bifidobacterium, Clostridium, and Streptococcus in the three types of samples collected (P<0.05); and higher unique operational taxonomic units (OTUs), especially in the systemic tissues. Further, the multivariate analysis between microbiota and immunologic data showed that 38 OTUs at the genus level correlated (r2≤0.5 or ≥-0.5; P<0.05) with at least one host immune response parameter (regulatory [Tregs and transforming growth factor-ß], effectors [interferon (IFN)-γ+ CD4+ and CD8+ T cells, IFN-γ and interleukin (IL)-12], and inflammatory [tumour necrosis factor-α, IL-17 and IL-22]) and with opposite trends between diet groups. Differences described above were increased after HRV challenge. We demonstrated that a protein deficient diet affects the composition of the gut microbiota and those changes may further correlate with immune responses induced by HRV and perturbed by the deficient diet. Thus, our findings suggest that the reduced efficacy of HRV vaccine observed in Gn pig model is in part attributed to the altered microbiota composition.

Effect of antibiotic, probiotic, and human rotavirus infection on colonisation dynamics of defined commensal microbiota in a gnotobiotic pig model.

We developed a gnotobiotic (Gn) pig model colonised with defined commensal microbiota (DMF) to provide a simplified and controlled system to study the interactions between intestinal commensals, antibiotics (ciprofloxacin, CIP), probiotics (Escherichia coli Nissle 1917, EcN) and virulent human rotavirus (VirHRV). The DMF included seven gut commensal species of porcine origin that mimic the predominant species in the infant gut. Gn piglets were divided into four groups: DMF control (non-treated), DMF+CIP (CIP treated), DMF+CIP+EcN (CIP/EcN treated), DMF+EcN (EcN treated) and inoculated orally with 105 cfu of each DMF strain. The pig gut was successfully colonised by all DMF species and established a simplified bacterial community by post-bacteria colonisation day (PBCD) 14/post-VirHRV challenge day (PCD) 0. Overall, Bifidobacterium adolescentis was commonly observed in faeces in all groups and time points. At PCD0, after six days of CIP treatment (DMF+CIP), we observed significantly decreased aerobic and anaerobic bacteria counts especially in jejunum (P<0.001), where no DMF species were detected in jejunum by T-RFLP. Following HRV challenge, 100% of pigs in DMF+CIP group developed diarrhoea with higher diarrhoea scores and duration as compared to all other groups. However, only 33% of pigs treated with EcN plus CIP developed diarrhoea. EcN treatment also enhanced the bacterial diversity and all seven DMF species were detected with a higher proportion of Bifidobacterium longum in jejunum in the DMF+CIP+EcN group on PBCD14/PCD0. Our results suggest that EcN increased the proportion of B. longum especially in jejunum and mitigated adverse impacts of antibiotic use during acute-infectious diarrhoea. The DMF model with a simplified gut commensal community can further our knowledge of how commensals and probiotics promote intestinal homeostasis and contribute to host health.

[Reassortment and gene interactions in the crossing of low-pathogenic avian influenza H5 virus with human influenza virus].

The reassortants obtained via the crossing of highly productive influenza virus A/Puerto Rico/8/34 (H1N1) strain and the low pathogenic avian influenza virus A/Duck/Primorie/2621/2001 (H5N2) strain were genotyped and characterized. The H5N2 reassortant having 6 genes from A/Puerto Rico/8/34 virus has the high level of reproduction in chick embryos, while slightly more moderate than in the parent A/Puerto Rico/8/34 strain. The reproduction of the H5N1 reassortant that had 7 genes from A/Puerto Rico/8134 virus was very low. The serial passage selection allowed the investigators to obtain the H5N1 strain that was reproductively close to the H5N2 reassortant. This variant had one amino acid substitution in hemagglutinin (N244D, H3 numbering) and a lower affinity for fetuin. By the level of virulence to mice, the H5N1 and H5N2 reassortants were close to A/Puerto Rico/8/34 virus and greatly differed in this respect from low virulent A/Duck/Primorie/2621/2001 (H5N2). The results are discussed in connection with the problem of vaccination when there is a threat for H5N1 virus subtype-caused pandemic.

[Sensitivity of methods of titration of the vaccine strain of porcine fever virus].

Methods of titration of the CS vaccine strain of classical swine fever virus were compared in vitro and vivo. The titration in the TL and PK-15 cell culture without cytopathic effect is based on the detection of virus antigen by labeled antibodies. The infection intensity in the cell culture virtually correlated with the antigenic and immunogenic activity of dry vaccine used for swine.

Detection and genetic characterization of porcine group A rotaviruses in asymptomatic pigs in smallholder farms in East Africa: predominance of P[8] genotype resembling human strains.

