The characterization of VP7 (G type) and VP4 (P type) genes of bovine group A rotaviruses from field samples using RT-PCR and RFLP analysis

1996 ◽  
Vol 141 (9) ◽  
pp. 1727-1739 ◽  
Author(s):  
K. O. Chang ◽  
A. V. Parwani ◽  
L. J. Saif
Keyword(s):  
Rflp Analysis ◽  
Rt Pcr ◽  
Field Samples ◽  
Group A Rotaviruses ◽  
Group A ◽  
P Type

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.

scholarly journals A Survey of G6 and G10 Serotypes of Group a Bovine Rotaviruses from Diarrheic Beef and Dairy Calves using Monoclonal Antibodies in ELISA

1994 ◽  
Vol 6 (2) ◽  
pp. 175-181 ◽  
Author(s):  
A. Lucchelli ◽  
S. Y. Kang ◽  
M. K. Jayasekera ◽  
A. V. Parwani ◽  
D. H. Zeman ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
South Dakota ◽  
Dairy Herd ◽  
Dairy Calves ◽  
Enzyme Linked Immunosorbent Assay ◽  
Positive Stool ◽  
Field Samples ◽  
Group A Rotaviruses ◽  
Beef Herds ◽  
Group A

Group A bovine rotaviruses (BRV) have been identified worldwide as a major cause of diarrhea in the young of many species, including humans. Group A rotaviruses are classified into serotypes on the basis of the outer capsid proteins, VP7 (G types) and VP4 (P types). To date, there are 14 G types of group A rotaviruses, with G1, G6, G8, and G10 described for BRV isolates. In this study, G6- and G lo-specific monoclonal antibodies (MAbs) were used in an enzyme-linked immunosorbent assay (ELISA) for the G typing of BRV-positive stool samples from diarrheic beef and dairy calves from South Dakota, Ohio, Michigan, Nebraska, and Washington, USA, and Ontario, Canada. ELISA plates were coated using a broadly reactive VP7 MAb (Common 60) or with G6- or G10-specific MAbs. BRV-positive fecal samples were diluted and added to duplicate wells, followed by the addition of polyclonal guinea pig anti-group A rotavirus serum as the secondary antibody. Several reference G6 and G10 BRV strains as well as other G types previously reported in cattle (G1, G2, G3, G8) and BRV-negative samples were included as G type specificity and negative controls. From a total of 308 field samples analyzed, 79% (244/308) tested positive by the broadly reactive VP7 MAb; of these, 54% (131/244) were G6 positive, 14% (35/244) were G10 positive, 4% (9/244) were both G6 and G10 positive, and 28% (69/244) were G6 and G10 negative. The negative samples may represent additional or undefined serotypes. The 89 samples from South Dakota were further subdivided into samples from beef ( n = 43) or dairy ( n = 46) herds. G6 was more prevalent in beef herd samples (67%) than in dairy herd samples (47.5%). In addition, dairy herds had higher percentages of G10-positive samples (17.5%) G6-G10 double positives (10%), and untypable samples (25%) than did beef herds, in which the prevalence of G10 positive samples was 5.5%, G6-G10 double positives was 5.5%, and untypable samples was 22%. Application of the serotype ELISA for the analysis of additional BRV samples will provide further epidemiologic data on the distribution of BRV serotypes in beef or dairy cattle, an important consideration for the development of improved BRV vaccines.

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.

Molecular characterization of the first human G15 rotavirus strain of zoonotic origin from the bovine species

2021 ◽  
Vol 102 (4) ◽  
Author(s):  
Takeshi Tsugawa ◽  
Yoshiki Fujii ◽  
Yusuke Akane ◽  
Saho Honjo ◽  
Kenji Kondo ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Molecular Characterization ◽  
Human Infection ◽  
Potential Reservoir ◽  
Group A Rotaviruses ◽  
Paediatric Outpatient ◽  
Group A ◽  
Human Infections ◽  
Bovine Species

Group A rotaviruses (RVAs) infect a wide variety of mammalian and avian species. Animals act as a potential reservoir to RVA human infections by direct virion transmission or by contributing genes to reassortants. Here, we report the molecular characterization of a rare human RVA strain Ni17-46 with a genotype G15P[14], isolated in Japan in 2017 during rotavirus surveillance in a paediatric outpatient clinic. The genome constellation of this strain was G15-P[14]-I2-R2-C2-M2-A13-N2-T9-E2-H3. This is the first report of an RVA with G15 genotype in humans, and sequencing and phylogenetic analysis results suggest that human infection with this strain has zoonotic origin from the bovine species. Given the fact that this strain was isolated from a patient with gastroenteritis and dehydration symptoms, we must take into account the virulence of this strain in humans.

