First report of group A rotavirus and bovine coronavirus associated with neonatal calf diarrhea in the northwest of Argentina.

Group A rotavirus (RVA) and bovine coronavirus (BCoV) are the two main viral enteropathogens associated with neonatal calf diarrhea. The aim of the present survey was to investigate the epidemiology and the role of RVA and BCoV in the presentation of dairy and beef calf diarrhea in Lerma Valley of Salta province, within the Northwest region of Argentina. Stool samples of calves with or without diarrhea younger than 2 months of age were collected from 19 dairy farms and 20 beef farms between the years 2014 and 2016. Stool samples were screened for RVA and BCoV detection by ELISA. Heminested multiplex RT-PCR was used for RVA typing and RT-PCR to confirm BCoV. Positive samples were submitted to sequencing analysis. Bovine RVA and BCoV were circulating in 63% (12/19) and 10.52% (2/19) of the dairy farms, respectively, where 9.5% (46/484) of the calves were positives to RVA and 0.4% (2/484) to BCoV. In beef herds, RVA was detected in 40% (8/20) of the farms and in 6.75% (21/311) of the calves, without positives cases of BCoV. Molecular analysis showed that in dairy farms, G6P[11] and G10P[11] were the prevalent RVA strains, while in beef farms, G10P[11] was the prevalent. The main finding was the detection for the first time of a G15P[11] causing diarrhea in beef calves of Argentina that represents a new alert to be consider for future vaccine updates. Analysis of detected BCoV showed that it is related to the other circulating strains of Argentina.

Epidemiological study of Middle East respiratory syndrome coronavirus infection in dromedary camels in Saudi Arabia, April-May 2015.

A cross-sectional study was conducted in five regions in Saudi Arabia to investigate the epidemiology of Middle East respiratory syndrome coronavirus (MERS-CoV) infection in dromedary camels (Camelus dromedarius) during April and May2015. Serum and nasal swab samples were tested for MERS-CoV antibodies andribonucleic acid (RNA) using a recombinant enzyme-linked immunosorbent assay (rELISA) and real-time reverse-transcription polymerase chain reaction (rRT-PCR), respectively. The overall MERS-CoV antibody seroprevalence was 80.5%, whereas the overall viral RNA prevalence was 2.4%. The associations of risk factors with each prevalence were quantified using univariate and multivariate analyses. The multivariate models identified region, age, grazing system, exposure to wild animals and dung removal as factors significantly associated with seroprevalence (p ??0.05). A higher seroprevalence was more likely to occur in camels from the Riyadh, Eastern, Northern and Makkah regions than those from the Jazan region; camels ??4 and 1-3 years of age (marginally significant) than calves < 1 year; and camels raised in zero grazing and semi-open grazing systems than those raised in an open grazing system. However, the presence of wild animals and daily dung removal were negatively associated with seroprevalence. On the other hand, region and sex were significantly associated with MERS-CoV RNA prevalence(p ??0.05). A higher viral RNA prevalence was more likely to occur in camels from the Riyadh region and Eastern region (marginally significant) than in those from the Makkah region, and in male camels than female camels. In conclusion, the risk factors identified in this study can be considered to be predictors of MERS-CoV infection in camels and should be taken into account when developing an efficient and cost-effective control strategy.

