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Virus reprogramming of host cellular function is a critical strategy for viral survival and replication. A better understanding of virus-host interaction may provide new potential avenues for the treatment of viral diseases. It has been reported that hypoxia-inducible factor-1 (HIF-1) pathway is activated by a range of pathogens via different mechanisms, but the impact of Influenza A virus on HIF-1 signaling is still unclear. In this study, we observed H1N1 infection stabilized HIF-1α under normoxic conditions. In detail, H1N1 did not increase HIF-1α mRNA transcription, nor impaired posttranslational prolyl hydroxylation or ubiquitination of HIF-1α, but inhibited the function of proteasome, resulting in HIF-1α accumulation. Furthermore, a decreased expression of factor inhibiting HIF-1 (FIH-1), which hydroxylates asparagine 803 within HIF-1α to repress HIF-1α activity, was seen after H1N1 infection. Taken together, these findings reveal a previously unrecognized mechanism of viral activation of the HIF-1 pathway, resembling a hypoxic response in normoxia.

Objective: To investigate viral infection in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) in Shanghai, and to analyze the clinical characteristics and biomarkers in viral infection. Methods: This study included all consecutive patients who were admitted for a diagnosis of AECOPD during June 2013 to May 2015. 31 stable COPD patients and 31 healthy controls were also recruited. Oropharyngeal samples were assessed, PCR for respiratory viruses were performed. Patients were divided into AECOPD virus-positive (+) group and AECOPD virus-negative (-) group according to viral detection. Luminex was used to detect the concentrations of inflammatory cytokines in the serum. Results: A total of 264 patients were included with a mean age of 75 ± 0.5 years. There were 72 patients (27.3%) identified with viral positive, of whom two patients were detected with double viral infections (FluA+FluB and RSVA+HRV, respectively). The rate of viral detection was associated with season, highest in winter. Comparisons of clinical characteristics showed no significant differences between AECOPD virus+ group and AECOPD virus- group. However, serum concentrations of interferon-inducible protein-10 (IP-10) and interferon-gamma (IFN-γ) in virus+ AECOPD patients were significantly higher than those in the virus- AECOPD, stable COPD and healthy control groups (P

Summary IFITM3 and TLR3 SNPs are associated with fatal clinical outcome of Chinese patients with avian (H7N9) or pandemic (H1N1pdm09) influenza virus infections, and the risks are cumulative. Our findings pose important public health and clinical implications in the at-risk populations., Background. We examined associations between single-nucleotide polymorphisms (SNPs) of IFITM3, TLR3, and CD55 genes and influenza clinical outcomes in Chinese. Methods. A multicenter study was conducted on 275 adult cases of avian (H7N9) and pandemic (H1N1pdm09) influenza. Host DNA was extracted from diagnostic respiratory samples; IFITM3 rs12252, TLR3 rs5743313, CD55 rs2564978, and TLR4 rs4986790/4986791 were targeted for genotyping (Sanger sequencing). The primary outcome analyzed was death. Results. IFITM3 and TLR3 SNPs were in Hardy–Weinberg equilibrium; their allele frequencies (IFITM3/C-allele 0.56, TLR3/C-allele 0.88) were comparable to 1000 Genomes Han Chinese data. We found over-representation of homozygous IFITM3 CC (54.5% vs 33.2%; P = .02) and TLR3 CC (93.3% vs 76.9%; P = .04) genotypes among fatal cases. Recessive genetic models showed their significant independent associations with higher death risks (adjusted hazard ratio [aHR] 2.78, 95% confidence interval [CI] 1.29–6.02, and aHR 4.85, 95% CI 1.11−21.06, respectively). Cumulative effects were found (aHR 3.53, 95% CI 1.64−7.59 per risk genotype; aHR 9.99, 95% CI 1.27−78.59 with both). Results were consistent for each influenza subtype and other severity indicators. The CD55 TT genotype was linked to severity. TLR4 was nonpolymorphic. Conclusions. Host genetic factors may influence clinical outcomes of avian and pandemic influenza infections. Such findings have important implications on disease burden and patient care in at-risk populations.

