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Rotavirus is the major cause of infectious diarrhoea in infants and children throughout the world. Several studies have indicated that some milk fractions may protect against rotavirus infection. The present study evaluates the effect of some heat treatments on the antirotaviral activity of bovine and ovine whey by an immunofluorescence assay validated in this work. Neutralising activity of diverse proteins present in whey has also been verified individually. Bovine and ovine whey, at 1 mg mL−1 protein, inhibit MA104 cells infection with bovine rotavirus strain WC3, with values of 89.2 and 76.6% inhibition, respectively. Furthermore, the inhibitory activity was influenced by temperature and duration of heat treatment. The ability of bovine and ovine whey to inhibit rotavirus infection was not affected by treatment at 75 °C for 20 s; after 85 °C for 10 min bovine and ovine whey still maintained 16.1 and 32.5%, respectively, of neutralisation activity.

Neonatal calf diarrhea is still one of the most important diseases in calf rearing, and severe diarrhea has a marked effect on animal welfare. Furthermore, significant economic losses can result from this disease due to high mortality rates, high medical costs, and low weight gain. To avoid a fatal outcome of the disease, it is crucial that vulnerable calves are identified as early as possible. Interleukin-6 is described as an early and reliable prognostic marker in several diseases. In this study, 20 scouring calves were tested by ELISA for their IL-6 serum concentrations. Samples were collected twice, at the beginning of diarrhea and 7 to 10 d later. Regarding the clinical outcome after 7 to 10 d, calves were classified as recovered or nonrecovered. A receiver operating characteristic analysis was conducted to determine the prognostic value of IL-6 for the progress of clinical symptoms. At the beginning of diarrhea, the IL-6 concentration was significantly higher in nonrecovering calves compared with those that recover 7 to 10 d after the onset of diarrhea. Interleukin-6 proved to be a useful additional parameter in the clinical examination. High initial IL-6 values can support the decision for closer monitoring and an adapted therapeutic strategy for the respective calves. This may help to prevent unnecessary animal suffering and reduce economic losses.

RESUMO O objetivo deste estudo foi detectar rotavírus em fezes de bezerros com diarreia em Uberaba, MG, e caracterizar os genes VP7 e VP4 por meio da genotipagem e da análise filogenética. Setenta e quatro amostras foram coletadas entre novembro de 2008 e setembro de 2009. A detecção do vírus foi feita por teste de aglutinação e as amostras positivas foram submetidas à transcrição reversa, seguida de reação em cadeia da polimerase (RT-PCR), tipagem por PCR e sequenciamento. A taxa de detecção de rotavírus foi de 6,8% e todas as amostras apresentaram o genótipo G6P[5]. A análise filogenética mostrou que as amostras do genótipo G6 pertencem à linhagem IV e que, para ambos os genes (VP7 e VP4), as amostras deste estudo compõem um sub-cluster à parte daquele das cepas referências e das amostras campo mais similares. O alinhamento das sequências de aminoácidos deduzidas mostrou substituições em regiões antigênicas quando comparadas com as sequências das cepas bovinas UK e NCDV, presentes nas vacinas disponíveis no Brasil. Uma nova sublinhagem genética de G6P[5] foi evidenciada neste estudo. Substituições de aminoácidos nas regiões antigênicas dos rotavírus e a circulação de novas variantes podem representar desafios para as vacinas utilizadas atualmente. O presente estudo contribui para a compreensão da epidemiologia dos rotavírus bovinos no Brasil.

Made available in DSpace on 2018-12-11T17:29:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-01-01. Added 1 bitstream(s) on 2019-10-09T18:32:38Z : No. of bitstreams: 1 S0102-09352016000400865.pdf: 216222 bytes, checksum: 94e08d25e7aaedfc3c14b4fd596e153b (MD5) The aim of the present study was to characterize changes in acute phase protein levels according to the occurrence of rotavirus diarrhea in calves in the first month of life. Blood and fecal samples were taken before colostrum intake and at 1, 2, 7, 15, 21 and 30 days of age from 24 Holstein calves allotted in three experimental groups: calves that did not present diarrhea (group A), calves that presented diarrhea, but tested negative for rotavirus in feces (group B), and calves that presented diarrhea and tested positive for rotavirus in feces (group C) (experiment 1). When the animals presented episodes of diarrhea, blood and fecal samples were taken at 24-hour intervals until the end of clinical signs (experiment 2). Serum proteins were separated by SDS-PAGE technique and rotavirus in feces was detected by PAGE. Data of experiment 1 were analyzed by ANOVA and Tukey's test, considered significant at P

Rotavirus was recognized as the virus that responsible for causing acute gastroenteritis, especially young livestock. Taiwan Center for Disease Control (CDC) confirms the majority cases of acute gastroenteritis in Taiwan on February 2015 were caused by rotavirus. In this study, we report the incidence and zoonotic impact of rotavirus strain from Taiwan. This study examined 90 (swine) and 60 (bovine) fecal samples collected from south of Taiwan in March 2015. Detection of rotavirus using VP6 gene by RT-PCR technique with amplicons 379 bp. Zoonotic potential analysis based on nucleotide sequence and phylogenetic analysis. RT-PCR utilizing the primers specific for VP6 gene detected rotavirus with positive reactions 3/30 (10%) in piglets and 1/20 (5%) in the calf. Based on the nucleotide sequences and phylogenetic analysis indicated that 1 of 3 wild strains from swine rotavirus had 85.0% - 91.1% and 1 wild strain from bovine had 78.7% - 85.9% identity relations with human strains. These findings indicated that the wild strains of swine and bovine rotavirus may broadly spread and contribute to zoonotic transmission. Keywords: Bovine, Rotavirus, RT-PCR, Swine, Zoonotic.

