Novel Rotavirus VP7 Typing Assay Using a One-Step Reverse Transcriptase PCR Protocol and Product Sequencing and Utility of the Assay for Epidemiological Studies and Strain Characterization, Including Serotype Subgroup Analysis

Abstract

Rotavirus is the most common cause of severe dehydrating gastroenteritis in infants. To date, 10 different serotypes of rotavirus have been identified in human stools. While four or five serotypes dominate, serotype circulation varies with season and geography. Since our laboratory has been involved in the development of a multivalent rotavirus vaccine, it is important to identify the serotypes of rotavirus encountered during our clinical trials. We have developed methodologies for the molecular identification of rotavirus strains based on VP7 gene segment sequence. A 365-bp reverse transcriptase PCR product was generated from the VP7 gene segment using a pair of novel degenerate primers. All serotypes tested (both animal and human) yielded an identically sized product after amplification. Sequencing of these products is performed using truncated versions of the original primers. The sequence generated is compared against a database of rotavirus VP7 sequences, with the G type determined, based on the sequence homology. Using this assay, we have correctly identified human VP7 strains from a panel of available serotypes, as well as numerous animal strains. The assay was qualified using rotavirus positive stool samples, negative stool samples, and rotavirus-spiked stool samples. In addition, samples from cases of acute gastroenteritis collected at Children's Hospital of Philadelphia have been evaluated and indicate that the assay is able to discriminate subtle differences within serotypes. The assay has been utilized in the testing of >3,000 antigen-positive (enzyme immunoassay) samples collected during clinical trials of a rotavirus vaccine (RotaTeq) and identified a serotype in ϳ92% of samples (3, 17, 19).

Analysis by rotavirus gene 6 reverse transcriptase-polymerase chain reaction assay of rotavirus-positive gastroenteritis cases observed during the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST)

“…This molecular assay utilized methodology similar to the previously validated rotavirus gene 9 (encoding VP7) 24 and rotavirus gene 4 (encoding VP4) 26 RT-PCR assays, used to determine the rotavirus genotypes in stool samples trial participants. The rotavirus gene 6 RT-PCR assay was then applied to the RVGE stool samples collected during the vaccination phase of REST to identify the species of origin of the rotavirus gene 6 of the rotavirus strain present.…”

“…For EIA rotavirus-positive stool samples, the neutralization antigens VP7 (G type) and VP4 (P type) genotypes were determined utilizing RT-PCR followed by amplicon sequencing, 24 while a microbiological plaque assay was utilized to determine whether the stool samples contained infectious vaccine virus strains. 25 Given that few wild-type human rotaviruses are cytopathic in cell culture unless adapted to culture by several blind passages, 19 failure to form plaques in the microbiological plaque assay categorized, for REST, the rotavirus present in the stool sample as wild-type rotavirus.…”

“…Amplicon purification, direct sequencing with dye-terminator chemistry, and nucleotide and amino acid sequence analysis were performed as described previously. 24 Sequences were evaluated for identity to known gene 6 sequences utilizing MacVector™ (MacVector, Inc.). The final gene 6 designation (wild-type [human strain] or vaccinetype [bovine rotavirus WC3 strain] type) was determined using a reference database containing sequences of diverse gene 6 sequences from known infected species (human, bovine [other than that of strain WC3], porcine, simian, lapine, canine, feline, equine, avian, ovine, and murine), as well as the gene 6 sequence of the RV5 strains.…”

Viral Etiology Acute Intestinal Infections Molecular Monitoring in Children’s Hospital

“…Генотипирование кишечных вирусов мето-дом секвенирования проводили путем опре-деления нуклеотидной последовательности фрагментов кДНК, полученных при амплифи-кации типоспецифических участков вирусных геномов размером 365, 349 и 449 нуклеотидов для ротавирусов, норовирусов и астровирусов соответственно [8,10,13,16]. Ампликоны выде-ляли из геля с применением наборов реагентов производства ООО «Цитокин» и ООО «Фрак-талБио» (Санкт-Петербург).…”

Safety and immunogenicity of pentavalent rotavirus vaccine in a randomized, double-blind, placebo-controlled study in healthy elderly subjects

“…15 A VP6 reverse transcription-polymerase chain reaction (RT-PCR) assay was used as the confirmatory method on EIA-positive stool samples to identify presence of wildtypevirus or vaccine-virus strains. [22][23][24] In addition to the above assays, all AGE stool samples were assayed for Clostridium difficile toxin A and toxin B using a commercially available EIA (Meridian Biosciences, Inc., Cincinnati, OH) according to the manufacturer's instructions, and assayed for norovirus by RT-PCR as pre-specified in the protocol and previously reported.…”