Based on measles surveillance in Shanghai, People’s Republic of China, from 2006 to 2015, we found that measles virus isolates from 40 throat swab samples exhibited atypical cytopathic effects in Vero/hSLAM cells, which was found to be a result of coinfection with measles virus (MeV) and human herpes simplex virus type 1 (HSV-1). Serological and molecular approaches were used to confirm and characterize the coinfections in these patients. Among the 40 measles cases, measles-specific IgM was detected in 37 cases, while measles-specific IgG was detected in 27 cases. HSV-1-specific IgM and IgG were detected in 7 and 34 cases, respectively, suggesting that most of the MeV infections were primary, but that HSV-1 infection was due to the reactivation of latent virus in most cases. The titers of HSV-1 IgG in patients with either measles or measles-HSV-1 coinfection were significantly higher than those in the healthy group (P = 0.0026 and P

Background The pattern of epochal evolution of NoV is ongoing, while novel GII.4 variants emerge and cause new pandemics. Since, the emergence in March 2012, Sydney_2012 had replaced GII.4-2009 as the primary NoV strain in most countries in the northern hemisphere by November 2012. Objectives To determine the genotype distribution around the emergence of Sydney_2012 and to investigate the underlying evolution mechanisms of the contemporary GII.4 strains. Study design From January 2012 to December 2013, molecular epidemiology of norovirus in 846 adults (≥16 years) in Shanghai were conducted. The VP1 proteins of the contemporary GII.4 strains (Den_Haag_2006b, New_Orleans_2009 and Sydney_2012) were expressed in vitro and purified. Receptor binding patterns of these three epidemic strains were determined through histo-blood group antigen (HBGA) binding assays. Convalescent serum from patients infected with GII.4 epidemic strains were employed to investigate the role of antigenic drift in the persistence of GII.4 epidemic strains through receptor-binding blockade assays. Results Epidemiological studies revealed that Sydeny_2012 has completely replaced Den_Haag_2006b and New_Orleans_2009 and has been the dominant circulating strain in Shanghai since its emergence in October 2012. Interestingly, Den_Haag_2006b and New_Orleans_2009 have been co-circulating in Shanghai before the emergence of Sydeny_2012. The contemporary GII.4 epidemic norovirus strains displayed commonly high tropism to the histo-blood group antigen receptors, whereas Sydeny_2012 was antigenically different from Den_Haag_2006b and New_Orleans_2009. Conclusions Antigenic drift, rather than receptor switch, played a key role in the emergence and spreading of Sydney_2012. The contemporary GII.4 strains were evolving via epochal evolution without altered ligand binding profiles.

