Prevalence of antibodies to four different rotavirus strains in different age groups of cattle

Neutralizing antibody titers to four bovine rotavirus strains, representing three serotypes, were measured in 160 sera from cattle of different age groups. Age-specific seroprevalence analysis revealed serotype 6, represented by bovine rotavirus (BRV) NCDV, as the predominant rotavirus serotype infecting German cattle and serotype 10, represented by BRV V1005, as the least prominent. Infections with serotype 8, represented by BRV 678, occurred with intermediate frequency. Antibodies of young calves distinguished between NCDV and UK virus, two serotype 6 BRV strains differing in VP4 antige

Uniformity of rotavirus strain nomenclature proposed by the Rotavirus Classification Working Group (RCWG)

In April 2008, a nucleotide-sequence-based, complete genome classification system was developed for group A rotaviruses (RVs). This system assigns a specific genotype to each of the 11 genome segments of a particular RV strain according to established nucleotide percent cutoff values. Using this approach, the genome of individual RV strains are given the complete descriptor of Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx. The Rotavirus Classification Working Group (RCWG) was formed by scientists in the field to maintain, evaluate and develop the RV genotype classification system, in particular to aid in the designation of new genotypes. Since its conception, the group has ratified 51 new genotypes: as of April 2011, new genotypes for VP7 (G20-G27), VP4 (P[28]-P[35]), VP6 (I12-I16), VP1 (R5-R9), VP2 (C6-C9), VP3 (M7-M8), NSP1 (A15-A16), NSP2 (N6-N9), NSP3 (T8-T12), NSP4 (E12-E14) and NSP5/6 (H7-H11) have been defined for RV strains recovered from humans, cows, pigs, horses, mice, South American camelids (guanaco), chickens, turkeys, pheasants, bats and a sugar glider. With increasing numbers of complete RV genome sequences becoming available, a standardized RV strain nomenclature system is needed, and the RCWG proposes that individual RV strains are named as follows: RV group/species of origin/country of identification/common name/year of identification/G- and P-type. In collaboration with the National Center for Biotechnology Information (NCBI), the RCWG is also working on developing a RV-specific resource for the deposition of nucleotide sequences. This resource will provide useful information regarding RV strains, including, but not limited to, the individual gene genotypes and epidemiological and clinical information. Together, the proposed nomenclature system and the NCBI RV resource will offer highly useful tools for investigators to search for, retrieve, and analyze the ever-growing volume of RV genomic data.Fil: Matthijnssens, Jelle. Katholikie Universiteit Leuven; BélgicaFil: Ciarlet, Max. Novartis Vaccines & Diagnostics; Estados UnidosFil: McDonald, Sarah M.. National Institute Of Allegry & Infectious Diseases (niaid) ; National Institutes Of Health;Fil: Attoui, Houssam. Animal Health Trust.; Reino UnidoFil: Bányai, Krisztián. Hungarian Academy of Sciences; HungríaFil: Brister, J. Rodney. National Library Of Medicine; Estados UnidosFil: Buesa, Javier. Universidad de Valencia; EspañaFil: Esona, Mathew D.. Centers for Disease Control and Prevention; Estados UnidosFil: Estes, Mary K.. Baylor College of Medicine; Estados UnidosFil: Gentsch, Jon R.. Centers for Disease Control and Prevention; Estados UnidosFil: Iturriza Gómara, Miren. Health Protection Agency; Reino UnidoFil: Johne, Reimar. Federal Institute for Risk Assessment; AlemaniaFil: Kirkwood, Carl D.. Royal Children's Hospital; AustraliaFil: Martella, Vito. Università degli Studi di Bari; ItaliaFil: Mertens, Peter P. C.. Animal Health Trust.; Reino UnidoFil: Nakagomi, Osamu. Nagasaki University; JapónFil: Parreño, Gladys Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Rahman, Mustafizur. International Centre For Diarrhoeal Disease Research; BangladeshFil: Ruggeri, Franco M.. Istituto Superiore Di Sanita; ItaliaFil: Saif, Linda J.. Ohio State University; Estados UnidosFil: Santos, Norma. Universidade Federal do Rio de Janeiro; BrasilFil: Steyer, Andrej. University of Ljubljan; EsloveniaFil: Taniguchi, Koki. Fujita Health University School of Medicine; JapónFil: Patton, John T.. National Institute Of Allegry & Infectious Diseases (niaid) ; National Institutes Of Health;Fil: Desselberger, Ulrich. University of Cambridge; Estados UnidosFil: van Ranst, Marc. Katholikie Universiteit Leuven; Bélgic