Purpose: Bovine Rotavirus and Bovine Coronavirus are the most important causes of diarrhea in newborn calves and in some other species such as pigs and sheep. Rotavirus VP8 subunit is the major determinant of the viral infectivity and neutralization. Spike glycoprotein of coronavirus is responsible for induction of neutralizing antibody response. Methods: In the present study, several prediction programs were used to predict B and T-cells epitopes, secondary and tertiary structures, antigenicity ability and enzymatic degradation sites. Finally, a chimeric antigen was designed using computational techniques. The chimeric VP8-S2 antigen was constructed. It was cloned and sub-cloned into pGH and pET32a(+) expression vector. The recombinant pET32a(+)-VP8-S2 vector was transferred into E.oli BL21CodonPlus (DE3) as expression host. The recombinant VP8-S2 protein was purified by Ni-NTA chromatography column. Results: The results of colony PCR, enzyme digestion and sequencing showed that the VP8-S2 chimeric antigen has been successfully cloned and sub-cloned into pGH and pET32a(+).The results showed that E.coli was able to express VP8-S2 protein appropriately. This protein was expressed by induction of IPTG at concentration of 1mM and it was confirmed by Ni–NTA column, dot-blotting analysis and SDS-PAGE electrophoresis. Conclusion: The results of this study showed that E.coli can be used as an appropriate host to produce the recombinant VP8-S2 protein. This recombinant protein may be suitable to investigate to produce immunoglobulin, recombinant vaccine and diagnostic kit in future studies after it passes biological activity tests in vivo in animal model and or other suitable procedure.

The viability of murine norovirus (MNV-1), bovine rotavirus (boRV), and hepatitis A virus (HAV) was evaluated at 21 °C, 4 °C, and −20 °C on stainless steel surfaces, in bottled water, and on blueberries for up to 21 days. After 14 days of incubation at 21 °C on stainless steel, a viability loss >4 log for MNV-1, >8 log for boRV, and >1 log for HAV was observed. Losses were observed for MNV-1 (>1 log) and HAV (>2 log) incubated in water at 21 °C for 21 days. No significant loss was detected for MNV-1 and HAV at 4 °C and −20 °C and for boRV at 21 °C, 4 °C, and −20 °C. On blueberries incubated at 4 °C and −20 °C, they all maintained their infectivity. After 7 days at 21 °C, a loss >2 log, a loss of 3 log, and no loss were observed for boRV, MNV-1, and HAV, respectively. After RNase pretreatment, the detection of extracted RNA from infectious and noninfectious samples suggested the protection of RNA inside the capsid. Even though they all are enteric viruses, their persistence varied with temperature and the nature of the commodity. It is therefore important to use more than one viral surrogate, during inactivation treatments or implementation of control measures.

VI CHAPTER L GENERAL INTRODUCTION CHAPTER 2. LITERATURE REVIEW 6 CHAPTERS. BOVINE ROTAVIRUSES 26 CHAPTER 4. THE VP4 AND VP7 OF BOVINE ROTAVIRUS VMRI ARE ANTIGENICALLY AND GENETICALLY CLOSELY RELATED TO P-TYPE 5, G-TYPE 6 STRAINS 51 CHAPTER 5. RELATIVE FREQUENCY OF P AND G TYPES OF ROTAVIRUSES ASSOCIATED WITH CALF DIARRHEA AS DETERMINED BY NESTED REVERSE TRANSCRIPnON POLYMERASE CHAIN REACTION 78 CHAPTER 6. COMPARATIVE ANALYSIS OF THE VPS* SEQUENCES OF P[5] AND P[11] ROTAVIRUSES ISOLATED FROM CALVES WITH DIARRHEA 95 CHAPTER 7. SEQUENCE AND PHYLOGENETIC ANALYSIS OF THE VP7 GENE OF A BOVINE ROTAVIRUS WTTH G6 SUBTYPE 107 CHAPTERS. GENERAL CONCLUSIONS 112

Rotaviruses (RV) are the leading cause of acute infantile gastroenteritis, associated with elevated mortality in low-income countries. Morbidity and mortality, length and rates of hospitalization due to RV gastroenteritis are dropping. Improving the quality of newborns life is an ongoing challenge for health-care providers. In this study, homemade reassortant human-bovine rotavirus was developed and biological activity and molecular characterization of candidate vaccine were evaluated for the vaccine stability. Virus titration and purification of reassortant rotavirus strains were evaluated by plaque assays, electropherotyping. The genetic stability after first, third and sixth passage was by sequencing. Due to WHO recommendation, developingment of national capacity for vaccine production in appropriate quantities and at affordable prices is the cornerstone of developing global vaccination policies. Such studies are critical to producing national vaccines and modeling herd protection.

There are a number of diseases which are important globally in terms of the effect they have on livestock but for which the development of recombinant plant-produced vaccines is preliminary. For many of these diseases such as bovine viral diarrhoea (BVD), bovine rotavirus (BRV), bovine herpes (BoH), transmissible gastroenteritis (TGE) in pigs, infectious bronchitis (IB) in chickens, bluetongue (BT) in sheep, Rift Valley fever (RVF) in sheep and coccidiosis in chickens, commercially available live-attenuated or killed vaccines are available. Although most are effective to varying degrees, there are numerous issues with manufacture and potential reassortment of the vaccine strains. For some diseases such as bovine papillomavirus (BPV) infections and Crimean-Congo haemorrhagic fever (CCHF), there are no commercially available vaccines, and limited studies have been conducted on their development. This chapter discusses some of the research developments in plant-produced vaccine candidates which have potential for further development towards commercialisation.