scholarly journals Molecular characterization of group A rotaviruses detected in children with gastroenteritis in Ireland in 2006–2009

2011 ◽  
Vol 140 (2) ◽  
pp. 247-259 ◽  
Author(s):  
O. CASHMAN ◽  
P. J. COLLINS ◽  
G. LENNON ◽  
B. CRYAN ◽  
V. MARTELLA ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Irish Population ◽  
Surveillance Activity ◽  
Human Rotavirus ◽  
Group A Rotaviruses ◽  
Group A ◽  
Hospital Acquired ◽  
Rotavirus Infections ◽  
Pcr Genotyping

SUMMARYCommunity and hospital-acquired cases of human rotavirus are responsible for millions of gastroenteritis cases in children worldwide, chiefly in developing countries, and vaccines are now available. During surveillance activity for human rotavirus infections in Ireland, between 2006 and 2009, a total of 420 rotavirus strains were collected and analysed. Upon either PCR genotyping and sequence analysis, a variety of VP7 (G1–G4 and G9) and VP4 (P[4], P[6], P[8] and P[9]) genotypes were detected. Strains G1P[8] were found to be predominant throughout the period 2006–2008, with slight fluctuations seen in the very limited samples available in 2008–2009. Upon either PCR genotyping and sequence analysis of selected strains, the G1, G3 and G9 viruses were found to contain E1 (Wa-like) NSP4 and I1 VP6 genotypes, while the analysed G2 strains possessed E2 NSP4 and I2 VP6 genotypes, a genetic make-up which is highly conserved in the major human rotavirus genogroups Wa- and Kun-like, respectively. Upon sequence analysis of the most common VP4 genotype, P[8], at least two distinct lineages were identified, both unrelated to P[8] Irish rotaviruses circulating in previous years, and more closely related to recent European humans rotaviruses. Moreover, sequence analysis of the VP7 of G1 rotaviruses revealed the onset of a G1 variant, previously unseen in the Irish population.

Characterization of human skeletal muscle in weight-losing pancreatic cancer patients

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 9571-9571
Author(s):  
J. M. Brell ◽  
J. Hardacre ◽  
J. Schulak ◽  
R. Onders ◽  
T. Stellato ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Weight Loss ◽  
Body Mass ◽  
Caspase 3 ◽  
Expression Patterns ◽  
Rt Pcr ◽  
Group A ◽  
Median Weight Loss

9571 Background: Decreased body mass (cachexia) is a common cause of functional decline in pancreas carcinoma (PC) and other malignancies. The etiology is unknown. Characterization of human PC skeletal muscle, in regard to proteolysis and gene expression, compared to control muscle may reveal information about pathophysiology. Methods: Biopsies of rectus abdominus muscle were performed in weight-losing PC patients all stages (A) during cancer-related surgery and in cancer-free controls undergoing ventral hernia repair (B). Caspase-3, pAkt, and urinary 3-methylhistidine (u3-MH) were assessed by Western blot and high-performance liquid chromatography. Fat-free mass (FFM), body mass index (BMI), and time to progression were recorded. Muscle from five patients (median weight loss 21%) and five controls were analyzed for gene expression patterns using Affymetrix Human Genome U133 A 2.0 array chip. Two hundred differentially over- and under-expressed genes were examined in group A for potential association with cachexia. RT-PCR confirmation of six candidate genes was performed. Results: Thirty-eight patients were enrolled. Median weight loss in group A (N=27) was 14.5% (5% - 34%). No differences were noted between groups in caspase-3 and pAkt expression. Baseline u3-MH (p=0.86) and FFM (p= 0.28) did not differ; baseline BMI was lower in group A (p=0.04). BMI follow-up measurements (N=17) were significantly decreased (p=0.0005). In 65% patients, progressive disease was noted within median time of 3 months. RT-PCR established up-regulation of CHRNA1 and LMO7, but not GDF8. mRNA down-regulation for TRIM63, IGF-BP6, and MYH-1 was confirmed. Conclusions: Muscle proteolysis in human PC skeletal muscle was not demonstrated, perhaps due to unmeasurable proteolysis or use of non-informative endpoints. BMI decreased in group A with PD; further studies need tight control of BMI variables. New hypotheses about cachexia include neuromuscular junction dysfunction, as CHRNA1 has specific role in ion channel gating; this is disrupted in the paraneoplastic Eaton-Lambert syndrome. This is first study analyzing human muscle in weight-losing PC and proves symptom management multidisciplinary research is feasible in academic setting. Supported by American Cancer Society pilot grant. No significant financial relationships to disclose.

