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Hepatitis B virus exposure in children usually develops into chronic hepatitis B (CHB). Although hepatitis B surface antigen (HBsAg)–specific CD8+ T cells contribute to resolve HBV infection, they are preferentially undetected in CHB patients. Moreover, the mechanism for this rarely detected HBsAg-specific CD8+ T cells remains unexplored. We herein found that the frequency of HBsAg-specific CD8+ T cells was inversely correlated with expansion of monocytic myeloid-derived suppressor cells (mMDSCs) in young rather than in adult CHB patients, and CCR9 was upregulated by HBsAg on mMDSCs via activation of ERK1/2 and IL-6. Sequentially, the interaction between CCL25 and CCR9 mediated thymic homing of mMDSCs, which caused the cross-presentation, transferring of peripheral HBsAg into the thymic medulla, and then promoted death of HBsAg-specific CD8+ thymocytes. In mice, adoptive transfer of mMDSCs selectively obliterated HBsAg-specific CD8+ T cells and facilitated persistence of HBV in a CCR9-dependent manner. Taken together, our results uncovered a novel mechanism for establishing specific CD8+ tolerance to HBsAg in chronic HBV infection.

Enterovirus 71 (EV71) is a major pathogen that causes hand, foot and mouth disease (HFMD), which is a life threatening disease in certain children. The pathogenesis of EV71-caused HFMD is poorly defined due to the lack of simple and robust animal models with severe phenotypes that recapitulate symptoms observed in humans. Here, we generated the infectious clone of a clinical isolate from a severe HFMD patient. Virus rescued from the cDNA clone was infectious in cell lines. When administrated intraperitoneally to neonatal ICR, BALB/c and C57 immune competent mice at a dosage of1.4 × 104 pfu per mouse, the virus caused weight loss, paralysis and death in the infected mice after 4–5 days of infection. In the infected mice, detectable viral replication was detected in various tissues such as heart, liver, brain, lung, kidney, small intestine, leg skeletal muscle and medulla oblongata. The histology of the infected mice included massive myolysis, glomerular atrophy, villous blunting in small intestine, widened alveolar septum, diminished alveolar spaces and lymphocytes infiltration into the lung. By using the UV-inactivated virus as a control, we elucidated that the virus first amplified in the leg skeletal muscle tissue and the muscle tissue served as a primary viral replication site. In summary, we generated a stable EV71 infectious clone that is capable of infecting neonatal immune competent mice without adaptive mutations and provide a simple, valuable animal model for the studies of EV71pathogenesis and therapy.

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.

In this study, chitosan (CH) coating with different number-average molecular weight (MW, ca. 5, 19 and 61 kDa) was electrostatic sprayed on strawberry. The effects of MW on strawberry quality changes were evaluated during 15 days of storage at 4 °C. The qualities of strawberry included mold growth, weight loss, firmness, total soluble solids (TSS), pH, flavonoids content, superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. Results showed that CH coating could significantly maintain the strawberry qualities during storage compared to uncoated treatment. CH coating with 61 kDa was more effective in retarding the increases of pH and MDA, and could better maintain flavonoids content. However, MW had no significant impact on mold growth, weight loss, firmness, SOD activity of coated strawberry. According to evaluation criteria, CH coating with 61 kDa had better performance on strawberry preservation with the highest synthetic value (6.93), and could be used to maintain quality and prolong the shelf life of strawberry during cold storage.

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

Zika virus (ZIKV) is associated with neonatal microcephaly and Guillain-Barre syndrome1,2. While progress has been made in understanding the causal link between ZIKV infection and microcephaly3-9, the life cycle and pathogenesis of ZIKV are less well understood. In particular, there are conflicting reports on the role of AXL, a TAM family kinase receptor that was initially described as the entry receptor for ZIKV10-22. Here, we show that while genetic ablation of AXL protected primary human astrocytes and astrocytoma cell lines from ZIKV infection, AXL knockout did not block the entry of ZIKV. We found, instead, that the presence of AXL attenuated the ZIKV-induced activation of type I interferon (IFN) signalling genes, including several type I IFNs and IFN-stimulating genes. Knocking out type I IFN receptor α chain (IFNAR1) restored the vulnerability of AXL knockout astrocytes to ZIKV infection. Further experiments suggested that AXL regulates the expression of SOCS1, a known type I IFN signalling suppressor, in a STAT1/STAT2-dependent manner. Collectively, our results demonstrate that AXL is unlikely to function as an entry receptor for ZIKV and may instead promote ZIKV infection in human astrocytes by antagonizing type I IFN signalling.