Determination and distribution of the G and P genotypes of group A bovine rotavirus was investigated on 386 fecal samples collected from calves with diarrhea using a semi-nested RT-PCR typing assay. Samples were collected from 11 provinces of Iran during 2010–2012. The provinces divided into 5 different groups based on geographical distance and climates. One hundred and nine samples (28.2%) were confirmed positive for rotavirus group A using ELISA. 75 positive samples were selected randomly and subjected to typing assay. G10 (50.6%) and P[11] (64%) were detected more than G6 (21.3%) and P[5] (9.3%). No any G8 and P[1] were observed. Of the 75 samples analyzed by RT-PCR in each geographical areas named as the south of Alborz mountain ranges area, the north-east area, the central area, the north-west area and the south-east area, number of samples with G10 genotype were 19, 9, 9, 0 and 1; G6 were 8, 0, 3, 5 and 0; P[11] were 25, 7, 11, 5 and 0 and finally P[5] were 5, 0, 2, 0 and 0 in each area, respectively. The most common VP7/VP4 combinations were G10P[11] (40%), G6P[11] (12%), G6P[5] (5.3%) and G10P[5] (2.6%). Phylogenetic analysis of one strain showed high identity with strain B223. Since the identification of G and P genotypes and their diversity is fundamental to development and use of effective vaccines, we determined the most prevalence G and P genotypes of bovine rotavirus group A (BRVA) in a broad area of Iran.

The study was aimed to determine the Rotavirus infection rates in diarrheic calves and effectiveness of some epidemiological factors on that rate in some provinces in Mid–Euphrates region in Iraq (Babylon, Al-Najaf, Karbala, Al-Qadisyia provinces). A total of 530 calves aged between 1-60 day were examined and fifty fecal (50) samples were collected from diarrheic calves only within six months period from November-2012 to April -2013. The fecal samples were examined for the presence of rotavirus by rapid rotavirus test (screening test), as well as the clinical examination for these diarrheic calves were done. The rapid test demonstrated that the rotavirus infection rate was 18/50 (36%). A 45.4% of rotavirus infection was found in calves less than 30 days of age which was significantly higher than 28.5% calves (30-60) days old. Male and female (37.5%, 40% respectively) were seen infected without any significant differences. The main clinical signs observed on rotavirus diarrheic calves were include, watery voluminous diarrhea with profound weakness and mild depression, so as temperature, respiration, heart rates could be decreased and appetite was changeable towards loss or anorectic. In Babylon, the highest rotavirus infection rate was recorded at 60%, and fewer rates in Karbala 20% and 40%, 33% in AL-Najaf and AL-Qadisyia provinces respectively. The rotavirus infection rate in diarrheic calves was affected by months significantly, which it was higher at January 75%, so as in November 50%, December 40% and February 33.3%, but not recorded in March and April.

Background Rotavirus Gastroenteritis (RVGE) is an important global public health problem. Recently a Lyophilized Pentavalent Human Bovine Reassortant Rotavirus vaccine (BRV-PV, Rotasiil) was licensed in India. A Liquid formulation of the same vaccine (LBRV-PV) was tested in a Phase I clinical trial. Methods Total 20 healthy adults were given a single oral dose of LBRV-PV and were followed for one month for safety outcomes: immediate reactions, solicited reactions, unsolicited adverse events and serious adverse events. Results All 20 adults completed the study without any major protocol deviations. No immediate reaction, solicited reactions and unsolicited adverse events were reported during the study. No clinically significant changes were seen in the vital parameters and safety laboratory test results. Conclusions LBRV-PV developed in India was safe and well tolerated in adults. Further clinical development of this vaccine in infants should be undertaken. Trial Registration – CTRI/2015/11/006,384.

A bovine rotavirus pentavalent vaccine (BRV-PV) containing rotavirus human-bovine (UK) reassortant strains of serotype G1, G2, G3, G4 and G9 has been developed by the Serum Institute of India Ltd, in collaboration with the National Institute of Allergy and Infectious Diseases (NIAID), USA. The vaccine underwent animal toxicity studies and Phase I and II studies in adults, toddlers and infants. It has been found safe and immunogenic and will undergo a large Phase III study to assess efficacy against severe rotavirus gastroenteritis.

Coordinated and synchronous virological surveillance for zoonotic viruses in both human clinical cases and animal reservoirs provides an opportunity to identify interspecies virus movement. Rotavirus is an important cause of viral gastroenteritis in humans and animals. We have documented the rotavirus diversity within co-located humans and animals sampled from the Mekong delta region of Vietnam using a primer-independent, agnostic, deep sequencing approach. A total of 296 stool samples (146 from diarrhoeal human patients and 150 from pigs living in the same geographical region) were directly sequenced, generating the genomic sequences of 60 human rotaviruses (all group A) and 31 porcine rotaviruses (13 group A, 7 group B, 6 group C and 5 group H). Phylogenetic analyses showed the co-circulation of multiple distinct rotavirus group A (RVA) genotypes/strains, many of which were divergent from the strain components of licensed RVA vaccines, as well as considerable virus diversity in pigs including full genomes of rotaviruses in groups B, C and H, none of which have been previously reported in Vietnam. Furthermore the detection of an atypical RVA genotype constellation (G4-P[6]-I1-R1-C1-M1-A8-N1-T7-E1-H1) in a human patient and a pig from the same region provides some evidence for a zoonotic event

The aim of the present study was to characterize changes in acute phase protein levels according to the occurrence of rotavirus diarrhea in calves in the first month of life. Blood and fecal samples were taken before colostrum intake and at 1, 2, 7, 15, 21 and 30 days of age from 24 Holstein calves allotted in three experimental groups: calves that did not present diarrhea (group A), calves that presented diarrhea, but tested negative for rotavirus in feces (group B), and calves that presented diarrhea and tested positive for rotavirus in feces (group C) (experiment 1). When the animals presented episodes of diarrhea, blood and fecal samples were taken at 24-hour intervals until the end of clinical signs (experiment 2). Serum proteins were separated by SDS-PAGE technique and rotavirus in feces was detected by PAGE. Data of experiment 1 were analyzed by ANOVA and Tukey's test, considered significant at P