Phylogenetic analyses of typical bovine rotavirus genotypes G6, G10, P[5] and P[11] circulating in Argentinean beef and dairy herds

Group A rotavirus (RVA) is one of the main causes of neonatal calf diarrhea worldwide. RVA strains affecting Argentinean cattle mainly possess combinations of the G6, G10, P[5] and P[11] genotypes. To determine RVA diversity among Argentinean cattle, representative bovine RVA strains detected in diarrheic calves were selected from a survey conducted during 1997–2009. The survey covered the main livestock regions of the country from dairy and beef herds. Different phylogenetic approaches were used to investigate the genetic evolution of RVA strains belonging to the prevalent genotypes. The nucleotide phylogenetic tree showed that all genotypes studied could be divided into several lineages. Argentinean bovine RVA strains were distributed across multiple lineages and most of them were distinct from the lineage containing the vaccine strains. Only the aminoacid phylogenetic tree of G6 RVA strains maintained the same lineages as observed at the nucleotide level, whereas a different clustering pattern was observed for the aminoacid phylogenetic trees of G10, P[5] and P[11] suggesting that the strains are more closely related at the aminoacid level than G6 strains. Association between P[5] and G6(IV), prevalent in beef herd, and between P[11] and G6(III) or G10 (VI and V), prevalent in dairy herds, were found. In addition, Argentinean G6(III), G10, P[5] and P[11] bovine RVA strains grouped together with human strains, highlighting their potential for zoonotic transmission. Phylogenetic studies of RVA circulating in animals raised for consumption and in close contact with humans, such as cattle, contribute to a better understanding of the epidemiology of the RVA infection and evolution.Fil: Badaracco, Alejandra. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Garaicoechea, Lorena Laura. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Matthijnssens, J.. University of Leuven. Rega Institute for Medical Research; BélgicaFil: Louge Uriarte, Enrique Leopoldo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Área de Investigación en Producción y Sanidad Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Odeón, Anselmo Carlos. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Área de Investigación en Producción y Sanidad Animal; ArgentinaFil: Bilbao, Gladys Noemí. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Fernandez, Fernando. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Parra, G. I.. National Institutes of Health; Estados UnidosFil: Parreño, Gladys Viviana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Molecular determinants of rotavirus virulence

Rotaviruses are the single most important etiological agent of severe diarrhoea in infants and young children in both developed and developing countries. The World Health Organisation has identified the development of a rotavirus vaccine as a priority area for routine childhood immunisation to control rotavirus infections. However, the candidate vaccine strains have not been very successful. The main aim of this project was to map rotavirus virulence to its gene segments. Such studies can help in developing better vaccines for the control of rotavirus induced diarrhoea. A three step approach was undertaken (i) development of an animal model, (ii) construction and characterisation of reassortants between rotavirus strains of different virulence, (iii) mapping virulence to rotavirus gene segments. The mouse model developed revealed that the outcome of rotavirus infection was influenced by viral dose and viral strain as well as by host age and host strain. Homologous murine rotavirus strain was found to be most virulent. Among the heterologous strains studied, the OSU strain was found to be most virulent and UKtc strain the least virulent. The CD- 1 strain of mouse was found to be the most susceptible to virus infection and C57/BL the least susceptible. A very simple and rapid nucleic acid extraction method has been developed that requires only one centrifugation step and circumvents the use of any hazardous organic chemicals, which can be applied to very large numbers of samples saving time and labour. Rotavirus reassortants were constructed in a variety of ways and their genotype determined from relative mobility of their gene segments on polyacrylamide gels and restriction enzyme digestion of PCR amplified products. Twenty two reassortants (2%) were identified out of more than 1100 progeny clones examined and these reassortants belonged to 15 different genotypes. Possible reasons for obtaining this low number of reassortants are discussed. No reassortant could be identified between a murine rotavirus and other heterologous rotavirus strains. Preliminary sequence of VP7 gene of murine rotavirus strains, EDIM and EBR, was found to be different to the published rotavirus sequences including the recently published five murine rotavirus strains. The virulence mapping studies conducted in mice with some of the 22 reassortants obtained in the present study showed that gene 4 of the OSU and UKtc strains was involved in virulence. Segment 5 of OSU strain and segments 5, and 8 of UKtc strain may also be involved in virulence