To the Editor: Since the first case of avian-origin influenza (H7N9) virus infection was reported in March 2013 in Shanghai, China, this virus has caused ≈400 confirmed cases (http://www.who.int/influenza/human_animal_interface/influenza_h7n9/Data_Reports/en/). According to the Chinese Influenza Weekly Report (http://www.cnic.org.cn/eng/show.php?contentid=699), during the winter of 2013–14, multiple influenza viruses that infect humans co-circulated in southern China; these viruses were influenza A(H1N1)pdm09, seasonal influenza A(H3N2), and influenza B viruses, together with avian influenza (H7N9) virus,. Co-infection of humans with avian virus subtype H7N9 and human virus subtypes were reported in Jiangsu Province in June 2013 (1) and in Zhejiang Province in January 2014 (2), respectively. We report a fatal case of co-infection with influenza (H7N9) and A(H1N1)pdm09 viruses, which was further confirmed by virus isolation and neutralization antibody detection. In a retrospective study, we identified co-infection with A(H1N1)pdm09 and novel avian-origin H7N9 virus in a 61-year-old female patient from the urban area of Shanghai, China. She had a 3-year history of thrombocythemia. In October 2013, she received an annual vaccine that included an A/California/7/2009(H1N1)pdm09–like virus, an A/Texas/50/2012(H3N2)–like virus, and an influenza B virus, as recommended by the World Health Organization (http://www.who.int/influenza/vaccines/virus/recommendations/2013_14_north/en/). On December 26, 2013, the patient experienced influenza-like symptoms; the next day, she was hospitalized. Despite oral administration of oseltamivir, a rapidly progressive pneumonia developed, and the patient died of multiple organ failure 9 days after admission. On December 31, 2013 (day 5), 1 serum and 2 throat swab samples were collected; both throat swab samples were positive for influenza A and A(H1N1)pdm09 virus, according to the universal and subtype-specific primers provided by the Chinese Center for Disease Control and Prevention (http://www.who.int/influenza/resources/documents/diagnostic_recommendations/en/). On January 3, 2014 (day 8), 1 bronchial secretion and 1 serum sample were also collected. Recent DNA sequencing analysis of the stored bronchial secretion specimen, performed on a Roche 454 platform (3) (Roche, Basel, Switzerland) showed co-existence of H1, H7, N1, and N9 genomic segments, which were further confirmed by real-time reverse transcription PCR (RT-PCR). The cycle threshold values of H7 and H1 were 35.65 and 29.94, respectively. We then determined antibody levels of the 2 serum samples collected on days 5 and 8 by using a microneutralization assay (4) with MDCK cells infected with 2 representative strains, A/Shanghai/4664T/2013(H7N9) and A/Shanghai/37T/2009(H1N1), respectively. The samples were serially diluted 2-fold (1:10 to 1:1,280) at a starting dilution of 1:10; titers were expressed as the reciprocal of the highest dilution of 50% neutralization. The microneutralization titers against novel subtype H7N9 virus were 160 and 320 for the samples collected on days 5 and 8, respectively. This finding indicated that the patient could have been infected by novel subtype H7N9 viruses (5); microneutralization titers against A/Shanghai/37T/2009(H1N1) were 320 for both samples. To determine whether the reassortment had occurred between the 2 subtypes of influenza A viruses during the co-infection, we isolated the virus strains from the bronchial secretion on either MDCK cells (A[H1N1]pdm09) (http://www.who.int/influenza/gisrs_laboratory/manual_diagnosis_surveillance_influenza/en/) or embryonated eggs (H7N9) through several passages. The hemagglutination titers of the isolated H7N9 (A/Shanghai/Mix1/2014[H7N9]) and A(H1N1)pdm09 viruses (A/Shanghai/Mix1/2014[H1N1]) in allantoic fluid were 32 and 8, respectively. Cultures of the 2 isolates were subjected to RT-PCR for detection of 8 genomic segments. The RT-PCR products were sequenced on an ABI 3730XL Automatic DNA Analyzer by using an ABI Prism BigDye Terminator v3.1 Cycle Sequencing Kit (both from Applied Biosystems, Foster City, CA, USA). The full-genome sequences were submitted to the National Center for Biotechnology Information Influenza Virus Resource (GenBank accession nos. {"type":"entrez-nucleotide","attrs":{"text":"KJ946415","term_id":"660304600","term_text":"KJ946415"}}KJ946415, {"type":"entrez-nucleotide","attrs":{"text":"KJ946416","term_id":"660304618","term_text":"KJ946416"}}KJ946416 and {"type":"entrez-nucleotide-range","attrs":{"text":"KP058484-KP058489","start_term":"KP058484","end_term":"KP058489","start_term_id":"723446113","end_term_id":"723446126"}}KP058484-KP058489 for A/Shanghai/Mix1/201[H7N9] and {"type":"entrez-nucleotide","attrs":{"text":"KM607860","term_id":"698322612","term_text":"KM607860"}}KM607860, {"type":"entrez-nucleotide","attrs":{"text":"KM607861","term_id":"698322614","term_text":"KM607861"}}KM607861 and {"type":"entrez-nucleotide-range","attrs":{"text":"KP058490-KP058495","start_term":"KP058490","end_term":"KP058495","start_term_id":"723446129","end_term_id":"723446141"}}KP058490-KP058495 for A/Shanghai/Mix1/2014[H1N1]). Phylogenetic trees were constructed by using the neighbor-joining method in MEGA 5.1 (http://www.megasoftware.net) to estimate relationships with selected influenza A virus reference sequences obtained from GenBank and the Global Initiative on Sharing Avian Influenza Data database. DNA sequence analysis showed that no genetic reassortment had occurred between the 2 subtypes because only H7N9 or A(H1N1)pdm09 genomic fragments were found in the culture of the respective isolate (Figure; Technical Appendix). Phylogenetic analyses of the hemagglutinin and neuraminidase genes revealed that the A/Shanghai/Mix1/2014(H7N9) and A/Shanghai/Mix1/2014(H1N1) viruses were clustered into the clade of A/Shanghai/01/2014(H7N9)-like viruses (6) (Figure, panels A and B) and A/Shanghai/6109/2014(H1N1)-like viruses (Figure, panels C and D), respectively. In addition, the phylogenetic trees of the 6 internal genes were close to those of H7N9 viruses or A(H1N1)pdm09-lineage viruses that had recently circulated in China (Technical Appendix). Figure Phylogenetic analyses of A/Shanghai/Mix1/2014(H7N9) and A/Shanghai/Mix1/2014(H1N1) viruses from Shanghai, China, January 2014. A) Hemagglutinin and B) neuraminidase of A/Shanghai/Mix1/2014(H7N9); C) hemagglutinin and D) neuraminidase of A/Shanghai/Mix1/2014(H1N1). ... Co-infections with H7N9 and other influenza subtypes have been reported from Jiangsu (1) and Zhejiang (2) Provinces as well as in our study, indicating the potential risk for co-infection during the influenza season in China. Although reassortment was not detected in this co-infection, a potential risk for emergence of a new pandemic strain by reassortment between these 2 viruses (with humans as mixing vessels) should not be ignored. To reduce the risk for emergence of new viral subtypes, the public health and scientific communities should enhance surveillance for co-infection with influenza (H7N9) virus and other influenza virus subtypes. Technical Appendix: Phylogenetic analysis of 12 internal genes of the co-infection viruses in Shanghai, China. Click here to view.(5.1M, pdf)