Lumazine synthase from Brucella spp. (BLS) is a highly immunogenic decameric protein which can accommodate foreign polypeptides or protein domains fused to its N-termini, markedly increasing their immunogenicity. The inner core domain (VP8d) of VP8 spike protein from bovine rotavirus is responsible for viral adhesion to sialic acid residues and infection. It also displays neutralizing epitopes, making it a good candidate for vaccination. In this work, the BLS scaffold was assessed for the first time in plants for recombinant vaccine development by N-terminally fusing BLS to VP8d and expressing the resulting fusion (BLSVP8d) in tobacco chloroplasts. Transplastomic plants were obtained and characterized by Southern, northern and western blot. BLSVP8d was highly expressed, representing 40% of total soluble protein (4.85 mg/g fresh tissue). BLSVP8d remained soluble and stable during all stages of plant development and even in lyophilized leaves stored at room temperature. Soluble protein extracts from fresh and lyophilized leaves were able to induce specific neutralizing IgY antibodies in a laying hen model. This work presents BLS as an interesting platform for highly immunogenic injectable, or even oral, subunit vaccines. Lyophilization of transplastomic leaves expressing stable antigenic fusions to BLS would further reduce costs and simplify downstream processing, purification and storage, allowing for more practical vaccines. Fil: Alfano, Edgardo Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Lentz, Ezequiel Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Bellido, Demian. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Dus Santos, María José. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Wigdorovitz, Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina Fil: Bravo Almonacid, Fernando Felix. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina

Summary Eighteen Caesarian-derived, colostrum-deprived calves were orally infected with either bovine rotavirus, enterotoxigenic Escherichia coli (ETEC) or with a combination of both enteropathogens within the first 24 h of life. Animals which received ETEC developed severe diarrhea when 1010organisms were given, but remained healthy after inoculation of 107organisms. Animals infected with four different field strains of bovine rotavirus developed mild, transient diarrhea after an average incubation time of 29 h. They usually recovered within a few hours after onset of the symptoms. A synergistic effect was observed after simultaneous application of rotavirus and 107ETEC, resulting in severe diarrhea after an average incubation time of 22 h. These calves had significantly more rotavirus in the intestinal samples than to samples from monoinfected calves. Those calves, which were infected with 107ETEC followed by rotavirus 15 to 36 hours later, had significantly less rotavirus in the intestinal samples and shed significantly less rotavirus in their feces than to monoinfected calves. The number of ETEC organisms demonstrated in the small intestine was more dependent on the time of sampling than on the mode of infection. Zusammenfassung Synergismus nach experimenteller Infektion neugeborener Kalber mit bovinem Rotavirus und enterotoxischen Escherichia coli (ETEC) Achtzehn Kalber wurden innerhalb der ersten 24 Lebensstunden oral entweder mit bovinem Rotavirus, enterotoxinbildenden E. coli (ETEC) oder mit einer Kombination aus beiden Erregern infiziert. Tiere, die 1010ETEC erhielten, entwickelten eine schwere Durchfallerkrankung, wahrend 107ETEC keine Durchfallsymptome mehr auslosten. Tiere, die mit vier verschiedenen Rotavirus-Feldstammen infiziert wurden, zeigten nach einer durchschnittlichen Inkubationszeit von 29 Stunden eine milde, kurzzeitige Diarrhoe. Sie erholten sich wenige Stunden nach Sistieren des Durchfalls. Nach gleichzeitiger Infektion von Rotavirus mit 107ETEC wurde eine synergistische Interaktion beider Erreger beobachtet. Diese Kalber entwickelten eine schwere Durchfallerkrankung nach einer durchschnittlichen Inkubationszeit von 22 Stunden. In den Darmabschnitten dieser Tiere wurden signifikant hohere Rotavirusinfektionsitatstiter gefunden als bei Tieren, die mit Rotavirus allein infiziert wurden. Dagegen war die Virusausscheidung im Kot und in den Darmabschnitten signifikant verringert bei Tieren, denen zuerst 107ETEC und 15 bis 36 Stunden spater Rotavirus verabreicht wurde. Die Zahl der im Dunndarm festgestellten ETEC-Keime war in allen Gruppen starker vom Zeitpunkt der Probengewinnung nach Infektion als von der Art der Infektion abhangig. Resume Synergisme apres une infection experimentale de veaux nouveaux-nes avec un Rotavirus bovin et Escherichia coli enterotoxique (ETEC) Des infections mixtes experimentales ont ete faites sur des veaux nouveaux-nes, prives de colostrum avec un Rotavirus bovin et E. coli enterotoxique (ETEC). Dix-huit veaux ont ete infectes oralement dans les premieres 24 heures de vie avec un Rotavirus bovin ou E. coli enterotoxique (ETEC) ou avec une combinaison des deux agents. Les animaux ayant recu 1010ETEC developperent une grave diarrhee alors que 107ETEC ne provoquerent aucun symptome de diarrhee. Les animaux qui furent infectes avec quatre souches differentes de Rotavirus du terrain presenterent une legere et breve diarrhee apres un temps d'incubation de 29 heures. Ils ont ete gueris quelques heures apres la cessation de la diarrhee. Une interaction synergique des deux agents a ete observee apres une infection simultanee de Rotavirus avec 107ETEC. Ces veaux developerent une grave diarrhee apres une periode d'incubation moyenne de 22 heures. On a trouve des titres infectieux de Rotavirus significativement plus eleves dans les portions d'intestin de ces animaux que chez les veaux infectes seulement avec du Rotavirus. L'excretion virale dans les matieres fecales et dans les portions intestinales fut par contre significativement diminuee chez les animaux qui avaient recu d'abord 107ETEC et Rotavirus 15 a 36 heures plus tard. Le nombre des germes ETEC etablis dans l'intestin grele avec tous les groupes a dependu plus du moment des prelevements apres l'infection que du genre de l'infection. Resumen Sinergismo tras infeccion experimental de terneros recien nacidos con rotavirus bovino y Escherichia coli enterotoxica (ETEC) Dieciocho terneros que no recibieron calostro fueron infectados dentro de las 24 horas primeras de vida por via oral bien con un rotavirus bovino, E. coli enterotoxinogena (ETEC) o bien con una combinacion de ambos agentes etiologicos. Los animales que recibieron 1010ETEC desarrollaron una enteritis grave, mientras que 107ETEC no desencadenaron sintomas diarreicos. Los animales que fueron infectados con cuatro estirpes campales diferentes de rotavirus mostraron tras un tiempo medio de incubacion de 29 horas una diarrea benigna de duracion breve. Los mismos se recuperaron pocas horas despues del cese de la descomposicion. Tras la infeccion simultanea de rotavirus con 107ETEC se observo una interaccion sinergica de ambos agentes etiologicos. Estos terneros desarrollaron una enteritis grave tras un periodo medio de incubacion de 22 horas. En los tramos entericos de estos animales se hallaron titulos de infecciosidad significantemente mas elevados que en los animales que habian sido infectados solo con rotavirus. Sin embargo, la eliminacion de virus con las heces y en los tramos intestinales se hallaba reducida de forma significativa en los animales que recibieron primero 107ETEC y 15 hasta 36 horas despues rotavirus. La cuantia de los germenes ETEC comprobada en el intestino delgado dependia en todos los grupos mas del momento en que se recogia la muestra tras la infeccion que del tipo de infeccion.

