Molecular characterization of enterovirus 71 and coxsackievirus A16 using the 5′ untranslated region and VP1 region

Abstract

Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are members of the species Human enterovirus A, and are both major and independent aetiological agents of hand-foot-and-mouth disease. The human enterovirus (HEV) 59 untranslated region (UTR) is fundamentally important for efficient virus replication and for virulence, whilst the VP1 region correlates well with antigenic typing by neutralization, and can be used for virus identification and evolutionary studies. A comparison was undertaken of the 59UTR and VP1 nucleotide sequences of five EV71 clinical isolates and 10 CVA16 clinical isolates from one laboratory with the 59UTR and VP1 sequences of 104 EV71 strains and 45 CVA16 strains available in GenBank. The genetic relationships were analysed using standard phylogenetic methods. The EV71 phylogenetic analysis showed that the VP1 sequences were clustered into three genogroups, A, B and C, with genogroups B and C further divided into five subgenogroups, B1-B5 and C1-C5, respectively. All EV71 strains were clustered similarly in the 59UTR and VP1 trees, except for one Taiwanese strain, which demonstrated different clustering in the two trees, suggesting a recombination event in the phylogeny. The CVA16 phylogenetic analysis showed that the VP1 sequences were clustered into two genogroups, A and B, with genogroup B further divided into B1 (B1a and B1b), B2 and a possible B3; and that a similar pattern and grouping of all strains were displayed in the 59UTR tree. This study demonstrated that comparing the two regions provides evidence of epidemiological linkage of HEV-A strains, and that mutation in the two regions plays a vital role in the evolution of these viruses. The combination of molecular typing and phylogenetic sequence analysis will be beneficial in both individual patient diagnosis and public health measures. INTRODUCTIONEnterovirus 71 (EV71) and coxsackievirus A16 (CVA16), members of the species Human enterovirus A in the genus Enterovirus, family Picornaviridae, are both major and independent aetiological agents of large outbreaks of handfoot-and-mouth disease (HFMD). In contrast to CVA16, EV71-associated HFMD is more frequently associated with serious neurological complications and fatalities (McMinn et al., 2001b). Initial EV71 isolates were identified in the USA and Australia in the early 1970s, and in HFMD outbreaks in Sweden and Japan (Wong et al., 2010). Since 1997, the Asia-Pacific region has had several large EV71 epidemics, including in the Chinese mainland, Taiwan, Singapore, Malaysia and Japan. Several outbreaks have also been recorded in Australia (Gilbert et al., 1988;Sanders et al., 2006;McMinn et al., 2001b). Co-circulation of EV71 and CVA16 has contributed to serious HFMD outbreaks in China in (Zhang et al., 2009), Taiwan in 1998(Lin et al., 2003), and Malaysia in 1997(Podin et al., 2006.The enterovirus genome is a positive ssRNA molecule of approximately 7500 nt, comprising a single ORF flanked 59Abbreviations: CVA16, coxsackievirus A16; EV71, enterovirus 71; HEV, human enterovirus; ...

Coxsackievirus A16

“…41,42 The nucleotide variety between genotype B1 and B2 was 11.8%. 41 Genotypes B1a and B1b were the predominant circulating genotypes in Australia from 1999 to 2006, 43 and genotype B circulated in Malaysia from 1997-2006. 44 Phylogenetic analysis of CA16 isolated from 6 provinces in Mainland China from 1995 to 2008 showed that these strains were mainly subtypes B1a and B1b.…”

“…41,42 Therefore, more information on the molecular characterization of CA16 should be provided and a standard genotyping method basing on the VP1 nucleotide sequence should be established to properly study the molecular epidemiology of CA16. [42][43][44] Evolutionary rate analysis was performed using the sequence of VP1, and it was found that the annual evolution rate of CA16 was 0.91 × 10 -2 substitutions per synonymous nucleotide/year, 41 slightly lower than that of EV71 at 1.35 × 10 -2 . 45 A growing body of evidence supports the existence of recombination events involving EV71 and various HEV-A, including CA16 and CA8.…”

A comparative study of the characteristics of two Coxsackie A virus type 16 strains (genotype B)

“…Based on the analysis of different biological features of G20-D and G20-M and according to the basic principles of biological characteristics and related gene sequences of enteroviruses [19][20][21], we decided to sequence the 5′-noncoding region, the 3′ non-coding region, and the VP1 coding region. We repeated the sequencing three times and aligned the sequence ( Figure 4A).…”

Molecular Analysis of Enterovirus C Species Using the 5’ Untranslated Region and VP1 Region

“…PV2 EU566945-SPA-? PV2 EU566948- PCR primers and sequencing primers for 5' UTR and VP1: published primers for PCR amplification (5UTR-S and 5UTR-A1) and sequencing (5UTR-S and 5UTR-A2) of partial 5' UTR were used [15]. Published primers 494 to 497 for PCR amplification and sequencing of complete VP1 were obtained [16], as well as PCR amplification and sequencing primers 292 and 222 for analysis of partial VP1 sequences [16].…”

“…Reverse transcription (RT) was performed as described previously [17]. EV-specific 5' UTR PCR (using primers 5UTR-S and 5UTR-A1) for all 16 EV local isolates and partial VP1 PCR (using primers 292 and 222) for four isolates were performed as described previously [15]. The methods of complete VP1 PCR for 12 EV-C isolates were as described previously [17].…”