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

Replication capacity and adaptability of a severe fever with thrombocytopenia syndrome virus at different temperatures

<div><p>Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease caused by the SFTS virus (SFTSV). Although fever and thrombocytopenia are the typical manifestations of SFTS, a specific SFTS case with no fever was observed in Zhejiang, China. In this report, we aimed to explore the probable reason for the absence of fever by analyzing the genetic characteristics and temperature sensitivity (ts) of the SFTSV strain ZJ2013-06, which was isolated from the specific case. Phylogenetically, different clusters of SFTSV strains circulated in Zhejiang. ZJ2013-06 was farthest from ZJ2014-02, an isolate belonging to a Chinese dominant cluster, and nearest to the coastal strain NB24/CHN/2013. Ts tests, performed on Vero cells at 37°C and 39°C, indicated that ZJ2013-06 had restricted replication at 39°C. Its viral loads were substantially reduced at 39°C compared with that at 37°C (approximately 100-fold reduction) and were significantly lower than that of ZJ2014-02 at 39°C (<i>P</i> < 0.01). By adaptive culture at 39°C, the induced strain ZJ2013-06-P7 was obtained. Owing to a reverse mutation (S1616), ZJ2013-06-P7 lost the ts of the original strain, displaying similar replication processes with NB24/CHN/2013. The results indicated that the amino acid residue 1616 was related to the ts characteristics of ZJ2013-06. Our study revealed that ZJ2013-06 was temperature-sensitive and may be related to the absence of fever in our case.</p></div

Replication capacities of ZJ2013-06 at different temperatures.

<p>Replication capacities of ZJ2013-06 at different temperatures.</p

Temperature sensitivity of NB24/CHN/2013 and ZJ2013-06-P7.

<p>Temperature sensitivity of NB24/CHN/2013 and ZJ2013-06-P7.</p

Proliferation curves of ZJ2013-06 and ZJ2014-02 in Vero cells at different temperatures.

<p>Proliferation curves of ZJ2013-06 and ZJ2014-02 in Vero cells at different temperatures.</p

Specific amino acid sites possessed by the ZJ2013-06 strain.

<p>Specific amino acid sites possessed by the ZJ2013-06 strain.</p

Origin and Characteristics of Internal Genes Affect Infectivity of the Novel Avian-Origin Influenza A (H7N9) Virus

<div><p>Background</p><p>Human infection with a novel avian-origin influenza A (H7N9) virus occurred continuously in China during the first half of 2013, with high infectivity and pathogenicity to humans. In this study, we investigated the origin of internal genes of the novel H7N9 virus and analyzed the relationship between internal genes and infectivity of the virus.</p> <p>Methodology and Principal findings</p><p>We tested the environmental specimens using real-time RT-PCR assays and isolated five H9N2 viruses from specimens that were positive for both H7 and H9. Results of recombination and phylogeny analysis, performed based on the entire sequences of 221 influenza viruses, showed that one of the Zhejiang avian H9N2 isolates, A/environment/Zhejiang/16/2013, shared the highest identities on the internal genes with the novel H7N9 virus A/Anhui/1/2013, ranging from 98.98% to 100%. Zhejiang avian H9N2 isolates were all reassortant viruses, by acquiring NS gene from A/chicken/Dawang/1/2011-like viruses and other five internal genes from A/brambling/Beijing/16/2012-like viruses. Compared to A/Anhui/1/2013 (H7N9), the homology on the NS gene was 99.16% with A/chicken/Dawang/1/2011, whereas only 94.27-97.61% with A/bramnling/Beijing/16/2012-like viruses. Analysis on the relationship between internal genes and the infectivity of novel H7N9 viruses were performed by comparing amino acid sequences with the HPAI H5N1 viruses, the H9N2 and the earlier H7N9 avian influenza viruses. There were nine amino acids on the internal genes found to be possibly associated with the infectivity of the novel H7N9 viruses. </p> <p>Conclusions</p><p>These findings indicate that the internal genes, sharing the highest similarities with A/environment/Zhejiang/16/2013-like (H9N2) viruses, may affect the infectivity of the novel H7N9 viruses.</p> </div

Evolutionary process of the novel H7N9 virus isolated in China in 2013.

<div><p>Reassortment events on the six internal genes of the novel H7N9 viruses were analyzed based on the entire sequences of 221 influenza viruses using the RDP, GENECONV and MaxChi suites within the RDP3 software. In order to enhance the credibility of the results, the highest P value was set as 0.01. </p> <p>Note: A/brambling/Beijing/16/2012 (H9N2) -like strains including A/chicken/Shangdong/01/2009, A/chicken/Jiangsu/Q3/2010, A/chicken/Zhejiang/611/2011, A/chicken/Shanghai/C1/2012, A/chicken/Zhejiang/329/2011 and A/chicken/China/AH-10-01/2010. A/environment/Zhejiang/16/2013 (H9N2) -like strains including A/environment/Zhejiang/9/2013, A/environment/Zhejiang/13/2013, A/environment/Zhejiang/14/2013, A/environment/Zhejiang/16/2013. A/chicken/Dawang/1/2011 (H9N2)-like strains including A/chicken/Shuanggou/1/2011 (H9N2) and A/pigeon/Xuzhou/1/2011. The eight genes of different strains were shown in different color.</p></div

Phylogenetic trees for PB2 and NS genes of the novel H7N9 viruses.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081136#pone-0081136-g002" target="_blank">Figure 2A and 2B</a> was constructed based on the amino acid sequences of PB2 and NS genes respectively. These trees were all estimated using the Neighbor-joining (NJ) method of MEGA software (Version 5.0) with the bootstrap analysis of 1000 replications. The novel H7N9 isolates are marked with a triangle and the H9N2 virus strains isolated from environment specimens in Zhejiang province in 2013 are marked with a circle.</p