AIM: To determine the presence of Group A rotavirus G6, G8, and G10 genotypes in calves in the North Island of New Zealand. METHODS: Faecal samples from 730 calves (

In this study, bovine coronavirus (BCV) and bovine rotavirus (BRV) seroprevalances were detected by ELISA in 107 goat blood serum samples obtained from five different provinces of Northern Turkey. The results of the study reflected 41.12% and 82.24% seropositivity against BCV and BRV, respectively, in the goat sera. BCV seroprevalance in mature goats is determined for the first time with this study. Furthermore, this is the first statement of BRV and BCV seroprevalances in the mature goat population in Turkey.

The aims was to construct a prokaryotic expression plasmid and develop a subunit vaccine for bovine rotavirus (BRV), also to investigate the efficacy of this vaccine. BRV total RNA was extracted from M-A104 cell infected by the strain GSB01 of bovine rotavirus. Total RNA as template, BRV VP7 gene was amplified with real time fluorescence quantitative PCR (qPCR), PCR product was cloned into pEASY-T3 vector. The pEASY-T3-VP7 plasmid was recombined into the prokaryotic expression vector pET32a (+) after it was digested using double enzymes. The pET32a-VP7 and VP7-LTB used for prokaryotic expression were constructed, which were converted into Escherichia coli BL21 (DE3) competent cells. After IPTG induction and SDS-PAGE analysis were performed, the fusion proteins of pEASY-T3-VP7 and VP7-LTB with molecular weights of 42.2kD and 53.2kD were acquired respectively. Such a gene engineering bacteria for expressing VP7 protein within BL21(DE3) cells was constructed successfully. The mice immunized using both fusion proteins of pEASYT3-VP7 and VP7-LTB could promote VP7 IgG antibody production (8.33 vs 17.3). The immunization protection ratio of both fusion proteins in neonatal mice was 86.4% and 91.7%, respectively. These findings laid a foundation for further developing a high-efficiency subunit vaccine of BRV VP7 gene.

Bovine neonatal diarrhea is a disease, with etiology that can be infectious or non-infectious, which affects calves during the first three and causes serious economic losses in the dairy industry worldwide. This study describes the disease in a dairy herd in the Northern Antioquia Highlands. A cohort of 60 calves was evaluated during the first month of life through clinical examinations, haematological and serology analysis, and ELISA in fecal samples to detect Cryptosporidium parvum, bovine rotavirus type A, bovine coronavirus and E. coli K99. Additionally, the concentration of immunoglobulin in the colostrum administered and the passive transfer of immunoglobulin were estimated. Proportions of morbidity because of diarrhea were 94.92%, general mortality 17.58%, and lethality 15.79%. Among the group of sick animals 89.47%, 47.37%, 7.02% and 0% were found for each pathogenic agent respectively. A high percentage of animals with reactive lymphocytes were observed which indicates parasitic or viral infection. The results obtained from this study suggest that circulation of agents involved in bovine neonatal diarrhea can exist in the area, which generates concern because of the unknown sanitary status in the Northern Antioquia Highlands. RESUMEN: La diarrea neonatal bovina es una enfermedad cuya etiología puede ser infecciosa o no infecciosa, afecta los neonatos bovinos especialmente durante las tres primeras semanas de vida y causa serias pérdidas económicas en el sector lechero a nivel mundial. En este estudio se describió la enfermedad en un hato de producción lechera en el altiplano norte de Antioquia. Se evaluó una cohorte de 60 terneros durante su primer mes de vida mediante examen clínico, análisis hematológicos, de serología, y ELISA en materia fecal para Cryptosporidium parvum, rotavirus bovino tipo A, coronavirus bovino y E. coli K99. Adicionalmente, se estimó la concentración de inmunoglobulinas en el calostro suministrado y la transferencia pasiva de inmunoglobulinas. Se encontraron proporciones de morbilidad por diarrea de 94,92%, mortalidad general de 17,58% y letalidad del 15,79%. Entre los animales enfermos se encontró un porcentaje de 89,47%, 47,37%, 7,02% y 0% para cada agente patógeno respectivamente. Se evidenció un alto porcentaje de animales con linfocitos reactivos, lo que indica la infección con agentes parasitarios o virales. Los resultados obtenidos en este estudio sugieren que puede existir circulación de agentes involucrados en la diarrea neonatal bovina en la zona, lo que genera preocupación por el desconocimiento del estatus sanitario del altiplano norte de Antioquia en general.

