Characterisation of a rare, reassortant human G10P[14] rotavirus strain detected in Honduras

BACKGROUND Although first detected in animals, the rare rotavirus strain G10P[14] has been sporadically detected in humans in Slovenia, Thailand, United Kingdom and Australia among other countries. Earlier studies suggest that the strains found in humans resulted from interspecies transmission and reassortment between human and bovine rotavirus strains. OBJECTIVES In this study, a G10P[14] rotavirus genotype detected in a human stool sample in Honduras during the 2010-2011 rotavirus season, from an unvaccinated 30-month old boy who reported at the hospital with severe diarrhea and vomiting, was characterised to determine the possible evolutionary origin of the rare strain. METHODS For the sample detected as G10P[14], 10% suspension was prepared and used for RNA extraction and sequence independent amplification. The amplicons were sequenced by next-generation sequencing using the Illumina MiSeq 150 paired end method. The sequence reads were analysed using CLC Genomics Workbench 6.0 and phylogenetic trees were constructed using PhyML version 3.0. FINDINGS The next generation sequencing and phylogenetic analyses of the 11-segmented genome of the G10P[14] strain allowed classification as G10-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3. Six of the genes (VP1, VP2, VP3, VP6, NSP2 and NSP4) were DS-1-like. NSP1 and NSP5 were AU-1-like and NSP3 was T6, which suggests that multiple reassortment events occurred in the evolution of the strain. The phylogenetic analyses and genetic distance calculations showed that the VP7, VP4, VP6, VP1, VP3, NSP1, NSP3 and NSP4 genes clustered predominantly with bovine strains. NSP2 and VP2 genes were most closely related to simian and human strains, respectively, and NSP5 was most closely related to a rhesus strain. MAIN CONCLUSIONS The genetic characterisation of the G10P[14] strain from Honduras suggests that its genome resulted from multiple reassortment events which were possibly mediated through interspecies transmissions.

Study of the dynamic of rotavirus circulation by identification of their electropherotypes / Estudo da dinâmica de circulação de rotavírus através da identificação de seus eletroferótipos eletroferótipos

An electrophoretical study of rotavirus strains was carried out to determine the RNA migration pattern sand to follow the dynamics of virus circulation. Fifty five positive fecal samples for rotavirus, obtained from 0-5 year old children during 2005 and 2006, were subjected to the Polyacrylamide Gel Electrophoresis. It was possible to define the RNA migration pattern of 37 (62%) rotavirus strains, displaying eight distinct profiles, all of them compatible with group A rotavirus. All rotavirus were detected from May to September showing an outstanding seasonality. In 2005 were detected only rotavirus strains presenting long migration patterns (100%=15/15), named L1, L2, L3 or L4. The L2 profile was predominant. However, in 2006, a few rotavirus strains displaying long migration patterns (L1 and L2) were detected (14%=3/22) from May to June, although rotavirus strains presenting short migration patterns (S1, S2, S3 or S4) were detected between Julyand August. These viruses of short RNA migration patterns were the most predominant strains (86%=19/22). The variability of RNA migration patterns detected showed a great genomic heterogeneity of rotavirus strains. By monitoring viral nucleic acid electrophoretic characteristics was possible to follow the dynamics of rotavirus circulation between 2005 and 2006.

Foi realizado um estudo eletroforético de amostras de rotavírus, visando determinar os perfis de migração do RNA viral, bem como acompanhar a dinâmica da circulação dos mesmos. Para tanto, 55 amostras fecais diarréicas, positivas para rotavírus, obtidas de crianças de 0-5 anos, durante os anos de 2005 e 2006, foram submetidas à técnica de eletroforese em gel de poliacrilamida. Foi possível definir o perfil eletroforético de 37 (62%) delas, tendo sido observados oito perfis distintos, todos compatíveis com rotavírus do grupo A. Todas as amostras de rotavírus foram detectadas de maio a setembro, indicando uma sazonalidade na ocorrência destas infecções em Juiz de Fora. Em 2005 todas as amostras apresentaram perfis longos (100%=15/15), denominados de L1 a L4, com predominância de amostras de perfil L2. Em 2006, poucas amostras de perfil longo (14%=3/22) foram detectadas nos meses de maio e junho, tendo sido substituídas pelas amostras de perfil curto (C1-C4), que emergiram em julho, predominando neste ano (86%=19/22). A variabilidade dos perfis eletroforéticos detectados mostrou uma grande heterogeneidade genômica e através do monitoramento foi possível acompanhar a dinâmica de circulação das amostras de rotavírus entre 2005 e 2006.

Ultrastructural study of rotavirus replication and localization of the intermediate capsid protein VP6

Rotavirus, a triple-layered non-enveloped member of the Reoviridae family, obtained a transient membrane envelope when newly synthesized subviral particles bud into the endoplasmic reticulum [ER]. As rotavirus particles mature, they lose their transient membrane and obtain outer layer. It is mostly believed that only double layered particles bud into the ER. The present study describes that the single layered particles can also bud into the ER and become the immediate precursors of the mature virions. Virus replication was studied within a line of African green monkey kidney [BSC-1] cells infected with simian rotavirus SA11. The virus intermediate capsid protein [VP6] was localized within the infected cells using protein A-gold. Monospecific antibody to VP6 was the primary antibody. The electron micrographs of budding sites of ER showed two different sizes of subviral particles. The gold particles were seen on the double layered particles and very little in the cytoplasm and some on ER or very close to it. These results indicate that the single layered particles are also capable of being the precursor of the triple layered and obtain the VP6 while budding into the ER

Effect of isoprinosine on rotavirus replication in vitro

Isoprinosine (IPS) is a synthetic drug whose antiviral effect on rotavirus replication in vitro has been characterized in terms of the decrease in metachromasia after acridine orange staining. The present study describes the effect of IPS on the synthesis of viral RNA in vitro. MA-104 cell cultures infected with simian rotavirus strain SA-11 were incubated with zero, 250, 500 and 1,000 microg/ml IPS and 22, 24, 48, 52, 72 and 76 h after infection the cultures were submitted to a 1-h starvation period, followed by a 2-h pulse with 10 microCi/ml of [3H]-uridine. The homogenates of virus-infected cultures treated or not with IPS were submitted to phenol/chloroform extraction followed by polyacrylamide gel electrophoresis. The amount of radioactivity in viral RNA eluted from the gel strips was determined. Inhibition of viral RNA synthesis was highest at the IPS concentration of 1,000 microg/ml at 72 h after infection, corresponding to 78 percent inhibition. Although the results obtained in vitro suggest that IPS may be useful for the treatment of rotavirus infection, an in vivo demonstration of its efficacy is needed.