scholarly journals Sequence analysis of the guanylyltransferase (VP3) of group A rotaviruses

2004 ◽  
Vol 85 (4) ◽  
pp. 929-932 ◽  
Author(s):  
Jonathan P. Cook ◽  
Malcolm A. McCrae
Keyword(s):  
Mammalian Species ◽  
Sequence Divergence ◽  
Amino Acid Sequences ◽  
Rt Pcr ◽  
Human Virus ◽  
Sequence Identity ◽  
Group A Rotaviruses ◽  
Group A ◽  
Mammalian Origin ◽  
Virus Isolates

The RNA segment encoding the guanylyltransferase (VP3) from 12 group A rotavirus isolates has been sequenced following RT-PCR and molecular cloning of the full-length amplicons produced. Alignment of the derived amino acid sequences including those of the four VP3 sequences available from GenBank revealed two levels of sequence divergence. Virus isolates from humans showed greater than 94 % sequence identity, whereas those isolated from different mammalian species showed as low as 79 % sequence identity. The exceptions were avian virus isolates, which diverged ∼45 % from those of mammalian origin, and the human virus isolates DS1 and 69M, which showed much closer (over 90 %) identity to viruses of bovine origin, suggesting that these human isolates may have undergone recent reassortment events with a bovine virus. Analysis of the sequences for a putative enzymic active site has revealed that the KXTAMDXEXP and KXXGNNH motifs around amino acids 385 and 545, respectively, are conserved across both group A and C rotaviruses.

scholarly journals Electropherotypes and G-Types of Group A Rotaviruses Detected in Children with Diarrhea in Lagos, Nigeria

ISRN Virology ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Christianah Idowu Ayolabi ◽  
David Ajiboye Ojo ◽  
George Enyimah Armah
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Migration Pattern ◽  
Genomic Diversity ◽  
Rt Pcr ◽  
Group A Rotaviruses ◽  
Group A ◽  
Stool Samples ◽  
Health Care Centers ◽  
Virus Isolates

Approximately over 500,000 children die annually due to severe dehydrating diarrhea caused by rotaviruses. This work investigated rotavirus infection among children less than 5 years with diarrhea in Lagos and determined the circulating electropherotypes and genotypes of the virus isolates. Three hundred and two (n=302) stool samples from children below 60 months were collected from different hospitals and health care centers in Lagos and subjected to enzyme immunoassay (EIA) to determine the presence of Group A rotavirus, RT-PCR to determine the G-types, and polyacrylamide gel electrophoresis (PAGE) to determine the electropherotypes. The results show that 60.3% of the samples showed distinct rotavirus RNA migration pattern, having long electropherotypes (55.3%) of seven variations dominating over the short electropherotypes (44.5%). Six different G-types were detected (G1, G2, G3, G4, G9, and G12). Serotypes G1 and G12 showed long electropherotypic pattern while G2, G3, and G9 exhibited either short or long electropherotype. All G4 detected show short electropherotypic pattern. In conclusion, information on the genomic diversity and RNA electropherotypes of rotaviruses detected in children with diarrhea in Lagos is reported in this study.

scholarly journals Isolation, immunochemical demonstration of field strains of porcine group A rotaviruses and electrophoretic analysis of RNA segments of group A and C rotaviruses

2012 ◽  
Vol 51 (No. 5) ◽  
pp. 288-295 ◽  
Author(s):  
R. Smitalova ◽  
L. Rodak ◽  
I. Psikal ◽  
B. Smid
Keyword(s):  
Cell Line ◽  
Electrophoretic Analysis ◽  
Elisa Test ◽  
Farm Animals ◽  
Economic Losses ◽  
Rt Pcr ◽  
Group C Rotavirus ◽  
Group A Rotaviruses ◽  
Group A ◽  
Virus Isolates