Viral enteritis is a serious problem accounting for deaths in neonatal animals and humans worldwide. The absence of surveillance programs and diagnostic laboratory facilities have resulted in a lack of data on rotavirus associated diarrheas in pigs in East Africa. Here we describe the incidence of group A rotavirus (RVA) infections in asymptomatic young pigs in East Africa. Of the 446 samples examined, 26.2% (117/446) were positive for RVA. More nursing piglets (78.7%) shed RVA than weaned (32.9%) and grower (5.8%) pigs. RVA incidence was higher in pigs that were either housed_free-range (77.8%) or tethered_free-range (29.0%) than those that were free-range or housed or housed-tethered pigs. The farms with larger herd size (>10 pigs) had higher RVA prevalence (56.5%) than farms with smaller herd size (24.1-29.7%). This study revealed that age, management system and pig density significantly (p<0.01) influenced the incidence of RVA infections, with housed_free-range management system and larger herd size showing higher risks for RVA infection. Partial (811-1604nt region) sequence of the VP4 gene of selected positive samples revealed that different genotypes (P[6], P[8] and P[13]) are circulating in the study area with P[8] being predominant. The P[6] strain shared nucleotide (nt) and amino acid (aa) sequence identity of 84.4-91.3% and 95.1-96.9%, respectively, with known porcine and human P[6] strains. The P[8] strains shared high nt and aa sequence identity with known human P[8] strains ranging from 95.6-100% to 92-100%, respectively. The P[13] strains shared nt and aa sequence identity of 83.6-91.7% and 89.3-96.4%, respectively, only with known porcine P[13] strains. No P[8] strains yielded RNA of sufficient quality/quantity for full genome sequencing. However analysis of the full genome constellation of the P[6], two P[13] and one untypeable strains revealed that the P[6] strain (Ke-003-5) genome constellation was G26-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H1, P[13] strains (Ug-049 and Ug-453) had G5-P[13]-I5-R1-C1-M1-A8-N1-T7-E1-H1 while the untypeable strain (Ug-218) had G5-P[?]-I5-R1-C1-M1-A8-N1-T1-E1-H? In conclusion, P[6] and P[8] genotypes detected were genetically closely related to human strains suggesting the possibility of interspecies transmission. Further studies are required to determine the role of RVA in swine enteric disease burden and to determine the genetic/antigenic heterogeneity of the circulating strains for development of accurate diagnostic tools and to implement appropriate prophylaxis programs.

[Genetic variability of the nucleocapsid protein of the virus of the porcine reproductive and respiratory syndrome]. / Variabel'nost' gena belka nukleokapsida virusa reproduktivnogo i respiratornogo sindroma svinei (RRSS).

The porcine reproductive and respiratory syndrome (PRRS) is a contagious viral pathology caused by PRRS virus. There are 2 types of the above virus--the European and American ones. Distribution patterns of the PRRS virus were studied for Russia and Byelorussia. Above 700 porcine sera obtained from 32 households of 21 Russia's administrative regions and from 19 households of 6 Byelorussia's administrative regions were tested for presence of antibodies to the PRRS virus. Simultaneously, the samples were tested for virus presence by polymerase chain reaction (PCR). It was proven serologically that the PRRS virus is widespread in the territories of Russia and Byelorussia. Noteworthily, all field isolates found in Russia and Byelorussia belong to the European type. Not a single viral isolate of the American PRRS type was found. The nucleocapsid (N) recombinant protein was obtained on the basis of the Russian field isolate of the PRRS virus by using the E. coli. expression system. Finally, it was shown as possible to use the recombinant protein in indirect immune enzyme assay for the sake of detecting the antibodies to the PRRS virus.

Prevalence and genetic heterogeneity of porcine group C rotaviruses in nursing and weaned piglets in Ohio, USA and identification of a potential new VP4 genotype.

Swine fecal samples collected from seven farms were screened for group C rotaviruses (RVCs) using a reverse transcription-polymerase chain reaction assay. A total of 380 samples were tested and 19.5% were positive. Of the 128 samples collected in 2012, 23.5% from nursing piglets and 8.5% from weaned piglets were RVC positive, with a higher RVC frequency in diarrheic (28.4%) than in non-diarrheic (6.6%) piglets. Two strains (RVC/Pig-wt/USA/RV0104/2011/G3PX and RVC/Pig-wt/USA/RV0143/2012/G6Px) from two different farms were characterized genetically to gain information on virus diversity based on full length sequences of the inner capsid VP6, enterotoxin NSP4 and the outer capsid VP7 and VP4 (partial for RV0104) genes. The VP6 gene of the two strains showed high (99%) nucleotide identity to one another, 84-91% identity to other porcine RVCstrains and 81-82% identity to human and bovine RVC strains. The NSP4 gene analysis revealed that RVC/Pig-wt/USA/RV0104/2011/G3PX and RVC/Pig-wt/USA/RV0143/2012/G6Px strains were not closely related to each other (87% identity), but shared higher identity with prototype RVC/Pig-wt/USA/Cowden/1980/G1Px strain (93% and 89%, respectively) and were more distantly related to human strains (72-76% identity). The VP7 gene analysis indicated that the two strains were distantly related to one another (72% identity). RVC/Pig-wt/USA/RV0143/2012/G6Px was most closely related to porcine RVC G6 strains (82-86% identity), whereas RVC/Pig-wt/USA/RV0104/2011/G3PX was most closely related to porcine HF (G3) strain (94% identity). Analysis of the full length nucleotide sequence of the VP4 gene revealed that RVC/Pig-wt/USA/RV0143/2012/G6Px was distantly related to porcine (75%), bovine (74%) and human (70%) strains. The deduced amino acid identities (69.5-75.6%) of VP4 between RVC/Pig-wt/USA/RV0143/2012/G6Px and other RVCs were low; hence, we propose that this strain comprises a new VP4 genotype. Our results indicate high genetic heterogeneity in RVCs genes and the concurrent co-circulation of different genotypes at the same time. Our findings are useful for the development of more accurate diagnostic tools, for basic research to understand gene function and to provide information for RVC diversity germane to vaccine development.

Isolation and characterization of full-length recombinant cattle PrPC protein.

Full-length Bos taurus PrPC protein was obtained in the eu- and prokaryotic expression systems. Immunoblotting and indirect enzyme immunoassay demonstrated high specificity and antigenic activity of full-length proteins in the reactions with monoclonal antibodies (anti-SAF-32 and VRQ-84). Membrane location of recombinant PrPC protein in insect cells was shown by immunofluorescent analysis.