The avian origin influenza A virus (IAV) H7N9 has caused a considerable number of human infections associated with high rates of death since its emergence in 2013. As a vital component of the host innate immune system, the nucleotide-binding domain leucine-rich repeat containing receptor, pyrin domain containing 3 (NLRP3) inflammasome plays a critical role against H1N1 viral infection. However, the function of NLRP3 inflammasome in host immunological responses to the lethal H7N9 virus is still obscure. Here, we demonstrated that mice deficient for NLRP3 inflammasome components, including NLRP3, caspase-1, and Apoptosis-associated speck-like protein containing a CARD (ASC), were less susceptible to H7N9 viral challenge than wild type (WT) controls. Inflammasome deficiency in these animals led to significantly milder mortality and less pulmonary inflammation compared with WT mice. Furthermore, IL-1 receptor deficient mice also exhibited a higher survival rate than WT controls. Thus, our study reveals that the NLRP3 inflammasome is deleterious for the host during H7N9 infection in mice, which is due to an overwhelming inflammatory response via caspase-1 activation and associated IL-1 signal. Therefore, fine-tuning the activity of NLRP3 inflammasome or IL-1 signaling may be beneficial for the host to control H7N9 associated lethal pathogenesis.

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.

Infection with the influenza A (H1N1) virus is a major challenge for public health because it can cause severe morbidity and even mortality in humans. The over-secretion of inflammatory cytokines (cytokine storm) is considered to be a key contributor to the severe pneumonia caused by H1N1 infection. It has been reported that hypoxia-inducible factor 1-alpha (HIF-1α) is associated with the production of proinflammatory molecules, but whether HIF-1α participates in the acute inflammatory responses against H1N1 infection is still unclear. To investigate the role of HIF-1α in H1N1 infection, the expression and nuclear translocation of HIF-1α in A549 and THP-1 cell lines infected with H1N1 virus were observed. The results showed that without altering the intracellular mRNA or protein expression of HIF-1α, H1N1 infection only induced nuclear translocation of HIF-1α under normal oxygen concentrations. The use of 2-methoxyestradiol (2ME2), a HIF-1α inhibitor that blocks HIF-1α nuclear accumulation, in H1N1-infected cells decreased the mRNA and protein expression of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 and increased the levels of IL-10. In contrast, H1N1-infected cells under hypoxic conditions had increased HIF-1α nuclear accumulation, increased expression of TNF-α and IL-6 and decreased levels of IL-10. In conclusion, our data implied that in vitro H1N1 infection induced nuclear translocation of HIF-1α without altering the expression of HIF-1α, which may promote the secretion of proinflammatory cytokines during H1N1 infection.

Zika virus (ZIKV) infection can cause fetal developmental abnormalities and Guillain-Barre syndrome in adults. Although progress has been made in understanding the link between ZIKV infection and microcephaly, the pathology of ZIKV, particularly the viral reservoirs in human, remains poorly understood. Several studies have shown that compared to serum samples, patients' urine samples often have a longer duration of ZIKV persistency and higher viral load. This finding suggests that an independent viral reservoir may exist in the human urinary system. Despite the clinical observations, the host cells of ZIKV in the human urinary system are poorly characterized. In this study, we demonstrate that ZIKV can infect renal proximal tubular epithelial cells (RPTEpiCs) in immunodeficient mice in vivo and in both immortalized and primary human renal proximal tubular epithelial cells (hRPTEpiCs) in vitro. Importantly, ZIKV infection in mouse kidneys caused caspase-3-mediated apoptosis of renal cells. Similarly, in vitro infection of immortalized and primary hRPTEpiCs resulted in notable cytopathic effects. Consistent with the clinical observations, we found that ZIKV infection can persist with prolonged duration in hRPTEpiCs. RNA-Seq analyses of infected hRPTEpiCs revealed a large number of transcriptional changes in response to ZIKV infection, including type I interferon signaling genes and anti-viral response genes. Our results suggest that hRPTEpiCs are a potential reservoir of ZIKV in the human urinary system, providing a possible explanation for the prolonged persistency of ZIKV in patients' urine.