Functional studies on the rotavirus non-structural proteins NSP5 and NSP6

The rotavirus replication cycle has not been fully characterised, one vital stage of virus replication involves large cytoplasmic occlusion bodies termed viroplasms. These are the sites of synthesis and replication of dsRNA, packaging of viral RNA into newly synthesized cores and the formation of double-shelled previrions. The detailed mechanism by which these events occur is poorly understood but is thought to be mediated by the non-structural proteins localised to these structures. Rotavirus gene segment 11 expresses two proteins NSP5 and NSP6 which are found in alternate open reading frames. NSP5 exists is several isoforms which differ on their level of phosphorylation. It has been shown to be essential for virus replication and localises to the viroplasms. The smaller NSP6 protein is the most uncharacterised of all of the rotavirus proteins. It has however been shown to interact with NSP5 and has been tentatively suggested to be localised to the viroplasms. To further investigate these two proteins the pET expression system was utilised to obtain purified protein which was subsequently used to generate mono specific polyclonal antisera. Studies into the function and localisation of these proteins found that both localised to the viroplasms and their relative distributions within these structures were defined. NSP6 was found to be expressed at a low level throughout the course of a rotavirus infection and in contrast to other non-structural proteins, to have a high rate of turnover. The RNA binding ability of both NSP5 and NSP6 was investigated using quantitative filter binding assays and these showed both have sequence independent nucleic acid binding ability. Studies were also conducted into the mechanism of NSP6 expression from the second open reading frame of gene 11, the results obtained being consistent with a leaky scanning mechanism of expression

Using species A rotavirus reverse genetics to engineer chimeric viruses expressing SARS-CoV-2 spike epitopes:Heterologous viral peptide expression by rotavirus A

Species A rotavirus (RVA) vaccines based on live attenuated viruses are used worldwide in humans. The recent establishment of a reverse genetics system for rotoviruses (RVs) has opened the possibility of engineering chimeric viruses expressing heterologous peptides from other viral or microbial species in order to develop polyvalent vaccines. We tested the feasibility of this concept by two approaches. First, we inserted short SARS-CoV-2 spike peptides into the hypervariable region of the simian RV SA11 strain viral protein (VP) 4. Second, we fused the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, or the shorter receptor binding motif (RBM) nested within the RBD, to the C terminus of nonstructural protein (NSP) 3 of the bovine RV RF strain, with or without an intervening Thosea asigna virus 2A (T2A) peptide. Mutating the hypervariable region of SA11 VP4 impeded viral replication, and for these mutants, no cross-reactivity with spike antibodies was detected. To rescue NSP3 mutants, we established a plasmid-based reverse genetics system for the bovine RV RF strain. Except for the RBD mutant that demonstrated a rescue defect, all NSP3 mutants delivered endpoint infectivity titers and exhibited replication kinetics comparable to that of the wild-type virus. In ELISAs, cell lysates of an NSP3 mutant expressing the RBD peptide showed cross-reactivity with a SARS-CoV-2 RBD antibody. 3D bovine gut enteroids were susceptible to infection by all NSP3 mutants, but cross-reactivity with SARS-CoV-2 RBD antibody was only detected for the RBM mutant. The tolerance of large SARS-CoV-2 peptide insertions at the C terminus of NSP3 in the presence of T2A element highlights the potential of this approach for the development of vaccine vectors targeting multiple enteric pathogens simultaneously. IMPORTANCE We explored the use of rotaviruses (RVs) to express heterologous peptides, using SARS-CoV-2 as an example. Small SARS-CoV-2 peptide insertions (<34 amino acids) into the hypervariable region of the viral protein 4 (VP4) of RV SA11 strain resulted in reduced viral titer and replication, demonstrating a limited tolerance for peptide insertions at this site. To test the RV RF strain for its tolerance for peptide insertions, we constructed a reverse genetics system. NSP3 was C-terminally tagged with SARS-CoV-2 spike peptides of up to 193 amino acids in length. With a T2A-separated 193 amino acid tag on NSP3, there was no significant effect on the viral rescue efficiency, endpoint titer, and replication kinetics. Tagged NSP3 elicited cross-reactivity with SARS-CoV-2 spike antibodies in ELISA. We highlight the potential for development of RV vaccine vectors targeting multiple enteric pathogens simultaneously

Ferrets exclusively synthesize Neu5Ac and express naturally humanized influenza A virus receptors

Mammals express the sialic acids ​N-acetylneuraminic acid (​Neu5Ac) and ​N-glycolylneuraminic acid (​Neu5Gc) on cell surfaces, where they act as receptors for pathogens, including influenza A virus (IAV). ​Neu5Gc is synthesized from ​Neu5Ac by the enzyme cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH). In humans, this enzyme is inactive and only ​Neu5Ac is produced. Ferrets are susceptible to human-adapted IAV strains and have been the dominant animal model for IAV studies. Here we show that ferrets, like humans, do not synthesize ​Neu5Gc. Genomic analysis reveals an ancient, nine-exon deletion in the ferret CMAH gene that is shared by the Pinnipedia and Musteloidia members of the Carnivora. Interactions between two human strains of IAV with the sialyllactose receptor (sialic acid—α2,6Gal) confirm that the type of terminal sialic acid contributes significantly to IAV receptor specificity. Our results indicate that exclusive expression of ​Neu5Ac contributes to the susceptibility of ferrets to human-adapted IAV strains