Neuraminidase inhibitors (NAIs) are the only available licensed therapeutics against human H7N9 influenza virus infections. The emergence of NAI-resistant variants of H7N9viruses with an NA R292K mutation poses a therapeutic challenge. A comprehensive understanding of the susceptibility of these viruses to clinically available NAIs, non-NAIs and their combinations is crucial for effective treatment. In this study, by using limited serial passage and plaque purification, an R292K variant of the Anhui1 lineage was isolated from a patient with clinical evidence of resistance to oseltamivir. In vitro and cell-based assays confirmed a high level of resistance conferred by the R292K mutation to oseltamivir carboxylate and a moderate level of resistance to zanamivir and peramivir. Non-NAI antivirals, such as T-705, ribavirin and NT-300, efficiently inhibited both the variant and the wild-type in cell-based assays. A combination of NAIs and non-NAIs did not exhibit a marked synergistic effect against the R292K variant. However, the combination of two non-NAIs (T-705 and ribavirin) exhibited significant synergism against the mutant virus. In experimentally infected mice, the variant showed delayed onset of symptoms, a reduced viral load and attenuated lethality compared with the wild-type. Our study suggested non-NAIs should be tested clinically for H7N9 patients with a sustained high viral load. Possible drug combination regimens, such as T-705 plus ribavirin, should be further tested in animal models. The pathogenicity and transmissibility of the R292K H7N9 variant should be further assessed with genetically well-characterized pairs of viruses and, most-desirably, with competitive fitness experiments.

We report a case of acute cytomegalovirus (CMV) infection with positive HEV and Epstein–Barr virus (EBV) serology. No patients have been reported positive for immunoglobulin (Ig)M antibodies to all three viruses. This patient had progressively increasing titres of IgM antibody for CMV, HEV and EBV. Only CMV DNA was detectable before antiviral treatment. After antiviral treatment, the patient recovered completely. At day 180 the CMV IgG test had converted to positive with CMV IgM (+), EBV IgM (-) and HEV IgM (-). Our report indicates that dependence upon serology alone is unreliable in the diagnosis of acute CMV, EBV and HEV infections. The diagnosis of CMV, HEV and EBV should be based on a combination of clinical features, serology and confirmatory PCR testing.

Infection of poultry with influenza A subtype H7 viruses occurs worldwide, but the introduction of this subtype to humans in Asia has not been observed previously. In March 2013, three urban residents of Shanghai or Anhui, China, presented with rapidly progressing lower respiratory tract infections and were found to be infected with a novel reassortant avian-origin influenza A (H7N9) virus.We obtained and analyzed clinical, epidemiologic, and virologic data from these patients. Respiratory specimens were tested for influenza and other respiratory viruses by means of real-time reverse-transcriptase-polymerase-chain-reaction assays, viral culturing, and sequence analyses.A novel reassortant avian-origin influenza A (H7N9) virus was isolated from respiratory specimens obtained from all three patients and was identified as H7N9. Sequencing analyses revealed that all the genes from these three viruses were of avian origin, with six internal genes from avian influenza A (H9N2) viruses. Substitution Q226L (H3 numbering) at the 210-loop in the hemagglutinin (HA) gene was found in the A/Anhui/1/2013 and A/Shanghai/2/2013 virus but not in the A/Shanghai/1/2013 virus. A T160A mutation was identified at the 150-loop in the HA gene of all three viruses. A deletion of five amino acids in the neuraminidase (NA) stalk region was found in all three viruses. All three patients presented with fever, cough, and dyspnea. Two of the patients had a history of recent exposure to poultry. Chest radiography revealed diffuse opacities and consolidation. Complications included acute respiratory distress syndrome and multiorgan failure. All three patients died.Novel reassortant H7N9 viruses were associated with severe and fatal respiratory disease in three patients. (Funded by the National Basic Research Program of China and others.).

Norovirus (NoV) is now recognized as a leading cause of nonbacterial acute gastroenteritis; however, the NoV GII.17 genotype has rarely been reported as the predominant genotype in clinical diarrhea cases. During the winter of 2014–2015, the GII.17 genotype, together with the NoV GII.4 genotype, dominated in sporadic adult patients with gastroenteritis in Shanghai. Phylogenetic analysis based on full-length VP1 amino acid sequences showed that the GII.17 strains that emerged in Shanghai have close evolutionary relationships with strains recently collected in the Hong Kong area, Guangdong province of China, and Japan during the same period. This cluster in the phylogenetic tree may represent a novel NoV GII.17 lineage recently circulating in East Asia. Pairwise distances between clusters also revealed the evolution of the NoV GII.17 genotype in previous decades. Our study emphasizes the importance of combined surveillance of NoV-associated infections.