This study used electrostatic spraying (ES) system as an efficient technique for strawberry preservation with chitosan (CH) coating. The effects of CH deacetylation degree (DD, 81.0, 88.1 and 95.2%) on the qualities (weight loss, pH, total soluble solids (TSS), firmness, flavonoids content, malondialdehyde (MDA) content and superoxide dismutase (SOD) activity and mold growth) of strawberry during cold storage were investigated. Results showed that CH coating by ES formed more continuous and uniform protective layer compared with conventional spraying (CS). CH coating effectively enhanced the overall quality and extended shelf life of strawberry at least 2 days. CH coating with 81.0% and 88.1% DD were more effective in reducing weight loss of strawberry, and CH coating with 88.1% DD could better maintain TSS content, while CH coating with 95.2% DD delayed the decline in flavonoids more obviously. However, DD had no significant impact on pH, firmness, SOD activity, MDA and mold growth of coated samples. The evaluation criteria indicated CH coating with 88.1% DD had better preservation performance with the highest synthetic value (6.17) during storage. These findings suggest that CH coating especially with 88.1% DD by ES, as a safe, cheap and efficient technique, has great potential for industrial application of fruit preservation.

Chronic hepatitis B virus (HBV) infection is characterized by T cell tolerance to virus. Although inhibition of T cell responses by myeloid-derived suppressor cells (MDSCs) has been observed in patients with chronic hepatitis B (CHB), the mechanism for expansion of MDSCs remains ambiguous. In this study, a significant increased frequency of monocytic MDSCs (mMDSCs) was shown positively correlated to level of HBsAg in the patients with CHB. We further found hepatitis B surface Ag (HBsAg) efficiently promoted differentiation of mMDSCs in vitro, and monocytes in PBMCs performed as the progenitors. This required the activation of ERK/IL-6/STAT3 signaling feedback. Importantly, the mMDSCs polarized by HBsAg in vitro acquired the ability to suppress T cell activation. Additionally, treatment of all-trans retinoic acid, an MDSC-targeted drug, restored the proliferation and IFN-γ production by HBV-specific CD4+ and CD8+ T cells in PBMCs from patients with CHB and prevented increase of viral load in mouse model. In summary, HBsAg maintains HBV persistence and suppresses T cell responses by promoting differentiation of monocytes into mMDSCs. A therapy aimed at the abrogation of MDSCs may help to disrupt immune suppression in patients with CHB.

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.

Background Enteroviruses cause hand, foot and mouth disease (HFMD). The host B-cells recognize the viral proteins and provoke humoral responses. Deciphering the B-cell responses to the viral epitopes helps diagnosis and vaccine development. Objectives The objective of the present study was to investigate for the first time the landscape of genome-wide linear B-cell epitopes of enterovirus 71 in HFMD population. Study design The peptides encompassing the entire coding region of EV71 were chemically synthesized and displayed on a microarray. The peptide microarray was used to screen serum samples from an HFMD population, including EV71-, CAV10-, CAV16- and CAV6-infected patients. We identified the dominant epitope-containing-peptides (DECPs) that react with the sera of more than 20% of the HFMD population and the common DECPs that cross-react with the sera from other enteroviruses-infected population. Results Ten DECPs reacting with IgM and 9 DECPs reacting with IgG antibodies were identified, of which, 6 IgM and 5 IgG common DECPs cross-reacted with the sera from other enteroviruses. Some DECPs preferentially reacted with IgG or IgM antibodies and some epitope-antibody interactions correlated with the severity of HFMD. Conclusions We uncovered the DECPs and the common DECPs among a group of enteroviruses in HFMD population and found that some epitope-antibody reactions were associated with the outcome of HFMD. These data may guide developing vaccines against the enteroviruses and help the diagnosis and prognosis of HFMD.