Rotaviruses are major cause of acute diarrhea in animals and humans which can result in huge economic losses in farm animals including pigs. We collected 195 samples of feces of diarrhoeic animals. Rotavirus was demonstrated by electron microscopy using the method of negative staining in 27 samples and by ELISA test using monoclonal antibodies to the group antigen VP6 in 44 samples. Nine samples were selected for virus isolation. Three virus isolates (P375/4, P410/4 and P646/1) were successfully adapted to growth in cell line MA-104. These isolates were allocated to group A rotaviruses based on ELISA, immunoperoxidase test and electropherotype analysis. Electropherotype analysis demonstrated changes during passage in cell line in two of the three isolates. The selected sample P543/1 proved negative in ELISA in a fecal sample. Electropherotype analysis of this sample revealed a “longer” electropherotype profile. The profile was suggestive of group C rotavirus. Rotavirus group C was confirmed by RT-PCR and by sequence analysis in this sample.

scholarly journals Human, porcine and bovine rotaviruses in Slovenia: evidence of interspecies transmission and genome reassortment

2008 ◽  
Vol 89 (7) ◽  
pp. 1690-1698 ◽  
Author(s):  
Andrej Steyer ◽  
Mateja Poljšak-Prijatelj ◽  
Darja Barlič-Maganja ◽  
Jožica Marin
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Zoonotic Transmission ◽  
Interspecies Transmission ◽  
Genotype Combination ◽  
Human Rotavirus ◽  
Group A Rotaviruses ◽  
Group A ◽  
Stool Samples ◽  
Genome Reassortment

A surveillance of human, porcine and bovine rotaviruses was carried out in Slovenia in 2004 and 2005. Stool samples were collected from a total of 406 pigs (373 from asymptomatic animals), 132 cattle (126 from asymptomatic animals) and 241 humans (all with diarrhoea), tested for group A rotaviruses using RT-PCR and analysed by sequencing. The aims of the study were to determine the incidence of asymptomatic rotavirus infection in animals, to look for evidence of zoonotic transmission and to detect reassortment among rotaviruses. The rates of asymptomatic shedding of rotaviruses in pigs and cattle were 18.0 % (67/373) and 4.0 % (5/126), respectively. Evidence for zoonotic transmission was detected in one human rotavirus strain, SI-MB6, with the G3P[6] genotype combination, as the nucleotide and predicted amino acid sequences of the VP6, VP7, VP8* and NSP4 genes of strain SI-MB6 and of porcine strains showed high nucleotide and amino acid sequence identity. Two porcine rotavirus strains carried VP7 of probable human origin, suggesting an interspecies reassortment event in the past.

scholarly journals Molecular Detection of Rotavirus in Mollusks from the Oued El Maleh Estuary of Mohammedia, Morocco

2021 ◽  
Author(s):  
Abderrahim Hatib ◽  
Najwa Hassou ◽  
Abdelouahab Benani ◽  
Jamal Eddine Hafid ◽  
Moulay Mustapha Ennaji
Keyword(s):  
Real Time ◽  
Enteric Bacteria ◽  
Human Populations ◽  
Rt Pcr ◽  
Wet Season ◽  
Rotavirus A ◽  
Viral Loads ◽  
Wide Range ◽  
Group A Rotaviruses ◽  
Group A

Viral outbreaks can result from the consumption of contaminated bivalve mollusks. However, despite the regulation related to enteric bacteria in food products, the consumption of raw and undercooked mollusks remains linked to viral epidemics in human populations. Real-time RT-PCR is a highly sensitive approach for detecting and quantifying enteric viruses, and after eliminating enzymatic amplification inhibitors from samples of interest, sensitive and specific tests, like real-time RT-PCR, can facilitate the detection and quantification of a wide range of viruses that are concentrated in mollusk digestive tissues. The aim of the present study was to evaluate the prevalence of Group-A rotaviruses in mussel (Mytilus edulis Linnaeus, 1758) specimens (n=576) collected downstream of the Oued El Maleh Estuary, which is along the coast of Mohammedia City in Morocco, using real-time RT-PCR. Rotavirus A RNA was detected in 37.5% (n=18) of the 48 sample batches, and viral loads ranged from 0.42×101 to 1.8603×104 genomic copies per g digestive tissue. Most (72.22%) of the positive samples were collected during the wet season (September-April), and the probability of detecting rotaviruses was significantly greater during the wet season than during the dry season (P<0.001). Monitoring Rotavirus A and similar viruses in shellfish may help prevent viral contamination and preserve public health.