Our data demonstrated that the inoculation with vaccine derived from the 2009 pandemic influenza raised vigorous neutralization antibodies against both cognate H1N1 and heterotypic influenza viruses. This observation has important implication for vaccine development., Background. A mass vaccination has been implemented to prevent the spread of 2009 pandemic influenza virus in China. Highly limited information is available on whether this vaccine induces cross-reactive neutralization antibodies against other subtypes of influenza viruses. Methods. We employed pseudovirus-based assays to analyze heterosubtypic neutralization responses in serum samples of 23 recipients of 2009 pandemic influenza vaccine. Results. One dose of pandemic vaccine not only stimulated good neutralization antibodies against cognate influenza virus 2009 influenza A (H1N1), but also raised broad cross-reactive neutralization activities against seasonal H3N2 and highly pathogenic avian influenza virus H5N1 and lesser to H2N2. The cross-reactive neutralization activities were completely abolished after the removal of immunoglobin G (IgG). In contrast, H1N1 vaccination alone in influenza-naive mice elicited only vigorous homologous neutralizing activities but not cross-reactive neutralization activities. Conclusions. Our data suggest that the cross-reactive neutralization epitopes do exist in this vaccine and could elicit significant cross-reactive neutralizing IgG antibodies in the presence of preexisting responses. The exposure to H1N1 vaccine is likely to modify the hierarchical order of preexisting immune responses to influenza viruses. These findings provide insights into the evolution of human immunity to influenza viruses after experiencing multiple influenza virus infections and vaccinations.

Sensitive molecular techniques are needed for rapid detection of the R292K oseltamivir-resistant mutant of influenza A(H7/N9) virus strain to monitor its transmission and guide antiviral treatment. We developed a real-time reverse transcription PCR and single nucleotide polymorphism probes to differentiate this mutant strain in mixed virus populations in human specimens.

A novel recombinant coxsackievirus A6 (CVA6) strain was isolated during a coxsackievirus A6 outbreak in Shanghai, China, in 2013. Genomic sequence and similarity plot analysis showed that the novel CVA6 strain shared higher similarity with a recent CVA4 strain rather than the recent CVA6 strain in the 2C and 3′ untranslated regions (UTRs).

Serologic studies are urgently needed to assist in understanding an outbreak of influenza A(H7N9) virus. However, a biosafety level 3 laboratory is required for conventional serologic assays with live lethal virus. We describe a safe pseudovirus-based neutralization assay with preliminary assessment using subtype H7N9-infected samples and controls.

To the Editor: Yellow fever is disease caused by a flavivirus that is transmitted to humans and nonhuman primates through the bites of infected mosquitoes. In 2013, an estimated 130,000 persons in Africa experienced fever with jaundice or hemorrhage associated with yellow fever; ≈78,000 of these infections were fatal (1).

Human rhinovirus (HRV) is the virus most often associated with acute upper respiratory tract infections. Advances in molecular detection have shown that HRV is also the major viral cause of asthma exacerbations. Genotypic assignment and identification of HRV types are of significant value in the investigation of type-associated differences in disease outcomes, transmission, and epidemiology. Here, we describe a genotyping process involving two separate RT-PCR assays, targeted to VP4/VP2 and 5' UTR regions of HRV genome, respectively. Together with the reference sequences of each HRV species, the generated sequences are used to construct phylogenetic tree for genotyping.

