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.
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 approximately 92% of samples (3, 17, 19).
DiStefano
Daniel J
DJ
Vaccine & Biologics Research, Merck Research Laboratories, Merck & Co. Inc., West Point, PA 19486, USA. daniel_distefano@merck.com
Kraiouchkine
Nikolai
N
Mallette
Laura
L
Maliga
Marianne
M
Kulnis
Gregory
G
Keller
Paul M
PM
Clark
H Fred
HF
Shaw
Alan R
AR
D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics*; D002675:Child, Preschool; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000453:epidemiology / Q000821:virology*; D006801:Humans; D007223:Infant; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000145:classification* / Q000235:genetics; D012400:Rotavirus Infections / Q000453:epidemiology / Q000821:virology*; D022243:Rotavirus Vaccines; D012703:Serotyping
D000956:Antigens, Viral; D036022:Capsid Proteins; D012367:RNA, Viral; D022243:Rotavirus Vaccines; C050779:VP7 protein, Rotavirus