The highly related LIM factors, LMO1, LMO3 and LMO4, play different roles in the regulation of the pituitary glycoprotein hormone α-subunit (αGSU) gene

2009 ◽  
Vol 30 (1) ◽  
pp. 51-58 ◽  
Author(s):  
Takao Susa ◽  
Akio Ishikawa ◽  
Li-yi Cai ◽  
Takako Kato ◽  
Kaori Matsumoto ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Species ◽  
Amino Acid Sequences ◽  
Gh3 Cells ◽  
Small Interfering Rnas ◽  
Rt Pcr ◽  
Glycoprotein Hormone ◽  
Homologous Proteins ◽  
Α Subunit ◽  
Pituitary Glands

LMO1, LMO3 and LMO4 were cloned from the adult porcine pituitary cDNA library. Amino acid sequences of porcine LMO1, LMO3 and LMO4 were highly conserved among mammalian species. Transfection assay of the pituitary-derived cell line LβT2 was carried out using the pituitary αGSU (glycoprotein hormone α-subunit) promoter (−1059/+12 b) fused to pSEAP2-Basic vector as a reporter gene. The results demonstrated that, whereas LMO4 showed no apparent effect, αGSU promoter activity was markedly repressed by LMO1 but activated by LMO3, indicating the different roles of the three highly homologous proteins, LMO1, LMO3 and LMO4. Knockdown assay by LMO siRNAs (small interfering RNAs) confirmed the above results for LMO1 and LMO3, whereas that by LMO4 siRNA increased the expression, indicating different modes of action. RT–PCR (reverse transcription–PCR) for total RNAs of several cell lines showed that LMO1 and LMO4 mRNAs were present ubiquitously in all cell lines, except for LMO1 in L929 cells. In contrast, LMO3 mRNA was abundant only in LβT4 and GH3 cells with only small amounts in LβT2 and MtT/S cells, indicating the cell-type-specific function of this protein. Real-time analyses of porcine pituitary ontogeny revealed that the three LMO genes are expressed during the fetal period and decline immediately afterwards, followed by a remarkably low level of LMO3 and LMO4 after birth. RT–PCR of the porcine tissues examined showed ubiquitous expression of LMO4, whereas LMO1 and LMO3 are expressed tissue specifically. Thus the present study demonstrated that three highly related LIM cofactors, LMO1, LMO3 and LMO4, have different effects on αGSU gene expression in the pituitary glands.

scholarly journals Characterization of Group C Rotaviruses Associated with Diarrhea Outbreaks in Feeder Pigs

1999 ◽  
Vol 37 (5) ◽  
pp. 1484-1488 ◽  
Author(s):  
Yunjeong Kim ◽  
Kyeong-Ok Chang ◽  
Barbara Straw ◽  
Linda J. Saif
Keyword(s):  
Amino Acid ◽  
Clinical Signs ◽  
Watery Diarrhea ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Fecal Samples ◽  
Group C Rotavirus ◽  
Group A Rotaviruses ◽  
Group A ◽  
Cowden Strain

Feces and serum specimens were collected from three farms in Michigan on which ∼50-lb (8- to 9-week-old) pigs experienced diarrhea just after placement into all-in-all-out finishing barns. The clinical signs (profuse watery diarrhea lasting about 2 weeks and no vomiting) were similar on all farms, and the morbidity rate was high (ranging from 60 to 80%) but without mortality. Eleven diarrheic fecal samples from the farms were tested for group A and C rotaviruses by immune electron microscopy (IEM) and various assays. IEM indicated that the fecal samples reacted only with antiserum against group C rotaviruses, and polyacrylamide gel electrophoresis indicated that the samples had characteristic genomic electropherotypes for group C rotavirus. Group C rotavirus was detected by cell culture immunofluorescence (CCIF) tests in nine fecal samples, but no group A rotavirus was detected by enzyme-linked immunosorbent assay or CCIF. By reverse transcription (RT)-PCR, all 11 fecal samples were positive for group C rotaviruses, with only 2 samples positive for group A rotaviruses. However, a second amplification of RT-PCR products using nested primers detected group A rotaviruses in all samples. Analysis of nucleotide and deduced amino acid sequences of the RT-PCR product (partial-length VP7) of the group C rotavirus showed 87.2 to 91% nucleotide identity and 92.6 to 95.9% amino acid identity among two strong samples from the different farms and the Cowden strain of porcine group C rotavirus. All nine convalescent-phase serum samples tested had neutralizing antibodies to the Cowden strain, and the majority of them had neutralizing antibody against group A rotaviruses (OSU or/and Gottfried strains) by fluorescent focus neutralization tests. Although group C rotaviruses have been reported as a cause of sporadic diarrhea in suckling or weanling pigs, to our knowledge, this is the first report of epidemic diarrhea outbreaks associated with group C rotavirus in older pigs.