AIM: To optimize the viral persistence rate in a hydrodynamic injection (HI) based hepatitis B virus (HBV) transfection mouse model. METHODS: (1) 5-6-wk-old male C3H/HeN and C57BL/6 mice were hydrodynamically injected with 10 μg endotoxin-free pAAV/HBV1.2 plasmid DNA via the tail vein. Hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and HBV DNA, both in the serum and liver, were detected at different time points post HI by ELISA, immunohistochemical staining or quantitative polymerase chain reaction (PCR); (2) male C3H/HeN and C57BL/6 mice, either hydrodynamically injected mice at 10 wk post HI or naive mice, were all immunized subcutaneously with 5 μg HBsAg formulated in complete Freund’s adjuvant three times at a 2-wk interval. Two weeks after the final immunization, splenocytes were isolated for T cell function analysis by ELISPOT assay; and (3) five weeks post HI, C3H/HeN mice were intragastrically administered 0.1 mg/kg entecavir once a day for 14 d, or were intraperitoneally injected with 1 mg/kg interferon (IFN)-α twice a week for 2 wk, or were treated with PBS as controls. The sera were collected and assayed for HBV DNA on days 0, 7 and 14 after drug treatment. RESULTS: (1) Approximately 90% (22/25) of the injected C3H/HeN mice were still HBsAg-positive at 46 wk post HI, whereas HBsAg in C57BL/6 mice were completely cleared at 24 wk. Serum levels of HBeAg in C3H/HeN mice were higher than those in C57BL/6 mice from 4 wk to 46 wk. HBV DNA levels in the hydrodynamically injected C3H/HeN mice were higher than those in the C57BL/6 mice, both in the serum (from 4 wk to 46 wk) and in the liver (detected at 8 wk and 46 wk post HI). Histology showed that hepatitis B core antigen and HBsAg were expressed longer in the liver of C3H/HeN mice than in C57BL/6; (2) HBsAg specific T cell responses after HBsAg vaccination in hydrodynamically injected C3H/HeN and C57BL/6 mice, or naive control mice were detected by ELISPOT assay. After stimulation with HBsAg, the frequencies of IFN-γ producing splenocytes in the hydrodynamically injected C3H/HeN mice were significantly lower than those in hydrodynamically injected C57BL/6 mice, control C3H/HeN and control C57BL/6 mice, which were 0, 17 ± 7, 18 ± 10, and 41 ± 10 SFCs/106 splenocytes, respectively, and the mean spot sizes showed the same pattern. Even just stimulated with PMA and ionomysin, T-cell responses elicited in the vaccinated control C3H/HeN were much higher than those in hydrodynamically injected C3H/HeN mice; and (3) For drug treatment experiments on the hydrodynamically injected C3H/HeN mice, serum HBV DNA levels in the entecavir treatment group declined (131.2 folds, P < 0.01) on day 7 after treatment and kept going down. In the group of IFN-α treatment, serum HBV DNA levels declined to a lowest point (6.42 folds, P < 0.05) on 7 d after treatment and then rebounded. CONCLUSION: We have developed a novel HI-based HBV transfection model using C3H/HeN mice, which had a higher HBV persistence rate than the classic C57BL/6 mouse model.

We analyzed the complete genome sequence of the A/Shanghai/01/2014 (H7N9) strain, which will provide a better understanding of the evolution of influenza A(H7N9) virus.

To the Editor: Norovirus (NoV) has been identified as one of the major causal agents of nonbacterial, acute gastroenteritis in humans (1). The genetic diversity among NoVs is great, and human strains have been classified into 3 genogroups (GI, GII, and GIV). Despite this diversity, in recent years only a few strains, primarily those of genogroup II, genotype 4 (GII.4), have been responsible for most cases and outbreaks worldwide (1,2). The pattern of epochal evolution of NoV is ongoing, and novel GII.4 variants emerge, which replace previously dominant strains and cause new pandemics. Surveillance systems worldwide showed an increase in NoV activity in late 2012 (3). Molecular data shared through NoroNet (www.rivm.nl/en/Topics/Topics/N/NoroNet) suggest that this increase is related to the emergence of a new GII.4 variant, termed Sydney_2012 (3). We found that this novel GII.4 variant also emerged in Shanghai, China, and caused increased levels of NoV activity during October–December 2012. During July 2011–December 2012, fecal specimens from 748 outpatients (≥16 years of age) with acute gastroenteritis who visited 1 of the 2 sentinel hospitals in Shanghai were collected and stored at Shanghai Public Health Clinical Center at −70°C. Molecular detection of GI and GII NoV was performed by using conventional reverse transcription PCR as described (4). Full-length viral protein 1 and 639 bp of the 3′ RNA-dependent RNA polymerase gene of 4 randomly selected GII-positive strains were amplified (5–7). NoV genotypes were classified on the basis of a 280-bp region for GI and a 305-bp region for GII by using the Automated Genotyping Tool (www.rivm.nl/mpf/norovirus/typingtool). A total of 77 patients showed positive results for GII NoV. An increase in GII NoV activity was observed during October–December in 2012; the detection rate was 46.08% (47 cases in 102 outpatients). The prevalence of GII NoV during the same period in 2011 was low; the detection rate was 6.90% (8 cases in 116 outpatients). Genotyping analysis of the strains detected in these 3 months in 2012 (39 strains were sequenced) showed that except for 1 GII.6 strain and 3 GII.4 2006b strains, the other 35 strains sequenced all belong to the new established cluster of GII.4, termed Sydney_2012. Retrospective analysis indicated that the novel GII.4 variant had already been detected in 2 outpatients during September 2011 in Shanghai. Phylogenetic analysis of full-length capsid nucleotide sequences for 4 strains randomly selected from the new cluster indicated a novel GII.4 pattern, and new strains clustering separately from previously identified GII.4 pandemic strains (Figure). On the basis of BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) searches, the most closely related NoVs (98%–100% nucleotide identity) were 4 GII.4 viruses recently detected in Australia and Hong Kong. The new GII.4 strains detected in Shanghai also clustered with these strains, a finding that was supported by bootstrap values >70% (Figure). The 3′ end of RNA-dependent RNA polymerase gene sequences also confirmed that the new GII.4 strains were recombinants, with a GII.e polymerase and GII.4 capsid (3). Figure Phylogenetic tree of norovirus GII.4 capsid nucleotide sequences, Shanghai, China. The dendrogram was constructed by using the neighbor-joining method in MEGA version 5.0 (8). Bootstrap resampling (1,000 replications) was used, and bootstrap values ≥70% ... Despite improved control measures to combat NOV, this highly infectious agent continues to cause a large number of epidemics of gastroenteritis globally (approximately every 2 years), and most epidemics have been associated with emergence of a novel GII.4 cluster (9). The new cluster reported in the present study was first detected in Australia in March, 2012, followed by detection in France, New Zealand, Japan, the United Kingdom, the United States, and Hong Kong, where increased levels of NoV activity in late 2012 compared with previous seasons were also observed (3). This novel GII.4 strain has also emerged in Shanghai, China, and caused increased levels of sporadic cases during October–and December 2012. This new variant has common ancestors, dominant NoV GII.4 variants Osaka_2007 and New Orleans_2009, but is phylogenetically distinct from them. Amino acid changes are present in major epitopes located in the P2 domain, a finding that is consistent with observations from previous epidemics (3).

