Structural basis for dsRNA recognition by NS1 protein of influenza A virus

Influenza A viruses are important human pathogens causing periodic pandemic threats. Nonstructural protein 1 （NS1） protein of influenza A virus （NS1A） shields the virus against host defense. Here, we report the crystal structure of NS1A RNA-binding domain （RBD） bound to a double-stranded RNA （dsRNA） at 1.7A. NS1A RBD forms a homodimer to recognize the major groove of A-form dsRNA in a length-independent mode by its conserved concave surface formed by dimeric anti-parallel a-helices, dsRNA is anchored by a pair of invariable arginines （Arg38） from both monomers by extensive hydrogen bonds. In accordance with the structural observation, isothermal titration calorimetry assay shows that the unique Arg38-Arg38 pair and two Arg35-Arg46 pairs are crucial for dsRNA binding, and that Ser42 and Thr49 are also important for dsRNA binding. Agrobacterium co-infiltration assay further supports that the unique Arg38 pair plays important roles in dsRNA binding in vivo.

Studies on Antiviral and Immuno-Regulation Activity of Low Molecular Weight Fucoidan from Laminaria japonica

The antiviral activity in vitro and in vivo and the effect of the immune system of two fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica(LMW fucoidans) were investigated in order to examine the possible mechanism. In vitro, I-type influenza virus, adenovirus and Parainfluenza virus I were used to infect Hep-2, Hela and MDCK cells, respectively. And 50% tissue culture infective dose was calculated to detect the antiviral activity of two LMW fucoidans. The results indicated that compared with the control group, 2 kinds of LMW fucoidans had remarkable antiviral activity in vitro in middle and high doses, while at low doses, the antiviral activity of 2 kinds of LMW fucoidans was not statistically different from that in the blank control group. And there was no statistically difference between two LMW fucoidans in antiviral activity. In vivo, LMW fucoidans could prolong the survival time of virus-infected mice, and could improve the lung index of virus-infected mice significantly, which have statistical differences with the control group significantly(p < 0.01). However, the survival time of the two LMW fucoidans was not statistically significant(p > 0.05). In this study, it was shown that both of two LMW fucoidans(LF1, LF2) could increase the thymus index, spleen index, phagocytic index, phagocytosis coefficient and half hemolysin value in middle and high doses, which suggested that LMW fucoidans could play an antiviral role by improving the quality of immune organs, improving immune cell phagocytosis and humoral immunity.

Anti-Influenza A Virus Effect of Hypericum perforatum L. Extract

To study the antiviral effect of Hypericum perforatum L. extract （HPE） on influenza A virus （IAV） （H1N1） in vitro and in vivo. Cytopathic effect （CPE） and neutral red （NR） dye uptake were used to examine the antiviral effect of HPE on Madin Darby Canine Kidney （MDCK） cells which were infected with IAV in vitro HPE was effective against influenza A virus （IAV） in vitro, with a 50% effective concentration （EC50） of 40 ug/mL, The mean 50% cytotoxic concentration （CC50） in the MDCK used in these experiments was 1.5 mg/mL. Ribavirin was run in parallel with EC50 values of 5.0 ug/mL; the mean CC50 for ribavirin was 520 ug/mL. Oral gavage administrations of HPE or ribavirin to mice infected with the IAV were highly effective in preventing death, slowing the decline of arterial oxygen saturation, inhibiting lung consolidation and reducing lung virus titers. The minimum effective dose of HPE in these studies was 31.25 mg/kg/day, which was administered twice daily for 5 d beginning 4 h prior to virus exposure. Below a dosage of 2000 mg/kg/day, almost all treated mice survived, which suggests that HPE is of low toxicity. Ribavirin＇s minimum effective dose was 40 mg/kg/day with the LDso determined to be 200 mg/kg/day. Delay of the initiation of either HPE or ribavirin therapy, using approximately 1/3 LD50 dose each time, could still be protective as late as 48 h after exposure to the IAV. While both agents appeared to have similar efficacy against IAV infections, HPE was considered to be less toxic and may warrant further evaluation as a possible therapy for influenza.

Severe human infection with a novel avian-origin influenza A(H7N4) virus

Human infections with influenza H7 subtypes, such as H7Ng, have raised concerns worldwide. Here, we report a human infection with a novel influenza A（HTN4） virus. A 68 years-old woman with cardiovascular and cholecystic comorbidities developed rapidly progressed pneumonia with influenza-like-illness as initial symptom, recovered after 23 days-hospitalization including 8 days in ICLI. Laboratory indicators for liver and blood coagulation dysfunction were observed. Oseltamivir phosphate, glucocorticoids and antibiotics were jointly implemented, with nasal catheterization of oxygen inhalation for this patient. We obtained the medical records and collected serial respiratory and blood specimens from her. We col- lected throat, cloacal and/or feces samples of poultry and wild birds from the patient＇s backyard, neigh- borhood, local live poultry markets （LPMs） and the nearest lake. All close contacts of the patient were followed up and sampled with throat swabs and sera. Influenza viruses and other respiratory pathogens were tested by real-time RT-PCR, viral culturing and/or sequencing for human respiratory and bird sam- ples. Micro-neutralizing assay was performed for sera. A novel reassortant wild bird-origin H7N4 virus is identified from the patient and her backyard poultry （chickens and ducks） by sequencing, which is dis- tinct from previously-reported avian H7N4 and H7N9 viruses. At least four folds increase of neutralizing antibodies to H7N4 was detected in her convalescent sera. No samples from close contacts, wild birds or other poultry were tested positive for H7N4 by real-time RT-PCR.