Une étude transversale a été réalisée au cours des mois d'avril et de mai 2015 dans cinq régions d'Arabie saoudite afin d'élucider l'épidémiologie de l'infection par le coronavirus responsable du syndrome respiratoire du Moyen-Orient(MERS­CoV) chez les dromadaires (Camelus dromedarius). Des échantillons de sérum et des écouvillons nasaux prélevés de dromadaires ont été analysés afin de détecter la présence d'anticorps dirigés contre le MERS-CoV ou d'ARN de ce même virus, en utilisant respectivement une épreuve immuno-enzymatique recombinante (ELISAr) et une amplification en chaîne par polymérase couplée à une transcription inverse (PCR­RT) en temps réel. La prévalence sérologique globale des anticorps dirigés contre le MERS-CoV s'élevait à 80,5 %, tandis que la prévalence globale de l'ARN viral était de 2,4 %. Les corrélations entre les facteurs de risque et les prévalences obtenues ont été quantifiées au moyen d'analyses à une seule et à plusieurs variables. Les modèles à plusieurs variables ont fait apparaître une association significative (p ??0,05) entre la prévalence sérologique et les facteurs suivants : la région, l'âge des animaux, le système pastoral pratiqué, l'exposition à la faune sauvage et l'élimination du fumier. La probabilité d'une forte prévalence sérologique était plus élevée chez les dromadaires provenant des régions de Riyad, de l'Est, du Nord et de la Mecque que chez ceux de la région de Jizan ; chez les dromadaires âgés de plus de quatre ans, ou âgés d'un à trois ans (différence marginalement significative) plutôt que chez les jeunes de moins d'un an ; et enfin chez les dromadaires nourris en stabulation (zéro pâturage) ou en pâturage semi-ouvert plutôt que chez ceux nourris dans des systèmes de pâturage ouvert. En revanche, une corrélation négative a été constatée entre la prévalence sérologique d'une part et la présence d'animaux sauvages et/ou l'élimination quotidienne du fumier, d'autre part. En ce qui concerne la détection virale, une corrélation significative (p ??0,05) a été constatée entre la région et le sexe des animaux et la prévalence de l'ARN du MERS-CoV. La probabilité d'une prévalence plus élevée de l'ARN viral était plus prononcée chez les dromadaires des régions de Riyad et de l'Est (différence marginalement significative) que chez ceux de la région de La Mecque, et chez les mâles que chez les chamelles. En conclusion, les facteurs de risque identifiés dans cette étude peuvent servir d'annonciateurs de l'infection par le MERS-CoV chez les dromadaires et devraient être pris en compte pour élaborer une stratégie efficace et rentable de lutte contre cette maladie.

Los autores describen un estudio transversal efectuado en abril y mayo de 2015 en cinco regiones de Arabia Saudí con objeto de investigar la epidemiologia de la infección de dromedarios (Camelus dromedarius) por el coronavirus del síndrome respiratorio de Oriente Medio (MERS­CoV). A tal efecto se analizaron muestras de suero y exudado nasal para detectar en ellas anticuerpos contra el MERS­CoV y ácido ribonucleico (ARN) del virus, empleando para ello, respectivamente, una técnica de ensayo inmunoenzimático recombinante (ELISAr) y una de reacción en cadena de la polimerasa acoplada a transcripción inversa en tiempo real (rRT­PCR, por sus siglas en inglés). Se calculó que la seroprevalencia global de anticuerpos contra el virus era del 80,5% y que la prevalencia global de ARN vírico era del 2,4%. Utilizando análisis multifactoriales y de una sola variable se cuantificó también la correlación de cada una de esas prevalencias con una serie de factores de riesgo. Con los modelos multifactoriales se observó que la región, la edad, el régimen de pastoreo, la exposición a animales salvajes y la retirada de estiércol eran factores que presentaban una asociación significativa con la seroprevalencia (p ??0,05): era más probable encontrar niveles elevados de seroprevalencia en dromedarios de las regiones de Riad y La Meca y las regiones oriental y septentrional del país que en los de la región de Jizán; en los de 4 o más años y entre 1 y 3 años de edad (correlación ligeramente significativa) que en las crías menores de 1 año; y en los animales estabulados o criados en sistemas de pasto semiabierto que en los criados con regímenes de pasto al aire libre. La presencia de animales salvajes y la retirada cotidiana del estiércol, por su parte, presentaban una correlación negativa con la seroprevalencia. Por otro lado, los factores asociados significativamente con la prevalencia de ARN vírico (p ??0,05) eran la región y el sexo: había mayor probabilidad de encontrar niveles elevados de prevalencia de ARN vírico en dromedarios de la región de Riad y la región oriental (correlación ligeramente significativa) que en los de la región de La Meca, y en machos más que en hembras. En conclusión, los factores de riesgo detectados con este estudio pueden ser considerados predictivos de la infección de dromedarios por el MERS­CoV y deben ser tenidos en cuenta para elaborar una estrategia de lucha que ofrezca a la vez eficacia y rentabilidad.

Seroepidemiology of Middle East respiratory syndrome (MERS) coronavirus in Saudi Arabia (1993) and Australia (2014) and characterisation of assay specificity.