A novel strain of influenza A(H7N9) virus has emerged in China and is causing mild to severe clinical symptoms in infected humans. Some case-patients have died. To further knowledge of this virus, we report the characteristics and clinical histories of 4 early case-patients.

Background Upper respiratory tract infection (URTI) is a major reason for hospitalization in childhood. More than 80% of URTIs are viral. Etiological diagnosis of URTIs is important to make correct clinical decisions on treatment methods. However, data for viral spectrum of URTIs are very limited in Shanghai children. Methods Nasopharyngeal swabs were collected from a group of 164 children aged below 3 years who were hospitalized due to acute respiratory infection from May 2009 to July 2010 in Shanghai. A VRDAL multiplex PCR for 10 common respiratory viruses was performed on collected specimens compared with the Seeplex® RV15 ACE Detection kit for 15 respiratory viruses. Results Viruses were detected in 84 (51.2%) patients by VRDAL multiplex PCR, and 8 (4.9%) of cases were mixed infections. Using the Seeplex® RV15 ACE Detection kit, viruses were detected in 129 (78.7%) patients, 49 (29.9%) were co-infected cases. Identified viruses included 37 of human rhinovirus (22.6% of cases), 32 of influenza A virus (19.5%), 30 of parainfluenzavirus-2 (18.3%), 23 of parainfluenzavirus-3 (14.0%), 15 of human enterovirus (9.1%), 14 each of parainfluenzavirus-1, respiratory syncytial virus B and adenovirus (8.5%), 8 of coronavirus 229E/NL63 (4.9%), 6 of human bocavirus (3.7%), 5 each of influenza B virus and respiratory syncytial virus A (3.0%), 3 of parainfluenzavirus-4 (1.8%), 2 of coronavirus OC43/HKU1 (1.2%), and 1 human metapneumovirus (0.6%). Conclusions A high frequency of respiratory infections (78.7%) and co-infections (29.9%) was detected in children with acute respiratory infection symptoms in Shanghai. The Seeplex® RV15 ACE detection method was found to be a more reliable high throughput tool than VRDAL method to simultaneously detect multiple respiratory viruses.

