Molecular Diagnosis of Human Enteroviruses by Phylogeny‐Based Classification by Use of the VP4 Sequence

Abstract

Human enteroviruses (EVs) are the major cause of a variety of acute and chronic illnesses. Virus isolation and neutralization tests are usually done to identify the causative virus, but these tests are labor intensive, time consuming, and sometimes require suckling mice from which certain viruses have been isolated. This study investigated a rapid and reliable method based on reverse-transcription polymerase chain reaction and phylogenetic analysis. The phylogenetic tree constructed by neighbor-joining on the basis of the VP4 sequence from 66 prototypes grouped all human EVs into 5 distinct clusters. These clusters correspond closely to the 5 newly designated species-human EV A-D and poliovirus. The VP4 sequences of 89 isolates from 26 serotypes obtained over >30 years plus those of 66 prototype strains were analyzed. Each isolate formed a monophyletic cluster along with its respective prototype strain, allowing for serotype identification (with the exception of E-8). VP4-based classification appears to be an effective tool for the molecular epidemiology study of EVs.

Molecular Epidemiology of Adenoviral Conjunctivitis in Hanoi, Vietnam

“…2 RT-PCR was performed to amplify enterovirus genome including the entire VP4 region as described. 3 …”

Genetic analysis of the VP4/VP2 coding region in human rhinovirus species C in patients with acute respiratory infection in Japan

“…RT-PCR of the HRV VP4/VP2 coding region was carried out using the following primers: forward primer EvP4 (59-CTACTTTGGGTGTCCGTGTT-39) and reverse primer OL68-1 (59-GGTAAYTTCCACCACCANCC-39). PCR was initiated at 94 uC for 3 min, followed by 40 cycles of 94 uC for 30 s, 55 uC for 30 s and 72 uC for 1 min, with a final extension at 72 uC for 10 min (Ishiko et al, 2002). The PCR product was purified with a Wizard SV Gel and PCR Clean-Up System (Promega).…”

“…Sequence analysis was performed on an ABI 3500 Genetic Analyzer (Applied Biosystems). We attempted to detect other respiratory viruses such as influenza virus, human parainfluenza virus (HPIV), adenovirus (AdV), respiratory syncytial virus (RSV), human metapneumovirus (HMPV), enterovirus (EV) and human bocavirus (HBoV) as described previously (Allander et al, 2005;Echevarría et al, 1998;Ishiko et al, 2002;Matsuzaki et al, 2009;Nakauchi et al, 2011;Sullender et al,1993;Xu et al, 2000;Zhang & Evans, 1991).…”

Detection and Characterization of Enteric Viruses in Flood Water from the 2011 Thai Flood

“…Detection of viral RNA: Reverse transcription (RT)-PCR was used to detect viral RNA in the flood water samples using a SuperScript III One-Step RT-PCR with Platinum Taq kit (Invitrogen). The primer sequences and references (13)(14)(15)(16) are listed in Table 1. One-step RT-PCR was performed for the detection of HEV, HAV, and EV, whereas nested RT-PCR was performed for the detection of NV and RV.…”

“…One-step RT-PCR was performed for the detection of HEV, HAV, and EV, whereas nested RT-PCR was performed for the detection of NV and RV. Previously reported RT-PCR conditions were followed (13)(14)(15)(16). Nested PCR conditions used for NV and RV are the same as those used for first-step RT-PCR, without the reverse transcription step.…”