Rotavirus ribonucleic acid (RNA) was extracted from ten faecal samples of diarrhoeic calves positive for group A rotavirus by enzyme-linked immunosorbent assay (ELISA). A portion of the extracted RNA was run in polyacrylamide gel to determine the presence of rotaviral RNA and the rest subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) to generate the near full length VP7 gene. Only six samples yielded the desired product. The amplified products were subjected to G-typing by PCR using a cocktail of G6, G8 and G10 typing primers. All of the six samples were characterised as G10 and none of the samples revealed mixed infection by twin G types. Four samples, despite possessing sufficient rotavirus particles as revealed by ELISA and polyacrylamide gel electrophoresis, did not yield any amplified product on RT-PCR. This could be due to non-specific inhibitors of the PCR reaction, present in the faecal samples, being carried through the extraction procedures.

The effect of colostral maternal antibodies (Abs), acquired via colostrum, on passive protection and development of systemic and mucosal immune responses against rotavirus was evaluated in neonatal calves. Colostrum-deprived (CD) calves, or calves receiving one dose of pooled control colostrum (CC) or immune colostrum (IC), containing an IgG1 titer to bovine rotavirus (BRV) of 1:16,384 or 1:262,144, respectively, were orally inoculated with 105.5 FFU of IND (P[5]G6) BRV at 2 days of age. Calves were monitored daily for diarrhea, virus shedding and anti-BRV Abs in feces by ELISA. Anti-rotavirus Ab titers in serum were evaluated weekly by isotype-specific ELISA and virus neutralization (VN). At 21 days post-inoculation (dpi), all animals were euthanized and the number of anti-BRV antibody secreting cells (ASC) in intestinal and systemic lymphoid tissues were evaluated by ELISPOT. After colostrum intake, IC calves had significantly higher IgG1 serum titers (GMT=28,526) than CC (GMT=1195) or CD calves (GMT

Rotavirus is a nonenveloped virus with a three-layered capsid. The inner layer, made of VP2, encloses the genomic RNA and two minor proteins, VP1 and VP3, with which it forms the viral core. Core assembly is coupled with RNA viral replication and takes place in definite cellular structures termed viroplasms. Replication and encapsidation mechanisms are still not fully understood, and little information is available about the intermolecular interactions that may exist among the viroplasmic proteins. NSP2 and NSP5 are two nonstructural viroplasmic proteins that have been shown to interact with each other. They have also been found to be associated with precore replication intermediates that are precursors of the viral core. In this study, we show that NSP5 interacts with VP2 in infected cells. This interaction was demonstrated with recombinant proteins expressed from baculovirus recombinants or in bacterial systems. NSP5-VP2 interaction also affects the stability of VP6 bound to VP2 assemblies. The data presented showed evidence, for the first time, of an interaction between VP2 and a nonstructural rotavirus protein. Published data and the interaction demonstrated here suggest a possible role for NSP5 as an adapter between NSP2 and the replication complex VP2-VP1-VP3 in core assembly and RNA encapsidation, modulating the role of NSP2 as a molecular motor involved in the packaging of viral mRNA.

バター製造時の副産物であるバターミルクをブロメライン消化することにより得られたバターミルク加水分解物 (HBM) に耐熱性の抗ウシロタウイルス活性を認めた. 活性部位をDEAEおよびゲル濾過 (Superdex 75) にて部分精製を行い, 抗ロタウイルス活性を有する4つの画分HBM-0.2-A, HBM-0.2-B, HBM-0.4-AおよびHBM-0.4-Bを得た. 各画分のED50 (mg/ml ) はHBM-0.2-A, HBM-0.2-B, HBM-0.4-AおよびHBM-0.4-Bの順にそれぞれ0.16, 1.75, 0.14, 3.25であった. 各フラクションをSDS-PAGEにより分析した結果, HBM-0.2-A, HBM-0.2-B, HBM-0.4-AおよびHBM-0.4-Bの順に分子量14～30, 14～21, 14～35および14～21kDaに強いバンドが検出された. ウエスタンブロット後に小麦胚芽レクチン (WGA) で検出したところ, 活性の高かったHBM-0.2-AとHBM-0.4-Aは同様のパターンを示し, 分子量20～25, 30～35および70～100kDaの3つの糖ポリペプチドから成るフラクションであることが認められた. シアル酸含量が高かったHBM-0.2-BおよびHBM-0.4-Bは抗ロタウイルス活性が弱く, WGAでも検出されなかったことから, 抗ロタウイルス活性はシアル酸の結合部位を中心とした糖鎖構造に依存していることが推測された.