The pseudoparticle virus neutralisation test (ppNT) and a conventional microneutralisation (MN) assay are specific for detecting antibodies to Middle East respiratory syndrome coronavirus (MERS-CoV) when used in seroepidemiological studies in animals. Genetically diverse MERS-CoV appear antigenically similar in MN tests. We confirm that MERS-CoV was circulating in dromedaries in Saudi Arabia in 1993. Preliminary data suggest that feral Australian dromedaries may be free of MERS-CoV but larger confirmatory studies are needed.

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.

Middle East Respiratory Syndrome (MERS) coronavirus seroprevalence in domestic livestock in Saudi Arabia, 2010 to 2013.

In Saudi Arabia, including regions of Riyadh and Al Ahsa, pseudoparticle neutralisation (ppNT) and microneutralisation (MNT) tests detected no antibodies to Middle East Respiratory Syndrome coronavirus (MERS-CoV) in sheep (n= 100), goats (n= 45), cattle (n= 50) and chickens (n= 240). Dromedary camels however, had a high prevalence of MERS-CoV antibodies. Bovine coronavirus (BCoV) infected sera from cattle had no cross-reactivity in MERS-CoV ppNT or MNT, while many dromedary camels' sera reacted to both BCoV and MERS-CoV. Some nevertheless displayed specific serologic reaction profiles to MERS-CoV.

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.

Pathogenesis and immune responses in gnotobiotic calves after infection with the genogroup II.4-HS66 strain of human norovirus.

We previously characterized the pathogenesis of two host-specific bovine enteric caliciviruses (BEC), the GIII.2 norovirus (NoV) strain CV186-OH and the phylogenetically unassigned NB strain, in gnotobiotic (Gn) calves. In this study we evaluated the Gn calf as an alternative animal model to study the pathogenesis and host immune responses to the human norovirus (HuNoV) strain GII.4-HS66. The HuNoV HS66 strain caused diarrhea (five/five calves) and intestinal lesions (one/two calves tested) in the proximal small intestine (duodenum and jejunum) of Gn calves, with lesions similar to, but less severe than, those described for the Newbury agent 2 (NA-2) and NB BEC. Viral capsid antigen was also detected in the jejunum of the proximal small intestine of one of two calves tested by immunohistochemistry. All inoculated calves shed virus in feces (five/five calves), and one/five had viremia. Antibodies and cytokine (proinflammatory, tumor necrosis factor alpha [TNF-alpha]; Th1, interleukin-12 [IL-12] and gamma interferon [IFN-gamma]; Th2, IL-4; Th2/T-regulatory, IL-10) profiles were determined in serum, feces, and intestinal contents (IC) of the HuNoV-HS66-inoculated calves (n = 5) and controls (n = 4) by enzyme-linked immunosorbent assay in the acute (postinoculation day 3 [PID 3]) and convalescent (PID 28) stages of infection. The HuNoV-HS66-specific antibody and cytokine-secreting cells (CSCs) were quantitated by ELISPOT in mononuclear cells of local and systemic tissues at PID 28. Sixty-seven percent of the HuNoV-HS66-inoculated calves seroconverted, and 100% coproconverted with immunoglobulin A (IgA) and/or IgG antibodies to HuNoV-HS66, at low titers. The highest numbers of antibody-secreting cells (ASC), both IgA and IgG, were detected locally in intestine, but systemic IgA and IgG ASC responses also occurred in the HuNoV-HS66-inoculated calves. In serum, HuNoV-HS66 induced higher peaks of TNF-alpha and IFN-gamma at PIDs 2, 7, and 10; of IL-4 and IL-10 at PID 4; and of IL-12 at PIDs 7 and 10, compared to controls. In feces, cytokines increased earlier (PID 1) than in serum and TNF-alpha and IL-10 were elevated acutely in the IC of the HS66-inoculated calves. Compared to controls, at PID 28 higher numbers of IFN-gamma and TNF-alpha CSCs were detected in mesenteric lymph nodes (MLN) or spleen and Th2 (IL-4) CSCs were elevated in intestine; IL-10 CSCs were highest in spleen. Our study provides new data confirming HuNoV-HS66 replication and enteropathogenicity in Gn calves and reveals important and comprehensive aspects of the host's local (intestine and MLN) and systemic (spleen and blood) immune responses to HuNoV-HS66.

