Spatiotemporal phylogenetic analysis and molecular characterization of coxsackievirus A4
Graphical abstract
Highlights
► A purifying (negative) selection was observed over the 3′ VP1 region. ► Spatiotemporal analysis of CV-A4 transmission indicated two prevalent genotypes. ► Co-circulation of positive selection sublineages implies adaptive radiation of CV-A4.
Introduction
Coxsackievirus A4 (CV-A4) is a member of the Picornaviridae family of human enteroviruses (HEVs). The highly contagious HEVs are among the most common viral infections in neonates and young children. By 1986, over 10 million HEV-symptomatic infections had been reported in the United States (Strikas et al., 1986), and the number of reported infections had substantially increased by the time of the next nationwide survey performed in 2000 (Khetsuriani et al., 2006). Previous studies indicate that dominant HEV serotypes vary by region, and even regularly detected serotypes may have highly variable prevalence rates. For example, sudden large outbreaks may follow long periods of inactivity. The prevalent HEV serotypes may alternate or co-circulate in certain areas because of newly emerging variants (Antona et al., 2007, Bahri et al., 2005, Blomqvist et al., 2008, Khetsuriani et al., 2006, Roth et al., 2007, Tseng et al., 2007). These observations suggest that the epidemic potential of an HEV outbreak correlates with the seasonality, geographic location, herd immunity, and serotype of prevalent HEVs.
The HEVs are a group of naked positive single-stranded RNA viruses with over 100 serotypes. The HEVs were initially classified into five subgroups: poliovirus (PV), coxsackievirus groups A (CV-A) and B (CV-B), echovirus (E), and enterovirus (EV). In 2007, however, HEVs were reclassified as HEV-A, B, C and D based on the molecular characterizations by the Picornavirus Study Group of the International Committee for the Taxonomy of viruses (http://www.picornastudygroup.com/proposals/2007/proposals_2007.htm). The CV-A4 belongs to the HEV-A species (Table A.1). In Taiwan, HEV-A is more prevalent than HEV-B (63.7% versus 36.7%, respectively) (Tseng et al., 2007) whereas HEV-B is relatively more prevalent than HEV-A in most countries elsewhere (Table A.2) (Antona et al., 2007, Bahri et al., 2005, Bingjun et al., 2008, Blomqvist et al., 2008, Jee et al., 2004, Khetsuriani et al., 2006, Roth et al., 2007). Although CV-A16 has been the serotype most frequently detected by the Taiwan Centers for Disease Control (CDC) Surveillance System since 1999, CV-A4 also raised concerns when it began co-circulating with CV-A16 in 2010 after its activities had peaked earlier in 2004 and 2006 (Fig. A.1).
The broad spectrum of HEV-A clinical manifestations include asymptomatic or flu-like febrile illness, herpangina, hand-foot-mouth disease (HFMD), and CNS involvement (Pallansch and Roos, 2007). The prevalence of HEV-A infections has likely been underestimated for many years due to the difficulty culturing CV-A (Witso et al., 2006). Additionally, CV-A16 and EV-71 are the only commercially available immunofluorescence assay (IFA) antibodies for HEV-A (Muir et al., 1998, Rigonan et al., 1998). Detection of HEV-A has improved since the human rhabdomyosarcoma (RD) cell became available (Muir et al., 1998) and after the CV-A IFA typing kit was developed to detect CV-A2, 4, 5, 6, and 10 in Taiwan (Lin et al., 2008). Moreover, sequences in the VP1 and 3′ VP1 regions have proven reliable for molecular typing of HEVs since the VP1 codon region of HEV comprises the major serotype-specific neutralization epitopes among the capsid structural protein (Cabrerizo et al., 2008, Caro et al., 2001, Chu et al., 2009, Oberste et al., 1999, Palacios et al., 2002).
The transmission of rapidly evolving RNA-viruses involves a continuous interaction between spatiotemporal dispersion and natural selection process; until now, however, phylogenetic data analyses have only revealed genetic divergence. Improved computational power and analytical methods now enable researchers to estimate the spatiotemporal origins of infectious viruses and to reconstruct their global transmission histories (Pybus and Rambaut, 2009). Pre-screened CV-A4 sequencing data obtained from GenBank indicate that the 3′ VP1 region of CV-A4 has a wider geographic distribution compared to other genes. Therefore, the 3′ VP1 region was selected for sequencing in the current study. To characterize the transmission dynamics and molecular characterization of CV-A4, this study analyzed 3′ VP1 region sequences in strains obtained from GenBank and in strains that had circulated in Taiwan from 2002 to 2009. Clinical presentations of CV-A4 infection in Taiwan were also examined.
Section snippets
Viruses and patient profiles
This study analyzed 31 randomly selected patients who had been treated for CV-A4 infection at Kaohsiung Medical University Hospital (KMUH), Taiwan during 2002–2009. Patient data collection was performed only after receiving approval from the KMUH ethics committee and signed consent forms from each patient. Data collection included gender, age, clinical symptoms, diagnosis, and complications. All viral isolates were confirmed by indirect fluorescence antibody test using an in-house IFA typing
Recombination detection of the VP1 region
The 420 nt (positions 2902–3321) of the 3′ VP1 region was analyzed in 58 worldwide CV-A4 sequences and in two outgroup strains. The permutation test results obtained p values greater than 0.05 for r-squared, D prime, and G4 (p = 0.453, 0.814, and 0.828, respectively). Therefore, the null hypothesis of “no recombination” was confirmed in this dataset. Evidence of 0 breakpoints was obtained using AICc in a GARD recombination analysis. These recombination analyses confirmed that no recombination
Discussion
The phylogenetic analysis of the partial VP1 nucleotide sequence included 58 strains of CV-A4 isolated worldwide in the past six decades (1950–2009). Most CV-A4 isolates were located on the most recent terminal parts of the tree (Table 1), possibly due to the difficulty of culturing cells before the RD cell was recommended or due to deficiencies in the commercial identification kit used before the improved molecular typing technique became available (Lin et al., 2008, Oberste et al., 1999,
Acknowledgements
The authors gratefully acknowledge the Centers for Disease Control and Prevention of Taiwan for their valuable technical assistance. This study is based in part on sequence data from the Taiwan Pathogenic Microorganism Genome Database provided by the Centers for Disease Control, Department of Health, Executive Yuan, Taiwan (supported by National Research Program for Genome Medicine 99-0324-01-F-12). The interpretation and conclusions contained herein do not represent those of Centers for
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