The bovine rotavirus (BRV) WC3 serves as the background strain in the development of a multivalent reassortant vaccine against rotavirus gastroenteritis in infants. The genes encoding the outer capsid spike protein VP4, the inner capsid protein VP6, the outer capsid glycoprotein VP7, and the viral enterotoxin NSP4 of BRV WC3 were sequenced. Comparative analysis of the deduced amino acids of the sequenced genes indicated that the BRV WC3 strain shares a high degree of amino acid identity with serotype P7[5] VP4 (93–96%), serotype G6 VP7 (91–97%), subgroup (SG) I VP6 (96–99%), and NSP4 genogroup A (96–98%) BRV strains. Our results confirm and extend previous studies which suggested that the VP4 of BRV WC3 was closely related to that of the P7[5] prototype, BRV UK. In addition, the VP6 and VP7 of BRV WC3 were very similar to the VP6 and VP7 of both SG I and G6 BRV NCDV and UK strains. However, the NSP4 of BRV WC3 was more closely related to that BRV NCDV, the P6[1] prototype, than to that of BRV UK.

Three doses of a live, oral bovine rotavirus pentavalent vaccine (BRV-PV) protected healthy infants in Niger from severe rotavirus gastroenteritis, reported a team of international investigators in the New England Journal of Medicine. Rotavirus is a leading cause of diarrhea-associated hospitalization and death in children younger than 5 years around the world, with the highest death rate from this virus occurring in sub-Saharan Africa. Currently there are 2 licensed vaccines in use against rotavirus, but both require refrigeration. The BRV-PV vaccine, which was designed specifically for use in remote areas with limited electricity or health clinics, is freeze-dried, does not require refrigeration, and is thermostable for 2 years at 37°C and for 6 months at 40°C. First Page Preview

Background: Worldwide, Group A bovine rotavirus (RVA boRV) is one of the main causes of neonatal calf diarrhoea. Currently, limited epidemiological and sequence data exists on the RVA disease in bovines in Southern Ireland only. The aim of the study was to generate epidemiological and sequence data of RVA boRV distributed over a wide geographical area in Ireland. Findings: 272 stool samples were obtained from symptomatic calves and analysed to identify the prevalent G and P genotypes. Viral type combinations including G6P[5], G6P[11] and G10P[11] genotype were the most frequently identified. The G6P[5] combination was predominant throughtout the study, accounting for 70% (n=191). Sequence analysis of the VP7 gene revealed that Irish G6 strains fell within Lineage IV, similiar to previous reports in Ireland. Conclusion: The detection of unusual G and P combinations may have an impact on rotavirus control programmes and current vaccines may need to incorporate new strains, as the current vaccine available may not offer protection against all of these circulating types.

To investigate the epidemical characteristics and genotypic distribution of bovine rotavirus in Punjab, 120 fecal samples were collected from calves exhibiting diarrhea and screened for the presence of rotavirus using RNA-PAGE and RT-PCR. Twenty-three samples were positive by RNA-PAGE having electrophoretic patterns that corresponded to mammalian group A rotaviruses. All the samples were (120) screened by RT-PCR for VP7 and VP4 genes and 16 samples were found to be positive for VP7. Out of these 16 samples, 4 samples were also positive for VP4. These were analyzed further by multiplex semi-nested PCR for G and P genotypes, and it was confirmed that 43.75% (7/16) were the G3 type, while 6.25% (1/16) was the G8 type and 50% (8/16) were dual G3G8 types. P genotyping classified all the samples 100% (4/4) as the P[5] genotype. It may be concluded that most prevalent combination of rotavirus infection in bovine was G3G8P[5] in Punjab., Radi istraživanja epizootioloških obilježja i proširenosti genotipova goveđeg rotavirusa u Punjabu, prikupljeno je bilo 120 uzoraka izmeta teladi s proljevom te pretraženo na prisutnost rotavirusa RNApoliakrilamid-gel-elekroforezom (RNA-PAGE) i lančanom reakcijom polimerazom uz prethodnu reverznu transkripciju (RT-PCR). Od toga su 23 uzorka bila pozitivna pretragom RNA-PAGE s elektroforetskim uzorkom koji je odgovarao skupini A rotavirusa sisavaca. Svih 120 uzoraka izmeta bilo je pretraženo RT-PCR-om na prisutnost gena za proteine VP7 i VP4. Ustanovljeno je 16 pozitivnih za VP7. Od tih 16, četiri su bila pozitivna i za VP4. Ti su dodatno bili pretraženi višestrukom poluugniježđenom lančanom reakcijom polimerazom za genotipove G i P. Utvrđeno je da je 43,75% (7/16) pripadalo genotipu G3, dok je 6,25% (1/16) bilo genotipa G8, a 50% (8/16) pripadalo je i G3 i G8 genotipu. Svi su uzorci (4/4) na osnovi P genotipizacije pripadali genotipu P[5]. Može se zaključiti da je infekcija goveda u Punjabu najčešće uzrokovana kombinacijom rotavirusa G3, G8 i P[5].

Objective To identify the distribution of G serotype and P genotype of bovine group A rotavirus in Japan. Design Detection, isolation, electropherotyping, G serotyping and P genotyping of bovine group A rotavirus in 167 diarrhoeal faecal samples. Procedure Bovine group A rotavirus was detected and isolated, and bovine group A rotavirus isolates were identified electropherotype by polyacrylamide gel electrophoresis and G serotype and P genotype by reverse transcription-polymerase chain reaction. Results Twenty-eight bovine group A rotavirus strains were isolated from 167 samples. Electropherotypes of the bovine group A rotavirus isolates were identified as long-genome electropherotype and the most prevalent combination of G serotype and P genotype of the bovine group A rotavirus isolates was G6P5 (25/28; 89.3%), followed by G6P11 (2/28; 7.1%) and G10P11 (1/28; 3.6%). Conclusion These results suggest that the bovine group A rotavirus exhibiting G6P5 is the most common in Japan.