A novel electronic cell sensor array technology, the real-time cell analysis (RTCA) system, was developed to monitor cell events. Unlike the conventional methods labeling the target cells with fluorescence, luminescence, or light absorption, the RTCA system allows for label-free detection of cell processes directly without the incorporation of labels. Here, we used this new format to measure the cytopathic effect (CPE) of the 2009 influenza A (H1N1) virus and the efficacy of neutralizing antibodies in human sera to this virus. The real-time dynamic monitoring of CPE was performed on MDCK cell cultures infected with the H1N1 virus, ranging from 5.50×10(2) to 5.50×10(7) copies/mL. The resulting CPE kinetic curves were automatically recorded and were both time and viral load dependent. The CPE kinetics were also distinguishable between different H1N1 stains, as the onset of CPE induced by the A/Shanghai/37T/2009 H1N1 virus was earlier than that of the A/Shanghai/143T/2009 H1N1 virus. Furthermore, inhibition of H1N1 virus-induced CPE in the presence of human specific anti-sera was detected and quantified using the RTCA system. Antibody titers determined using this new neutralization test correlated well with those obtained independently via the standard hemagglutination inhibition test. Taken together, this new CPE assay format provided label-free and high-throughput measurement of viral growth and the effect of neutralizing antibodies, illustrating its potential in influenza vaccine studies.

Several viral diseases exhibit measles-like symptoms. Differentiation of suspected cases of measles with molecular epidemiological techniques in the laboratory is useful for measles surveillance. In this study, a random PCR screening system was undertaken for the identification of isolates from patients with measles-like symptoms who exhibited cytopathic effects, but who had negative results for measles virus-specific reverse transcription (RT)-PCR and indirect immunofluorescence assays. Sequence analysis of random amplified PCR products showed that they were highly homologous to type 1 human herpes simplex virus (HSV-1). The results were further confirmed by an HSV-1-specific TaqMan real-time PCR assay. The random PCR screening system described in this study provides an efficient procedure for the identification of unknown viral pathogens. Measles-like symptoms can also be caused by HSV-1, suggesting the need to include HSV-1 in differential diagnoses of measles-like diseases.

Hepatitis C virus non-structural protein NS5A plays an important role in viral replication and various cellular events. To gain further insight into the function of NS5A, we screened a human fetal liver cDNA library for its interacting proteins using the yeast two-hybrid system. FKBP38, a 38kDa immunosuppressant FK506-binding protein, was identified and its interaction with NS5A was confirmed by both in vitro and in vivo. The interaction was mapped to the amino acids 148–236 of NS5A containing a BH domain (Bcl-2 homology domain). Besides, both NS5A and FKBP38 were found to localize in mitochondria and endoplasmic reticulum. Moreover, NS5A stably expressing Huh7 hepatoma cells showed more resistance to apoptosis and such inhibition of apoptosis could specifically be abrogated by depletion of FKBP38 using RNA interference. These results indicate that HCV NS5A inhibits apoptosis through interaction with FKBP38.

It has been suggested that cellular proteins are involved in hepatitis C virus (HCV) RNA replication. By using the yeast two-hybrid system, we isolated seven cDNA clones encoding proteins interacting with HCV RNA polymerase (NS5B) from a human liver cDNA library. For one of these, alpha-actinin, we confirmed the interaction by coimmunoprecipitation, immunofluorescent staining and confocal microscopic analysis. Experiments with deletion mutants showed that domains NS5B(84-95), NS5B(466-478), and alpha-actinin(621-733) are responsible for the interaction. Studies of the HCV subgenomic replicon system with small interference RNA indicate that alpha-actinin is essential for HCV RNA replication. Our results suggest alpha-actinin may be a component of the HCV replication complex.

To establish an efficient, sensitive, cell-based assay system for NS3 serine protease in an effort to study further the property of hepatitis C virus (HCV) and develop new antiviral agents.We constructed pCI-neo-NS3/4A-SEAP chimeric plasmid, in which the secreted alkaline phosphatase (SEAP) was fused in-frame to the downstream of NS4A/4B cleavage site. The protease activity of NS3 was reflected by the activity of SEAP in the culture media of transient or stable expression cells. Stably expressing cell lines were obtained by G418 selection. Pefabloc SC, a potent irreversible serine protease inhibitor, was used to treat the stably expressing cell lines to assess the system for screening NS3 inhibitors. To compare the activity of serine proteases from 1b and 1a, two chimeric clones were constructed and introduced into both transient and stable expression systems.The SEAP activity in the culture media could be detected in both transient and stable expression systems, and was apparently decreased after Pefabloc SC treatment. In both transient and stable systems, NS3/4A-SEAP chimeric gene from HCV genotype 1b produced higher SEAP activity in the culture media than that from 1a.The cell-based system is efficient and sensitive enough for detection and comparison of NS3 protease activity, and screening of anti-NS3 inhibitors. The functional difference between NS3/4A from 1a and 1b subtypes revealed by this system provides a clue for further investigations.

Analysis of complete capsid sequences of the emerging norovirus GII. 17 Kawasaki 308 from 13 countries demonstrated that they originated from a single haplotype since the initial emergence in China in late 2014. Global spread of a sublineage SL2 was identified. A new sublineage SL3 emerged in China in 2016.