Pretreatment of EV71 by CPZ had no effect on subsequent infection in A549 cells. EV71 was pretreated by DMSO or CPZ (20 μM), and then infected A549 cells (MOI=5). The “0h” indicated the infection in the presence or absence of CPZ (20 μM) without CPZ pretreatment. 12 h later, virus was removed and VP-1 expression was examined at 24 h. Means of three experiments are shown. *, p

Interferon-alpha (IFN-α) therapy of chronic hepatitis B (CHB) patients is constrained by limited response and side effects. We described a panel of circulating microRNAs (miRNAs) which could potentially predict outcome of IFN-α therapy. Here, we report development of a simplified scoring model for personalized treatment of CHB patients. 112 CHB patients receiving IFN-α treatment were randomly divided into a training (n = 75) or a validation group (n = 37). The expression of 15 candidate miRNAs was detected in training group with 5 miRNAs exhibiting significantly different levels (p

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)

Respiratory syncytial virus (RSV) infection induces activation and imbalance of the immune system; however, the role of T helper 17 cells (Th17) in the response to RSV infection remains unclear. Interleukin-23 (IL-23) is a key cytokine in Th17 cell differentiation. The aim of this study was to explore the function of IL-23 in determining the distribution of Th lymphocyte subsets (Th1, Th2, and Th17) after RSV infection in vitro. Human bronchial epithelial cell line BEAS-2B was infected with mock or RSV at various multiplicities of infection (MOI) and transcript expression of IL-6, IL-23p19, and transforming growth factor (TGF-β) was detected by real-time polymerase chain reaction; IL-6, IL-23, and TGF-β in the supernatant were measured by enzyme-linked immunosorbent assay. The Th subset distribution in lymphocytes was determined by flow cytometry after co-culture with supernatants from mock and 72-hr RSV infection cultures. The role of IL-23 in lymphocytes was assessed by specific receptor blockade (IL-23R) prior to co-culture with supernatants from RSV-infected BEAS-2B cells, followed by flow cytometry to analyze Th subset differentiation. Cytokine expression increased after RSV infection. IL-23R blockade suppressed the differentiation of Th1, Th2, and Th17 cells in the presence of supernatants from RSV-infected BEAS-2B cells. RSV infection may induce cytokine secretion, thus inducing Th1, Th2, and Th17 differentiation via an IL-23R-dependent process.

Aims To investigate the histopathological manifestations of two fatal cases of H7N9 influenza A virus infection. Methods Pulmonary and hepatic specimens from two fatal cases of H7N9 influenza virus infection were examined using H&E staining. Additionally, in situ hybridisation was performed with probes (ViewRNA) targeting H7N9 RNA and IP-10, interleukin (IL)-6 mRNA. The distribution of surfactant protein A (SP-A), surfactant protein B (SP-B), CD3, CD4, CD8, CD68 and C4d were determined with immunohistochemistry. Results Apart from the typical diffuse alveolar damage and hyaline membrane observed in severe influenza infection, we detected H7N9 RNA and massive intrapulmonary production of IP-10 and IL-6 mRNA using in situ hybridisation. Hyperplasia of type II pneumocytes was observed by H&E staining and immunohistochemistry. Proliferating macrophages and clustered neutrophils in the infected lungs were observed, whereas T lymphocytes, especially CD4T helper cells, were markedly depleted. No obvious complement deposition was found in lung tissues. Conclusions Our findings suggest that H7N9 influenza virus induced an immunological response towards overt pulmonary inflammation and systemic lymphopenia which led to intense alveolar damage and respiratory failure.