Human rotavirus type 2: cultivation in vitro.

A strain of type 2 human rotavirus (Wa) was grown to relatively high titer through 14 passages in primary cultures of African green monkey kidney (AGMK) cells. This passage series was initiated with virus that had been passaged 11 times serially in newborn gnotobiotic piglets. In contrast, virus present in the stool of patient Wa as well as virus from the first, second, or third passage in piglets could not be propagated successfully in African green monkey kidney cells. Prior to each passage in cell culture, the virus was treated with trypsin and the inoculated cultures were centrifuged at low speed. Cultivation of a type 2 human rotavirus should aid attempts to characterize this virus and to develop a means of immunoprophylaxis for a serious diarrheal disease of human infants.

Identification of a porcine calicivirus related genetically to human sapoviruses.

Whether animals may act as reservoirs for human caliciviruses is unclear. By sequence analysis of a short fragment of the RNA-dependent RNA polymerase (RdRp) region, porcine sapovirus (SaV) strains that genetically resemble human SaVs have been detected in piglets, but more-informative sequences (capsid gene) were not available for a precise characterization. In this study, the 3' terminus (the 3' end of open reading frame 1 [ORF1], including the polymerase complex and the complete capsid; ORF2; and the 3' untranslated region) of one such human SaV-like strain, 43/06-18p3/2006/It, was determined, revealing that these viruses are more related genetically to human (47.4 to 54.9% amino acid identity) than to animal (35.2 to 44.7% amino acid identity) SaVs in the capsid gene. In addition, the recombination-prone RdRp-capsid junction region was highly conserved with those of human SaVs of genogroup GI. The presence of porcine viruses similar to human SaVs is a significant finding because of the potential for zoonotic infections or generation of porcine/human recombinants.

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.

Seroprevalence in Household Raised Pigs Indicate High Exposure to GII Noroviruses in Rural Nicaragua.

Information about porcine norovirus (PoNoV), genetically similar to human NoV (HuNoV), is limited from rural areas where household-raised pigs are heavily exposed to faecal material which could facilitate transmission. Histo-blood group antigens (HBGAs) are known susceptibility factors to NoV in humans and in a germfree piglet model but their role in susceptibility in the porcine population remains unknown. This study reports: (i) the seroprevalence and antibody titres to human norovirus (NoV) VLPs in household raised pigs; (ii) the distribution of HBGAs in relation to NoV IgG antibody titres and further characterization by blocking of GII.4 VLP binding to pig gastric mucins (PGM). The majority of pigs were seropositive to all three VLPs tested (58-70%) with seropositivity and cross-reactivity increasing significantly with age. However, pig sera could not block the binding of NoV GII.4 VLPs (Dijon) to PGM suggesting no previous infection with this genotype. The majority of the pigs were H-positive (84%), a susceptibility factor for human infections. IgG antibody titres were however higher in H-negative (GMT = 247) as compared with H-positive (GMT = 57) pigs, but after age stratification, this difference in antibody titres was only observed in pigs ≤1 month of age. In conclusion, serological data show that the porcine population of Nicaragua is highly exposed to NoV infections, and the association to HBGAs warrants further investigation.

Egg yolk IgY antibodies: A therapeutic intervention against group A rotavirus in calves.

Bovine group A rotavirus (RVA) is considered the major cause of diarrhea in intensively reared neonatal calves. Chicken egg yolk antibodies (IgY) are efficient in protecting neonatal calves from RVA diarrhea; however, the value of this intervention in calves once diarrhea has appeared is unclear. The aim of the present study was to evaluate the application of RVA-specific IgY as a passive treatment in those cases. The experimental groups were: G1=RVA-specific IgY treatment; G2=no Ab treatment; and G3=colostrum deprived+no Ab treatment. IgY treatment significantly reduced virus shedding, diarrhea duration and severity compared to G2 and G3 calves. However, it caused a partial suppression of systemic Ab responses to RVA that could be associated with less severe diarrhea. The oral treatment with IgY for 7days was associated with significantly higher antibody secreting cell responses in the calves compared with other groups of animals.

Molecular and antigenic characterization of bovine Coronavirus circulating in Argentinean cattle during 1994-2010.