The performance characteristics of 2 enzyme immunoassays (ELISAs) and 4 latex agglutination assays (LXs) were evaluated for the detection of bovine rotavirus in faecal specimens of young calves with diarrhoea. A total of 26 specimens from calves less than 5 months of age were examined with different commercial assays and compared with electron microscopy (EM) as the gold standard and with polyacrylamide gel electrophoresis (PAGE) for the detection of atypical, non-group A rotaviruses. In the 2nd study, EIA (Dako) and LX (Murex), the assays of choice, were used to analyse 97 further faecal specimens from calves with diarrhoea. The ELISAs proved to be the most sensitive compared with the other tests used. The EM and PAGE are 100 % specific although slightly less sensitive than the commercial assays. The results show that all the commercial assays can accurately detect rotavirus in the stools of calves with gastroenteritis, although the suitability and choice of assay will depend upon the requirements of individual laboratories.

La diarrea neonatal bovina indiferenciada es una enfermedad multifactorial que afecta a una alta proporción de becerros (50% o más) causando hasta un 40% de mortalidad en los primeros 30 d de vida. Entre los agentes causales se incluyen muchos enteropatógenos (bacterias, virus, protozoarios), con predominio de infecciones combinadas. Entre los virus, los rotavirus constituyen los principales agentes causales de gastroenteritis aguda en diferentes especies animales. Su genoma, de 11 segmentos de ARN de doble cadena, puede rearreglarse genéticamente en coinfecciones de diferentes cepas, produciendo una progenie viral con un nuevo fenotipo. En la ganadería bovina venezolana, se han descrito los rotavirus con una prevalencia de 20-30% en animales con cuadros de diarrea entre 2-6 sem de edad. Los serotipos G6 y el genotipo P1 se han reportado con mayor frecuencia en becerros de la región central de Venezuela. Cepas de rotavirus bovino del grupo A fueron detectados por inmunoensayo enzimático (ELISA) y electroforesis en gel de poliacrilamida (PAGE), en 32 de 280 (11,43%) muestras de heces de becerros de 15 fincas del estado Yaracuy, Venezuela. La secuencia nucleotídica se logró para una de las variantes detectadas (A610). En el análisis comparativo de las secuencias de la VP8* (fragmento de la VP4) mostró corresponder al genotipo P[5]. Estos datos constituyen el primer reporte de una cepa de rotavirus bovino genotipo P[5], que circula en las fincas del estado Yaracuy, Venezuela, pudiendo servir de apoyo para la incorporación de este grupo viral en las vacunas que se utilizan en el país. The undifferentiated neonatal bovine diarrhea is a multifactorial disease that affects a high proportion of calves (50% or more) causing up to 40% of mortality in the first 30 d of life. Among the infectious agents known to cause diarrhea in calves are the rotavirus and coronavirus. Rotaviruses are the major causative agents of acute gastroenteritis in different animal species. Their 11-segment genome of double ARN chain can genetically re-arrange different strains, producing a viral progeny with new or atypical phenotype. Rotaviruses have been described in the bovine Venezuelan cattle, with a prevalence of 20-30% in animals between 2-6 weeks of age, with episodes of diarrhea. The serotypes G6 and the genotype P1 have been reported with higher frequency in calves from the central region of Venezuela. In this investigation, rotavirus strains of cattle from Group A were detected by the enzyme-linked immunosorbent assay (ELISA) and the polyacrylamide gel electrophoresis (PAGE) in 32 of 280 (11.43%) stool samples of calves in 15 farms in the State of Yaracuy, Venezuela. The nucleotide sequence was obtained for one of the variants detected (A610) and the comparative analysis of the sequences of the VP8* (the VP4 fragment) corresponded to the P [5] genotype. These data constitute the first report of a strain of bovine rotavirus genotype P[5], which circulates in the States of Yaracuy, Venezuela, and can serve as a support for the inclusion of this viral group to the vaccines that are being used in the country.

The purpose of this study was to monitor by negative stain electron microscopy the shedding of rotavirus in the feces of gnotobiotic calves orally inoculated with a commercial modified live bovine rotavirus-bovine coronavirus vaccine. Negative stain electron microscopic examination detected vaccine rotavirus in only 1 of 41 daily fecal specimens collected from 3 gnotobiotic calves during the 2 weeks following oral inoculation with a US Department of Agriculture-licensed modified live bovine rotavirus-bovine coronavirus vaccine. In contrast, rotavirus was demonstrable by the same negative stain electron microscopic examination procedure in 17 of 19 fecal specimens collected from diarrheic gnotobiotic or colostrum-deprived calves during the first 8 days after inoculation with virulent bovine rotavirus field strains. Rotavirus was also detected by this procedure in 4 enzyme-linked immunosorbent assay positive fecal specimens collected from naturally-infected diarrheic dairy calves. These results suggest that fecal shedding of vaccine rotavirus demonstrable by electron microscopic examination is uncommon following oral inoculation of calves with the bovine rotavirus-bovine coronavirus vaccine.

During the rotavirus strain surveillance in Slovenia, G6P[11] bovine rotavirus strain was detected in a 5 months old boy with gastroenteritis. The strain was enrolled in a whole genome sequence analysis to determine its genome segment composition and genetic characteristics. Genotype composition for the whole genome was G6-P[11]-I2-R2-C2-M2-A13-N2-T6-E2-H3, reflecting similarities with bovine rotavirus strains. The bovine origin of the strain was confirmed in all genome segments, showing the highest nucleotide identity with bovine rotavirus strains and clustering of the RVA/Human-wt/SVN/SI-R56/07/2007/G6P[11] together with bovine rotavirus strains in phylogenetic analysis. This is the first bovine G6P[11] rotavirus strain with the whole genome analysis and the first report on rotavirus G6P[11] genotype detected in humans.