scholarly journals Molecular Characterization of a Subgroup Specificity Associated with the Rotavirus Inner Capsid Protein VP2

2008 ◽  
Vol 82 (6) ◽  
pp. 2752-2764 ◽  
Author(s):  
Sarah M. McDonald ◽  
John T. Patton
Keyword(s):  
Monoclonal Antibody ◽  
Capsid Protein ◽  
Neutralizing Antibodies ◽  
Bovine Rotavirus ◽  
Single Region ◽  
Molecular Determinants ◽  
High Resolution Structure ◽  
Group A Rotaviruses ◽  
Group A

ABSTRACT Group A rotaviruses are classified into serotypes, based on the reactivity pattern of neutralizing antibodies to VP4 and VP7, as well as into subgroups (SGs), based on non-neutralizing antibodies directed against VP6. The inner capsid protein (VP2) has also been described as a SG antigen; however, little is known regarding the molecular determinants of VP2 SG specificity. In this study, we characterize VP2 SGs by correlating genetic markers with the immunoreactivity of the SG-specific monoclonal antibody (YO-60). Our results show that VP2 proteins similar in sequence to that of the prototypic human strain Wa are recognized by YO-60, classifying them as VP2 SG-II. In contrast, proteins not bound by YO-60 are similar to those of human strains DS-1 or AU-1 and represent VP2 SG-I. Using a mutagenesis approach, we identified residues that determine recognition by either YO-60 or the group A-specific VP2 monoclonal antibody (6E8). We found that YO-60 binds to a conformationally dependent epitope that includes Wa VP2 residue M328. The epitope for 6E8 is also contingent upon VP2 conformation and resides within a single region of the protein (Wa VP2 residues A440 to T530). Using a high-resolution structure of bovine rotavirus double-layered particles, we predicted these epitopes to be spatially distinct from each other and located on opposite surfaces of VP2. This study reveals the extent of genetic variation among group A rotavirus VP2 proteins and illuminates the molecular basis for a previously described SG specificity associated with the rotavirus inner capsid protein.

Molecular characterization of rotaviruses in mid-western Turkey, 2006–2007

Open Medicine ◽  
2010 ◽  
Vol 5 (5) ◽  
pp. 640-645
Author(s):  
Mustafa Altindis ◽  
Krisztián Bányai ◽  
Raike Kalayci ◽  
Cihangir Gulamber ◽  
Resit Koken ◽  
...  
Keyword(s):  
Immune Escape ◽  
Local Strain ◽  
Western Turkey ◽  
Vaccine Introduction ◽  
Rotavirus Vaccines ◽  
Group A ◽  
Stool Samples ◽  
Commercial Enzyme Immunoassay ◽  
P Type

AbstractVaccines against rotaviruses are now available in numerous countries, including Turkey. As the vaccines may show various efficiencies against different type specificities and routine vaccination in infants might result in selection and immune escape of wild-type rotavirus strains, strain surveillance has been initiated before and during the vaccine introduction. We aimed to provide corresponding information on local strain prevalence in Anatolia, mid-western Turkey during the introduction of rotavirus vaccines. Stool samples positive for group A rotavirus by commercial enzyme immunoassay were subjected to reverse transcription-polymerase chain reaction based genotyping of the outer capsid antigens, VP7 and VP4, determining G and P type specificities respectively. Among 36 fully and 5 partially typeable strains we detected genotype G1, G2, and G9 VP7 specificities and genotype P[4], P[6] and P[8] VP4 specificities in 5 individual and 4 mixed combinations. The most common strain was G2P[4] (n=17), followed by G9P[8] (n=9). Other strains were G1P[8] (n=2), G2P[8] (n=2), G1+2P[8] (n=2), G9P[4] (n=1), G2+9P[8] (n=1), G4+9P[6] (n=1), and G2P[4+8] (n=1). Partially typed strains included 2 G1P[NT] and 3 G2P[NT] strains. Our data may help determine a baseline of the rotavirus genotype prevalence in Turkey and see if changes in the incidence of individual strains will be observed after routine use of vaccine.

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.

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