Bovine coronavirus (BCoV) is an important viral pathogen associated with neonatal calf diarrhea. Our aim was to investigate the incidence of BCoV in diarrhea outbreaks in beef and dairy herds from Argentina during 1994-2010. A total of 5.365 fecal samples from diarrheic calves were screened for BCoV diagnosis by ELISA. The virus was detected in 1.71% (92/5365) of the samples corresponding to 5.95% (63/1058) of the diarrhea cases in 239 beef and 324 dairy farms. The detection rate of BCoV was significantly higher in dairy than in beef herds: 12.13% (29/239) vs. 4.32% (14/324) respectively. Phylogenetic analysis of the hypervariable S1 region of seven representative samples (from different husbandry systems, farm locations and years of sampling) indicated that BCoV strains circulating in Argentinean beef and dairy herds formed a cluster distinct from other geographical regions. Interestingly, Argentinean strains are distantly related (at both the nucleotide and amino acid levels) with the Mebus historic reference BCoV strain included in the vaccines currently available in Argentina. However, Mebus-induced antibodies were capable of neutralizing the BCoV Arg95, a field strain adapted to grow in vitro, and vice versa, indicating that both strains belong to the same CoV serotype reported in cattle. This work represents the first large survey describing BCoV circulation in Argentinean cattle.

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.

Passive immunity to transmissible gastroenteritis virus: intramammary viral inoculation of sows.

Sows were injected intramammarily with live-attenuated TGE virus, an enteric coronavirus--one sow during pregnancy and three sows during lactation. All sows were TGE antibody seronegative prior to inoculation except for one naturally infected sow inoculated during lactation. The animal injected during pregnancy had primarily IgG TGE antibodies in milk from all glands. By contrast, sows injected during lactation had IgA and IgM initially, and later IgA and IgG TGE antibodies in milk from injected and noninjected glands. The seropositive sow had elevated IgA TGE antibody titers in milk after IMm injection. Both seronegative sows inoculated intramammarily during lactation shed TGE virus in milk from injected glands, and their nursing piglets developed mild diarrhea and shed virus in their feces at three to nine DPE of the sows. Milk from IMm injected glands generally had higher TGE antibody titers than milk from noninjected glands. These results suggest that TGE virus replicates in lactating mammary gland tissue, thereby stimulating IgA immunocytes, leading to secretion of IgA antibodies in milk. Whether the intramammary route presents a natural route of enteric virus exposure in lactating animals (by way of infected nursing piglets), leading to IgA-antibody secretion in milk, requires further investigation.

The gnotobiotic piglet as a model for studies of disease pathogenesis and immunity to human rotaviruses.

Gnotobiotic piglets serve as a useful animal model for studies of human rotavirus infections, including disease pathogenesis and immunity. An advantage of piglets over laboratory animal models is their prolonged susceptibility to human rotavirus-induced disease, permitting cross-protection studies and an analysis of active immunity. Major advances in rotavirus research resulting from gnotobiotic piglet studies include: 1) the adaptation of the first human rotavirus to cell culture after passage and amplification in piglets; 2) delineation of the independent roles of the two rotavirus outer capsid proteins (VP4 and VP7) in induction of neutralizing antibodies and cross-protection; and 3) recognition of a potential role for a nonstructural protein (NSP4) in addition to VP4 and VP7, in rotavirus virulence. Current studies of the pathogenesis of group A human rotavirus infections in gnotobiotic piglets in our laboratory have confirmed that villous atrophy is induced in piglets given virulent but not cell culture attenuated human rotavirus (G1, P1A, Wa strain) and have revealed that factors other than villous atrophy may contribute to the early diarrhea induced. A comprehensive examination of these factors, including a proposed role for NSP4 in viral-induced cytopathology, may reveal new mechanisms for induction of viral diarrhea. Finally, to facilitate and improve rotavirus vaccination strategies, our current emphasis is on the identification of correlates of protective active immunity in the piglet model of human rotavirus-induced diarrhea. Comparison of cell-mediated and antibody immune responses induced by infection with a virulent human rotavirus (to mimic host response to natural infection) with those induced by a live attenuated human rotavirus (to mimic attenuated oral vaccines) in the context of homotypic protection has permitted an analysis of correlates of protective immunity. Results of these studies have indicated that the magnitude of the immune response is greatest in lymphoid tissues adjacent to the local site of viral replication (small intestine). Secondly, there was a direct correlation between the degree of protection induced and the level of the intestinal immune response, with significantly higher local immune responses and complete protection induced only after primary exposure to virulent human rotavirus. These studies thus have established basic parameters related to immune protection in the piglet model of human rotavirus-induced disease, verifying the usefulness of this model to examine new strategies for the design and improvement of human rotavirus vaccines.