scholarly journals Molecular characterization of a rare G1P[19] rotavirus strain from India: evidence of reassortment between human and porcine rotavirus strains

2009 ◽  
Vol 58 (12) ◽  
pp. 1611-1615 ◽  
Author(s):  
Shobha D. Chitambar ◽  
Ritu Arora ◽  
Preeti Chhabra
Keyword(s):  
Amino Acid Sequences ◽  
Rt Pcr ◽  
Coding Regions ◽  
Subgroup Ii ◽  
Human Rotavirus ◽  
Independent Segregation ◽  
Group A ◽  
Porcine Rotavirus ◽  
Porcine Origin

This study pertains to the characterization of a human rotavirus strain (NIV929893) with a rare specificity of G1P[19]. Three structural genes (VP4, VP6 and VP7) and one non-structural gene (NSP4) of strain NIV929893 were subjected to RT-PCR for amplification of entire coding regions. All of the amplicons were sequenced to carry out phylogenetic analysis. The complete amino acid sequences of the VP7 and VP4 gene products showed clustering of the VP7 gene with G1 strains of human origin and the VP4 gene with P[19] strains of porcine origin. The two viral proteins VP6 and NSP4, described previously as genetically linked proteins, were shown to be subgroup II and genotype B of human and porcine origins, respectively. The findings of this study provide evidence of reassortment between VP7/VP6 genes of humans and VP4/NSP4 genes of porcine species and an independent segregation of VP6 and NSP4 genes in a group A human rotavirus strain with G1P[19] specificity.

scholarly journals Molecular Characterization of VP6 Genes of Human Rotavirus Isolates: Correlation of Genogroups with Subgroups and Evidence of Independent Segregation

2002 ◽  
Vol 76 (13) ◽  
pp. 6596-6601 ◽  
Author(s):  
Miren Iturriza Gómara ◽  
Cecilia Wong ◽  
Sandra Blome ◽  
Ulrich Desselberger ◽  
Jim Gray
Keyword(s):  
Molecular Characterization ◽  
Rt Pcr ◽  
Genetic Lineages ◽  
Reverse Transcription Pcr ◽  
Human Rotavirus ◽  
Independent Segregation ◽  
Genes Encoding ◽  
Group A Rotaviruses ◽  
Group A

ABSTRACT A reverse transcription-PCR (RT-PCR) was established to amplify a 379-bp cDNA fragment (nucleotides 747 to 1126, coding for amino acids 241 to 367) of the VP6 gene of group A rotaviruses associated with subgroup (SG) specificity. Thirty-eight human rotavirus strains characterized with SG-specific monoclonal antibodies were subjected to VP6-specific RT-PCR, and PCR amplicons were used for sequencing. Nucleic acid sequencing and phylogenetic analysis of the VP6 amplicons revealed two clusters, or genogroups. Two genetic lineages were distinguished within genogroup I, consisting of strains serologically characterized as SG I, and three genetic lineages were distinguished within genogroup II, composed of strains serologically characterized as SG II, SG I + II, and SG non-I, non-II. Subgrouping of rotaviruses by means of serological methods may result in strains not being assigned the correct SG or in a failure of strains to subgroup. Molecular characterization of the SG-defining region of VP6 provided evidence for independent segregation of the rotavirus genes encoding VP4, VP6, and VP7.

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