The role of NSP1 in rotavirus pathogenesis

NSP1, a non-structural protein encoded by rotavirus gene segment 5, has been suggested as a virulence determinant for rotavirus and to function as an antagonist of the interferon signalling pathway. Although non-essential for rotavirus replication in cell culture, and is the least conserved in all rotavirus proteins, NSP1 from different rotavirus strains of different species has been demonstrated to interact with several cellular proteins involved in the IFNβ induction pathway. NSP1 from a bovine rotavirus strain (UKtc) has been shown to interact with and to degrade IRF3 in a proteasome dependent manner whereas NSP1 from a porcine rotavirus strain (OSU) fails to target IRF3 but is able to interfere with IFNβ production via similar targeting of β-TrCP. The research presented in this thesis sought to gain a better understanding of the molecular determinants of NSP1 specificity for targeting the IFNβ pathway by mapping the regions in NSP1 sequences responsible for targeting specific cellular proteins. NSP1 hybrid constructs with sequences from both UKtcNSP1 and OSUNSP1 were generated and their interactions with both IRF3 and β-TrCP were tested in a series of assays. The initial attempts to map interaction sites using the mammalian two-hybrid assay were not successful. No reporter plasmid signal was generated indicating the expected interaction. The failure of this assay might be due to the insufficient expression of the NSP1 proteins as subsequent modification of the expression vector\ud was shown to improve the expression level of NSP1 proteins in subsequent reporter assay analysis. Using IFNβ promoter reporter assays to demonstrate the functional consequence of NSP1 action in IRF3, it was found that the constructs containing the entire Cterminal part of UKtcNSP1 were able to reduce IRF3-induced IFNβ promoter activity. Such constructs also caused IRF3 degradation in a proteasome dependent manner in agreement with previous studies. However, the sequence containing the last 135 amino acids from UKtcNSP1 was not sufficient for these activities. Collectively, these data suggested that the sequence between amino acid position 165 and 135 from the C-terminus are required for this interaction and subsequent degradation of IRF3. Similar experiments focused on determining the interaction site for β-TrCP on NSP1 were more difficult to interpret according the data presented. Unexpectedly in the light of published data, not only OSUNSP1 was able to degrade β-TrCP but UKtcNSP1 appeared to have the similar effect, as well as two reciprocal pairs of NSP1 hybrid constructs. In summary, it appears that sequences from the C-terminal part of UKtcNSP1 can function in a heterogeneous NSP1 context to target IRF3 from human cells. Further analysis is clearly required to fulfil the understanding of the role of NSP1 in rotavirus pathogenesis, including its interaction with β-TrCP

Analysis of RNAs and Proteins of Rotaviruses With Rearranged Genomes: A Study of Molecular Variability

Genome heterogeneity among cocirculating human rotaviruses (hrv) has been described in different parts of the world and in this project was exemplified by a small collection of 100 specimens collected from infected children in Britain between 1975 and 1983. Pedley et al. (1984) described protracted hrv infections and virus shedding in children who suffered from severe combined immunodeficiency (SCID). The viruses isolated from the children showed abnormal genomes carrying extra bands of dsRNA which were found to have arisen by rearrangement of normal RNA segments to covalently linked concatemers. Rotaviruses with similar genomes were obtained after serial passage in-vitro at high m.o.i.: Hundley et al. (1985) isolated mutants D, A4, B4 and B5 of bovine rotavirus (brv) whose genomes had lost RNA segment 5 and instead carried additional RNA bands A-H. The main part of the project was to further characterise these mutants. The additional RNA bands migrated between genomic segments 1 and 7, and were not integral multiples c segment 5 length. Further characterisation by RNase T1 oligonucleotide mapping (Follett and Desselberger, 1983b) showed that band A of brv mutant D (Hundley et al., 1985) and band E of brv mutant A4 (this thesis) consisted of segment 5-related sequences. The brv mutants D, A4, B4 and B5 were not replication-defective as they could be passaged through multiple rounds of plaque-to-plaque purification without requiring parent virus (standard brv). Reduction in the degree of genome transcription and replication, plaque formation, suppression of host cell protein synthesis and development of CPE was observed in cells infected with the brv mutants A4, B4 and B5 when compared to standard brv. However, brv mutant D was similar to standard brv in all these parameters. Standard brv and the brv mutants grew to different titres at 3