Rotavirus subunit vaccines.

We evaluated rotavirus subunit vaccines for use in humans and animals. Insect cells were co-infected with combinations of individual baculovirus recombinants expressing human, bovine or simian rotavirus VP2, VP4, VP6 or VP7 to produce virus-like particles (VLPs). To determine whether immunization with VLPs could induce active protective immunity, VLPs were administered parenterally to rabbits, and the immune response and protection from rabbit ALA rotavirus challenge were evaluated. Complete or partial protection was attained, showing that parenteral immunization with VLPs induces active protective immunity. We also examined whether heterotypic immune responses could be induced with a limited number of broadly reactive VP7 proteins or with chimeric particles (multiple VP7 types on individual particles). The feasibility of this approach was determined by immunizing mice with VLPs containing a G3 VP7 or G1 VP7 and chimeric G1/G3 VLPs. Broadly reactive neutralizing antibody was induced by the G1 VLPs. VLPs also have been successfully used to boost lactogenic (colostral and milk) immunity in dairy cows. Taken together, these results show that VLPs can be effective immunogens in rabbits, mice and dairy cattle when administered parenterally, a limited number of VLPs may be sufficient to produce a broadly protective vaccine, and G3 VLPs may serve as an effective subunit vaccine for use in bovines.

Coronavirus immunogens.

Coronaviruses (CV) infect a variety of livestock, poultry and companion animals. They belong to at least five antigenic groups. CV cause localized infections of the respiratory and/or intestinal tracts, with the exception of feline infectious peritonitis virus (FIPV) and hemagglutinating encephalomyelitis (HEV) which cause systemic infections. The enteropathogenic CV infect the villous enterocytes resulting in villous atrophy leading to malabsorptive diarrhea. Several CV (bovine CV-BCV, porcine respiratory CV-PRCV, infectious bronchitis virus-IBV) cause respiratory disease. Current evidence indicates that protection against enteric and respiratory CV infections is mediated by passive or active immunity at the primary site of CV replication. Maternal vaccination approaches to induce passive immunity include the use of inactivated and modified live viral vaccines. Modified live viruses and a Ts mutant CV (FIPV) are also used as oral or intranasal vaccines to induce active mucosal immunity. The success of these vaccines in the field is often compromised by a number of potential problems. Coronaviruses are spherical, enveloped viruses, ranging from 80-160 nm in diameter and containing a positive-stranded RNA genome. They possess prominent surface spikes and some species display a fringe of smaller surface projections believed to be the hemagglutinin (HE). Coronaviruses possess 3 to 4 structural proteins: the spike (S) glycoprotein (150-200 kDa), the integral membrane glycoprotein (M; 20-30 kDa) and the nucleocapsid phosphoprotein (N; 43-50 kDa). A subset of CV (BCV, HEV, turkey CV) possess a third glycoprotein on the virion surface, the HE (60-65 kDa). These proteins can be quantitated using pooled monoclonal antibodies (mAb) to distinct epitopes of each protein in ELISA. Most research has focused on the S protein as a candidate antigen for CV vaccines since it induces virus neutralizing (VN) antibodies. However the HE protein stimulates the production of VN and HE inhibiting antibodies and the M protein induces antibodies that neutralize virus in the presence of complement. Attempts to correlate in vitro VN antibody activity with in vivo protection have shown that the passive transfer of VN mAb to the S or HE protein conferred passive protection against CV challenge in some studies, but not others. Additional research has implicated a possible role for other CV proteins in immunity. Studies of mAb to the M protein of transmissible gastroenteritis (TGEV) have provided evidence for a direct role of the M protein in the induction of alpha IFN by porcine blood leukocytes. The potential significance of this phenomenon to immunity to TGEV is unclear.(ABSTRACT TRUNCATED AT 400 WORDS)