One-step Quantitative RT-PCR Assays for Detecting, Genotyping and Differentiating Wild-Type Group a Rotaviruses and Vaccine (Rotarix® and RotaTeq®) Strains in Stool Samples

Background.Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time.Methods.In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostablerTthpolymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments.Results.The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8–100% sensitivity, 100% specificity, 86–89% efficiency and a limit of detection of 12–400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82–90% efficiency and limit of detection of 120–4000 copies in multiplex reaction.Discussion.The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains.

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Real-time RT-PCR assays to differentiate wild-type group A rotavirus strains from Rotarix®and RotaTeq®vaccine strains in stool samples

Human Vaccines & Immunotherapeutics ◽

10.4161/hv.27388 ◽

2013 ◽

Vol 10 (3) ◽

pp. 767-777 ◽

Cited By ~ 31

Author(s):

Rashi Gautam ◽

Mathew D Esona ◽

Slavica Mijatovic-Rustempasic ◽

Ka Ian Tam ◽

Jon R Gentsch ◽

...

Keyword(s):

Real Time ◽

Wild Type ◽

Rt Pcr ◽

Type Group ◽

Group A Rotavirus ◽

Group A ◽

Stool Samples ◽

Pcr Assays

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

ISRN Virology ◽

10.5402/2013/179871 ◽

2013 ◽

Vol 2013 ◽

pp. 1-5 ◽

Cited By ~ 2

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.

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Suspected zoonotic transmission of rotavirus group A in Danish adults

Epidemiology and Infection ◽

10.1017/s0950268811001981 ◽

2011 ◽

Vol 140 (6) ◽

pp. 1013-1017 ◽

Cited By ~ 21

Author(s):

S. E. MIDGLEY ◽

C. K. HJULSAGER ◽

L. E. LARSEN ◽

G. FALKENHORST ◽

B. BÖTTIGER

Keyword(s):

Genetic Relatedness ◽

Phylogenetic Analyses ◽

Geographical Area ◽

Nucleotide Sequences ◽

Zoonotic Transmission ◽

Adult Patients ◽

Epidemiological Features ◽

Group A Rotaviruses ◽

Group A ◽

Stool Samples

SUMMARYGroup A rotaviruses infect humans and a variety of animals. In July 2006 a rare rotavirus strain with G8P[14] specificity was identified in the stool samples of two adult patients with diarrheoa, who lived in the same geographical area in Denmark. Nucleotide sequences of the VP7, VP4, VP6, and NSP4 genes of the identified strains were identical. Phylogenetic analyses showed that both Danish G8P[14] strains clustered with rotaviruses of animal, mainly, bovine and caprine, origin. The high genetic relatedness to animal rotaviruses and the atypical epidemiological features suggest that these human G8P[14] strains were acquired through direct zoonotic transmission events.

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Immunological and molecular detection of rotavirus genotype in calves with gastroenteritis in Diyala-Iraq

10.53346/wjbpr.2021.1.1.0013 ◽

2021 ◽

Vol 1 (1) ◽

pp. 001-013

Author(s):

Ammar Talib Nasser ◽

Abdulrazak Shafiq Hasan ◽

Amer Khazaal Saleh ◽

Mohammad Kassem Saleh

Keyword(s):

Molecular Detection ◽

Cross Sectional Study ◽

Sectional Study ◽

Rt Pcr ◽

Cross Sectional ◽

Reverse Transcription Pcr ◽

Group A ◽

Stool Antigen ◽

Stool Samples ◽

Age Range

Aim: To explore the prevalence of rotavirus infection along with the molecular detection and genotyping of group A rotavirus (RVA) among bovine calves up to 5 months old in Diyala province-Iraq. Methods: This is a cross sectional study conducted in Diyala province-Iraq during the period of 2019-2020. One hundred bovine calves with age range of 1-5 months were included in the study. All were suffering acute gastroenteritis. Serum anti-rotavirus IgM and IgG plus fecal rotavirus Ag were tested for using ELISA techniques. Stool samples positive for rotavirus Ag were submitted for reverse transcription PCR (RT-PCR) for G and P genes, followed by sequencing and genotyping thereafter. Statistical analysis was done using SPSS version 25 and P values ≤ 0.05 were considered significant. Results: The positivity rate of anti-rotavirus IgM was 80% (P = 0.0001), and that of anti-rotavirus IgG was 79% (P = 0.0001). The rotavirus stool antigen was detected in 68% of calves (P = 0.01). A total of 45 stool samples which were positive for rotavirus Ag were submitted for RT-PCR; 13 (28.9%) were positive and 32 (71.1%) were negative (P = 0.084). 10 PCR positive samples were used for sequencing and genotyping and indicated that all investigated strains belonged to G1P[8] genotype. Conclusion: The current strains analyzed belonged to the G1P[8] RVA genotypes, affirming that employment of VP7 gene polymorphism accurately yielded uniform phylogenetic distances amongst investigated rotavirus strains and that there were no noticeable assortment events between human and animal rotavirus strains in Diyala province.

