Construction and Immunogenicity of a Recombinant Adenovirus Expressing the HA Gene of H5N1 Subtype Swine Influenza Virus

To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing the HA gene(rAd-H5HA-EGFP) was generated by co-transfecting the recombinant shuttle plasmid pDC315-H5HA-EGFP and the genomic plasmid pBHGlox△E1,E3Cre in HEK293 cells.The recombinant adenovirus was confirmed by PCR,RT-PCR and Western blot assay.These results demonstrated that HA protein was properly expressed by the rAd-H5HA-EGFP in HEK293 cells and had natural biological activities.The TCID50 of the rAd-H5HA- EGFP was assessed to be 2.26×1010/mL after propagation and purification.Immunization of BALB/ c mice indicated that rAd-H5HA-EGFP induced HI antibodies and protected mice from replication of the challenge virus in their lungs.

In silico modification of oseltamivir as neuraminidase inhibitor of influenza A virus subtype H1N1

This research focused on the modification of the functional groups of oseltamivir as neuraminidase inhibitor against influenza A virus subtype H1N1.Interactions of three of the best ligands were evaluated in the hydrated state using molecular dynamics simulation at two different temperatures.The docking result showed that AD3BF2 D ligand（N-[（1S,6R）-5-amino-5-{[（2R,3S,4S）-3,4-dihydroxy-4-（hydroxymethyl） tetrahydrofuran-2-yl]oxy}-4-formylcyclohex-3-en-1-yl]acetamide-3-（1-ethylpropoxy）-1-cyclohexene-1-carboxylate） had better binding energy values than standard oseltamivir.AD3BF2 D had several interactions,including hydrogen bonds,with the residues in the catalytic site of neuraminidase as identified by molecular dynamics simulation.The results showed that AD3BF2 D ligand can be used as a good candidate for neuraminidase inhibitor to cope with influenza A virus subtype H1N1.

Reduning plus ribavirin display synergistic activity against severe pneumonia induced by H1N1 influenza A virus in mice

OBJECTIVE:To investigate synergistic effect of Reduning(RDN)injection plus ribavirin against severe pneumonia induced by H1 N1 influenza A virus in mice.METHODS:We established a mouse model of severe pneumonia induced by influenza A virus by infecting Balb/c mice with CA07 virus.We randomly assigned the infected mice into four groups,and treated them with normal saline(NS group),RDN(injection,86.6 mg/kg),ribavirin(injection,66.6 mg/kg)or double Ribavirin plus RDN group,the same dosage as used in the single treatments)for 5 d.Lung index and lung pathology were recorded or calculated in terms of the curative effective.Cytokines,NOD-like receptor family pyrin domain containing 3(NLRP3)inflammasome related protein including caspase-associated recruitment domain(CARD)domain Apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC),caspase-1 and NOD-like receptor family,pyrin domain containing 3(NLRP3),and reactive oxygen species were simultaneously investigated.RESULTS:RDN plus ribavirin treatment,not RDN or ribavirin alone,provided a significant survival benefit to the influenza A virus-infected mice.The combination treatment protected the mice against severe influenza infection by attenuating the severe lung injury.The combined treatment also reduced the viral titers in mouse lungs and lung index,downregulated their immunocytokine levels,including IL-1βand IL-18,and down regulated the NLRP3,especially the transcription and translation of caspase-1.Meanwhile NS group had significantly higher reactive oxygen species(ROS)expression which could was dramatically reduced by the treatment of RDN plus ribavirin.CONCLUSION:Our study showed that RDN combined with ribavirin could protect the mice,and reduce the lung immunopathologic damage caused by severe influenza pneumonia.The mechanism could be that it reduced ROS produce and inhibited NLRP3 inflammasome activation so that mainly lower the downstream inflammatory cytokines IL-1βand IL-18.

Potential infections of H5N1 and H9N2 avian influenza do exist in Guangdong populations of China

Background Southeast China is one of the sites of influenza origin. During 2003-2004, nine avian influenza outbreaks took place in Guangdong Province. But no human case was reported. To examine the status of potential human infection by human influenza （H1N1, H3N2） and avian influenza （H5N1, H7N7, H9N2） in the avian influenza epidemic area of Guangdong Province, China, we conducted a seroepidemiologic survey in the people of this area from April to June of 2004.Methods Three out of 9 H5N1 avian influenza affected poultry areas in Guangdong were randomly selected, and the population living within 3 kilometers of the affected poultries were chosen as the survey subjects. One thousand two hundred and fourteen people were selected from 3 villages at random. Human and avian influenza antibody titers were determined by hemagglutination-inhibition （HI） test and microneutralization test （MNT）. Results The positive rate of antibody to H5N1 was 3.03% in the occupational exposure group and 2.34% in general citizens group; that of H9N2 was 9.52% in the occupational exposure group and 3.76% in the general citizens group. Moreover one case in the occupational exposure group was positive for H7N7. One year later, all previously positive cases had become negative except for one H5N1-positive case. Conclusion The observations imply that H5N1 and H9N2 avian influenza silent infections exist in Guangdong populations.