Avian influenza virus directly infects human natural killer cells and inhibits cell activity

Natural killer （NK） cell is a key component of innate immunity and plays an important role in host defense against virus infection by directly destroying infected cells. Influenza is a respiratory disease transmitted in the early phase of virus infection. Evasion of host innate immunity including NK cells is critical for the virus to expand and establish a successful acute infection. Previously, we showed that human influenza HIN1 virus infects NK cells and induces cell apoptosis, as well as inhibits NK cell activity. In this study, we further demonstrated that avian influenza virus also directly targeted NK cells as an immunoevasion strategy. The avian virus infected human NK cells and induced cell apoptosis. In addition, avian influenza virion and HA protein inhibited NK cell cytotoxicity. This novel strategy has obvious advantages for avian influenza virus, allowing the virus sufficient time to expand and subsequent spread before the onset of the specific immune response. Our findings provide an important clue for the immunopathogenesis of avian influenza, and also suggest that direct targeting NK cells may be a common strategy used by both human and avian influenza viruses to evade NK cell immunity.

Genomic characterizations of H4 subtype avian influenza viruses from live poultry markets in Sichuan province of China, 2014–2015

Dear Editor,Avian influenza viruses （AIVs） have posed a serious threat to poultry production and public health. To date, more than fourteen AIV subtypes that are able to infect human beings have been documented. Also, it is suggested that new subtypes may be reported in the future, owing to the migration of wild birds and live poultry transportation （Gao, 2018）.Poultry may act as a potential incubator for novel subtypes of avian influenza virus （Bi et al., 2016a; Bi et al., 2016b; Liu et al., 2014a; Su et al., 2017）. Up to date.

The S190R mutation in the hemagglutinin protein of pandemic H1N1 2009 influenza virus increased its pathogenicity in mice

Human influenza viruses preferentially bind to sialic acid-α2,6-galactose (SAα2,6Gal) receptors, which are predominant in human upper respiratory epithelia, whereas avian influenza viruses preferentially bind to SAα2,3Gal receptors. However, variants with amino acid substitutions around the receptor-binding sites of the hemagglutinin (HA) protein can be selected after several passages of human influenza viruses from patients’ respiratory samples in the allantoic cavities of embryonated chicken eggs. In this study, we detected an egg-adapted HA S190R mutation in the pandemic H1N1 virus 2009 (pdmH1N1), and evaluated the effects of this mutation on receptor binding affinity and pathogenicity in mice. Our results revealed that residue 190 is located within the pocket structure of the receptor binding site. The single mutation to arginine at position 190 slightly increased the binding affinity of the virus to the avian receptor and decreased its binding to the long human α2,6-linked sialic acid receptor. Our study demonstrated that the S190R mutation resulted in earlier death and higher weight loss in mice compared with the wild-type virus. Higher viral titers at 1 dpi (days post infection) and diffuse damage at 4 dpi were observed in the lung tissues of mice infected with the mutant virus.

Generation and application of replication-competent Venus-expressing H5N1,H7N9,and H9N2 influenza A viruses

The generation and application of replication-competent influenza A virus （IAV） expressing a reporter gene represent a valuable tool to elucidate the mechanism of viral pathogenesis and establish new coun- termeasures to combat the threat of influenza. Here, replication-competent 1AVs with a neuraminidase （NA） segment harboring a fluorescent reporter protein, Venus, were generated in the background of H5N1, H7N9, and H9N2 influenza viruses, the three subtypes of viruses with imminent pandemic poten- tial. All three reporter viruses maintained virion morphology, replicated with similar or slightly reduced titers relative to their parental viruses, and stably expressed the fluorescent signal for at least two pas- sages in embryonated chicken eggs. As a proof of concept, we demonstrated that these reporter viruses, used in combination with a high-content imaging system, can serve as a convenient and rapid tool for the screening of antivirals and host factors involved in the virus life cycle. Moreover. the reporter viruses demonstrated similar growth properties and tissue tropism as their parental viruses in mice, among which the HTN9 NA-Venus virus could potentially be used in ex vivo studies to better understand H7N9 pathogenesis or to develop novel therapeutics.

Clinical outcomes of influenza-like illness treated with Chinese herb- al medicine： an observational study

OBJECTIVE: To present and analyze treatmentsand clinical outcomes of Chinese patients with influenza-like illness.METHODS: We conducted a multi-site observational study from December 2009 to April 2010. Patients with influenza-like illness from 45 hospitals were enrolled. Patients received Chinese herbal medicine(CHM), conventional treatments, or CHM plus conventional treatments(combination treatment) according to the guidelines for influenza A/H1 N1 2009 in China. The primary outcomes were the time to alleviation of symptoms and the incidence of complications. The secondary outcomes were the time until becoming afebrile, incidence of severe illness, testing negative on an influenza A viral test, and total medical fees.RESULTS: In total, 5967 patients were enrolled. The percentages of patients prescribed CHM alone, conventional treatment, and combination treatment were 27.8%, 5.1%, and 67.7%, respectively. There were no significant differences in the time to alleviation of symptoms, incidence of complications,time to becoming afebrile, or rate of severe illness among the CHM, conventional, and combination treatment groups. The rates of testing negative on the influenza virus A rapid test and H1 N1 virus test were 90.3% and 76.3%, respectively. However,significant differences were found in the total medical fees among the three groups: CHM treatments were more economical than the other two treatments.CONCLUSION: The efficacy of CHM for influenza-like illness was not different from that of conventional treatments, but it was more economical.