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Effect of Intragenic Rearrangement and Changes in the 3′ Consensus Sequence on NSP1 Expression and Rotavirus Replication

Journal of Virology ◽

10.1128/jvi.75.5.2076-2086.2001 ◽

2001 ◽

Vol 75 (5) ◽

pp. 2076-2086 ◽

Cited By ~ 49

Author(s):

John T. Patton ◽

Zenobia Taraporewala ◽

Dayue Chen ◽

Vladimir Chizhikov ◽

Melinda Jones ◽

...

Keyword(s):

Cell Culture ◽

Consensus Sequence ◽

Active Role ◽

Serial Passage ◽

Selective Advantage ◽

Wild Type ◽

Reading Frame ◽

Group A Rotaviruses ◽

Group A ◽

Dsrna Genome

ABSTRACT The nonpolyadenylated mRNAs of rotavirus are templates for the synthesis of protein and the segmented double-stranded RNA (dsRNA) genome. During serial passage of simian SA11 rotaviruses in cell culture, two variants emerged with gene 5 dsRNAs containing large (1.1 and 0.5 kb) sequence duplications within the open reading frame (ORF) for NSP1. Due to the sequence rearrangements, both variants encoded only C-truncated forms of NSP1. Comparison of these and other variants encoding defective NSP1 with their corresponding wild-type viruses indicated that the inability to encode authentic NSP1 results in a small-plaque phenotype. Thus, although nonessential, NSP1 probably plays an active role in rotavirus replication in cell culture. In determining the sequences of the gene 5 dsRNAs of the SA11 variants and wild-type viruses, it was unexpectedly found that their 3′ termini ended with 5′-UGAACC-3′ instead of the 3′ consensus sequence 5′-UGACC-3′, which is present on the mRNAs of nearly all other group A rotaviruses. Cell-free assays indicated that the A insertion into the 3′ consensus sequence interfered with its ability to promote dsRNA synthesis and to function as a translation enhancer. The results provide evidence that the 3′ consensus sequence of the gene 5 dsRNAs of SA11 rotaviruses has undergone a mutation causing it to operate suboptimally in RNA replication and in the expression of NSP1 during the virus life cycle. Indeed, just as rotavirus variants which encode defective NSP1 appear to have a selective advantage over those encoding wild-type NSP1 in cell culture, it may be that the atypical 3′ end of SA11 gene 5 has been selected for because it promotes the expression of lower levels of NSP1 than the 3′ consensus sequence.

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Detection and Characterization of Human Enteroviruses, Human Cosaviruses, and a New Human Parechovirus Type in Healthy Individuals in Osun State, Nigeria, 2016/2017

Viruses ◽

10.3390/v11111037 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1037 ◽

Cited By ~ 3

Author(s):

Folakemi Abiodun Osundare ◽

Oladele Oluyinka Opaleye ◽

Akeem Abiodun Akindele ◽

Samuel Adeyinka Adedokun ◽

Olusola Anuoluwapo Akanbi ◽

...

Keyword(s):

Healthy Individuals ◽

Coding Region ◽

Rt Pcr ◽

Human Parechovirus ◽

Full Genome Sequencing ◽

Three Samples ◽

Two Samples ◽

Stool Samples ◽

Pcr Assays ◽

Species Specific

Human enteroviruses and human parechoviruses are associated with a broad range of diseases and even severe and fatal conditions. For human cosaviruses, the etiological role is yet unknown. Little is known about the circulation of non-polio enteroviruses, human parechoviruses, and human cosaviruses in Nigeria. A total of 113 stool samples were collected from healthy individuals in Osun State between February 2016 and May 2017. RT-PCR assays targeting the 5′ non-coding region (5′ -NCR) were used to screen for human enteroviruses, human parechoviruses, and human cosaviruses. For human enteroviruses, species-specific RT-PCR assays targeting the VP1 regions were used for molecular typing. Inoculation was carried out on RD-A, CaCo-2, HEp-2C, and L20B cell lines to compare molecular and virological assays. Ten samples tested positive for enterovirus RNA with 11 strains detected, including CV-A13 (n = 3), E-18 (n = 2), CV-A20 (n = 1), CV-A24 (n = 1), EV-C99 (n = 1), and EV-C116 (n = 2). Three samples tested positive for human parechovirus RNA, and full genome sequencing on two samples allowed assignment to a new Parechovirus A type (HPeV-19). Thirty-three samples tested positive for cosavirus with assignment to species Cosavirus D and Cosavirus A based on the 5′-NCR region. Screening of stool samples collected from healthy individuals in Nigeria in 2016 and 2017 revealed a high diversity of circulating human enteroviruses, human parechoviruses, and human cosaviruses. Molecular assays for genotyping showed substantial benefits compared with those of cell-culture assays.

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

Journal of General Virology ◽

10.1099/vir.0.2008/001206-0 ◽

2008 ◽

Vol 89 (7) ◽

pp. 1690-1698 ◽

Cited By ~ 79

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.

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