The sequences of the primers and probe for HEV, HEV71, and CVA16.

<p>Note:</p><p>1. “a” indicates the sequence position of the primers and probes referring to strain BrCr of HEV71 (GenBank accession number U22521); “b” indicates the sequence position of the primers and probe referring to strain G-10 of CVA16 (GenBank accession number U05876).</p><p>2. The lengths of real-time RT-PCR products for HEV71, CVA16, and HEV were 76, 104, and 194 nt, respectively.</p><p>3. The primers and probes of HEV71, CVA16, and HEV have been applied for national invention patent in China, and the numbers of the patents are ZL200810063097.5, ZL200810121454.9, and ZL200810121453.4, respectively.</p

The real-time RT-PCR results of HEV71, CVA16, and HEVs for different types of HFMD clinical specimens.

<p>Note:</p><p>1. *Among 48 HEVs-positive specimens, one specimen was positive for HEVs, but negative for HEV71 or CVA16, which was identified as Echo11 by virus isolation, RT-PCR, and sequencing for the VP1 gene.</p><p>2. For the positive specimens for HEV71 or CVA16, the results of real-time RT-PCR for HEVs were also positive.</p><p>3. A total of 85 HEV71 and 16 CVA16 were identified using RT-PCR method among the 213 HFMD clinical specimens, which were lower than those of the real-time RT-PCR assay.</p><p>4. The multiplex real-time RT-PCR assay was shown to exhibit 100% specificity in detecting HEV71 and CVA16, which was confirmed by sequencing.</p

Primers information in this study.

<p>Primers information in this study.</p

Demographic and clinical characteristics of patients presenting ARIs.

<p>Demographic and clinical characteristics of patients presenting ARIs.</p

Molecular Epidemiology of Coxsackievirus A16: Intratype and Prevalent Intertype Recombination Identified

<div><p>Coxsackievirus A16 (CVA16) is responsible for nearly 50% of all the confirmed hand, foot, and mouth disease (HFMD) cases in mainland China, sometimes it could also cause severe complications, and even death. To clarify the genetic characteristics and the epidemic patterns of CVA16 in mainland China, comprehensive bioinfomatics analyses were performed by using 35 CVA16 whole genome sequences from 1998 to 2011, 593 complete CVA16 <i>VP1</i> sequences from 1981 to 2011, and prototype strains of human enterovirus species A (EV-A). Analysis on complete <i>VP1</i> sequences revealed that subgenotypes B1a and B1b were prevalent strains and have been co-circulating in many Asian countries since 2000, especially in mainland China for at least 13 years. While the prevalence of subgenotype B1c (totally 20 strains) was much limited, only found in Malaysia from 2005 to 2007 and in France in 2010. Genotype B2 only caused epidemic in Japan and Malaysia from 1981 to 2000. Both subgenotypes B1a and B1b were potential recombinant viruses containing sequences from other EV-A donors in the 5’-untranslated region and P2, P3 non-structural protein encoding regions. </p> </div

Viral pathogens distribution in different season.

<p>Viral pathogens distribution in different season.</p

Viral pathogens distribution in different age group.

<p>Viral pathogens distribution in different age group.</p

The Neighbor-joining tree of all the 629 CVA16 viruses based on complete VP1 encoding sequences.

<p>The branches showed in red color highlight the sequences from China.</p

Phylogenetic dendrogram showing the relationships between the cluster B1a, B1b and EV-A prototypes.

<p>These were constructed by the different genomic regions of 35 complete CVA16 genomic sequences and EV-A prototype sequences. The neighbour-joining trees were reconstructed based on the P1 (a), P2 (b), P3 (c) genomic regions and complete genomes (d), respectively. Triangles indicated the subgenotype B1a. Rhombuses indicated the subgenotype B1b. Squares indicated the CVA16 prototype G-10 strain. The percentage of bootstrap (percentage of 1000 pseudoreplicate datasets) supporting the trees are indicated at the nodes; only values over 80% are shown.</p

Similarity plot and bootscanning analyses of all the two subgenotypes B1a, B1b of CVA16 and other EV-A prototype strains on the basis of full-length genomes.

<p>Two strains were chosen as the representative strains of each subgenotype, respectively. Each of the four viruses was used as the query sequence. A sliding window of 500 nucleotides moving in 20 nucleotides steps was used in this analysis. (a and b) Tainan/5079/98/Taiwan/1998; (c and d) BJ/11/11 /BJ/CHN/2011; (e and f) shzh00-1/GD/CHN/2000; (g and h) BJ11/12/BJ/CHN/2011. The a, c, e, g were the results of similarity plot analyses; the b, d, f, h were the results of bootscanning analyses.</p