Background The outbreak of novel avian H7N9 influenza virus infections in China in 2013 has demonstrated the continuing threat posed by zoonotic pathogens. Deciphering the immune response during natural infection will guide future vaccine development. Methods We assessed the induction of heterosubtypic cross-reactive antibodies induced by H7N9 infection against a large panel of recombinant hemagglutinins and neuraminidases by quantitative enzyme-linked immunosorbent assay, and novel chimeric hemagglutinin constructs were used to dissect the anti-stalk or -head humoral immune response. Results H7N9 infection induced strong antibody responses against divergent H7 hemagglutinins. Interestingly, we also found induction of antibodies against heterosubtypic hemagglutinins from both group 1 and group 2 and a boost in heterosubtypic neutralizing activity in the absence of hemagglutination inhibitory activity. Kinetic monitoring revealed that heterosubtypic binding/neutralizing antibody responses typically appeared and peaked earlier than intrasubtypic responses, likely mediated by memory recall responses. Conclusions Our results indicate that cross-group binding and neutralizing antibody responses primarily targeting the stalk region can be elicited after natural influenza virus infection. These data support our understanding of the breadth of the postinfection immune response that could inform the design of future, broadly protective influenza virus vaccines.

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.).

Rotavirus is a major cause of acute gastroenteritis in infants and young children, while its role as a pathogen in adults has long been underappreciated. In order to describe the epidemiological patterns and genetic characteristics of rotavirus causing sporadic acute gastroenteritis in adults, hospital-based surveillance of rotavirus infections was conducted in Shanghai, China, between June 2010 and May 2011. Stool specimens were collected from outpatients with acute gastroenteritis admitted to three local hospitals. Rotavirus was detected using a colloidal gold test device. G and P genotyping were performed by multiplex PCR assays, and the VP7 gene of G9 strains were sequenced for further genetic characterization. Of 1,479 adult diarrheal stool samples examined during the 1-year surveillance period, 138 (9.3 %) were found to be rotavirus positive. G1 appeared to be the predominant genotype (35.5 %), suggesting a shift from genotype G3 to G1 in the study population in Shanghai. Meanwhile, a high frequency of genotype G9 (27.5 %) was also observed, and G9 was also predominant (38.1 %) in the small number of children (n=123) involved in the present study. Other specificities detected in adults were G2 (12.3 %) and G3 (13.8 %). For P genotyping, only two types, P[8] and P[4], were detected. P[8] was dominant in both children and adults. Sequence and phylogenetic analysis revealed that these strains could be divided into two different groups, with clustering within G9 lineage 3 and the subcluster of Japanese and Chinese G9 strains, respectively. In comparison to the previous data, G9 strains established themselves in a short time span as an important genotype in Shanghai, China.

Enterovirus-71 (EV71) infections can cause life-threatening diseases with neurological symptoms. Currently, no direct targeting antivirals are available to combat severe EV71 infection. Rupintrivir (AG7088) is a compound originally designed for Rhinovirus 3C protease. Previous computational analyses by us and crystallography studies by others suggested that rupintrivir is also a high affinity inhibitor to EV71 3C. Thus, we aimed to further evaluate its anti-EV71 activity in vivo at clinically acceptable doses. It was observed that administration of rupintrivir in suckling mice largely protected them from limb paralysis and dramatically improved survival (38.5% DMSO vs. 90.9% at 0.1mg/kg, p=0.006). Histological, immunohistochemical and quantitative RT-PCR analyses confirmed that rupintrivir profoundly alleviated virus induced necrotizing myositis, suppressed viral RNA and blocked EV71 VP1 expression in various tissues. In conclusion, we established that rupintrivir can strongly contain the spread of EV71 infection in vivo at a clinically acceptable dose (as low as 0.1mg/kg). As its safety has been fully tested in previous clinical trials, rupintrivir is suitable for immediate evaluation of potential benefits in EV71-infected individuals with life-threatening neurological symptoms.