Hospitalized patients with novel influenza A （H1N1） virus infection： Shanghai, June-July 2009

Background From late May 2009, sporadic imported cases of novel influenza A （HIN1） were continuously confirmed in Shanghai, but there were few reports on its clinical presentation in China. The aim of the study was to investigate the demographic and clinical features of the laboratory-confirmed cases and the treatment with oseltamivir. Method We performed a retrospective study in the Shanghai Public Health Clinical Center （SHAPHC）, reviewing the medical records of the laboratory-confirmed patients derived from June 10 to July 20, 2009. Results A total of 156 cases were enrolled, of whom 152 had a history of recent travel. The mean age was 22.6 years and 89 cases （57.1%） were males. The most common symptoms were fever, cough, and sore throat, with children more likely to run a temperature above 38.5℃ than adults. The mean leucocyte count was 5.4×10^9/L, the mean neutrophil count 3.2×10^9/L and the mean lymphocyte count 1.4×10^9/L. Other findings included a normal range or elevated level of C-reactive protein （CRP） and glutamic-pyruvic transaminase and a normal or decreased level of prealbumin; the levels of prealbumin and CRP were significantly lower in the children than in the adults. Fifty-two patients had abnormal chest CT results, with small unilateral or bilateral pulmonary infiltrates, axillary and mediastinal lymphadenopathy and local pleural thickening, while no cases showed symptoms of hypoxia. All the patients received oseltamivir and recovered without complications, but the duration of fever and virus shedding were significantly longer in the children than in the adults. Conclusions Travel-related circulation may be an important reason for the H1N1 epidemic in the non-epidemic areas, and the virus caused mild respiratory symptoms. The infection in children was more severe in terms of prealbumin levels, temperature, the duration of fever and virus shedding. Oseltamivir was effective for H1N1, but more effective in the adults than in the children.

Influenza A pandemic （H1N1） 2009 virus infection

The clinical spectrum of the 2009 pandemic influenza A （H1N1） infection ranged from self-limited mild illness to progressive pneumonia, or even a fatal outcome. We summarize the clinical manifestations, risk factors for severe and fatal cases, pathologic findings and treatment of this disease in this paper based on current reports from different regions of the world.

Influenza A （H1N1） pneumonia： an analysis of 63 cases by chest CT

Background In early April 2009, cases of human infection with 2009 pandemic influenza A （H1N1） virus were identified in Mexico. The virus then spread rapidly to other regions of the world. From October 2009, sporadic imported cases of novel influenza A （HIN1） were continuously confirmed in Suzhou. The aim of the study was to review the chest CT findings in 63 patients with laboratory-confirmed novel swine-origin influenza A （H1N1） virus （S-OIV） infection. Methods Chest CT examinations were collected from 63 S-OIV infected patients during their hospital stay. Three experienced radiologists inspected images to qualitatively and quantitatively characterize S-OIV induced image changes. CT scores of lesion severity were calculated based on the percentage of affected area to determine severity of infectious lesions. Patients were divided into two groups based on the leukocyte counts. Lesion patterns, local distributions, and quantitative measures were investigated and compared between the two groups. Results Various degrees of bilateral multifocal lesions of ground-glass opacities were found with or without consolidations on the chest CT images. The lesions were both bronchocentric and centrilobular. Patients with elevated leukocyte counts had more extensive lesions, in terms of severity and affected area, than the patients with normal leukocyte counts. The lesion severity scores of patients in the elevated leukocyte group were significantly higher than those of the normal leukocyte group in terms of the entire lung area （P 〈0.01）, and upper （P 〈0.05） and lower （P 〈0.01） lobes as well. There were changes in the CT characteristics seen at follow-up as demonstrated by lesions absorption （P 〈0.01）, especially in the upper lobe of the lung （P〈0.01）, but less so in the middle lobe/lingual and lower lobe of the lung （m〉o.o5）. Conclusions The most common CT findings in S-OIV infection patients were bilateral multifocal distributed ground-glass opacities and consolidations. The lesions were located dominantly at bronchocentric and centrilobular areas. Lung lesions were more obviously absorbed in upper lobes between two examinations. The observations and analysis from this study provide information that may be useful in image understanding and patient management for future pandemic influenza.

Clinical features of initial cases of 2009 pandemic influenza A （H1N1） in Macao, China

Background The first case of pandemic influenza A （H1N1） virus infection in Macao Special Administrative Region （SAR） of the People＇s Republic of China was documented on June 18, 2009. Subsequently, persons with suspected infection or of contact with suspected cases received screening. All the confirmed cases were hospitalized and treated with oseltamivir. Their clinical features were observed. This may help for better management for later patients and be of benefit to the government of Macao SAR to adjust its strategy to combat the pandemic influenza A （H1N1） virus infection more efficiently. Methods From June to July 2009, the initial 72 cases of influenza A （H1N1） in Macao were hospitalized in Common Hospital Centre S. Januario （CHCSJ）. The infection was confirmed by real-time reverse-transcriptase polymerase chain reaction （RT-PCR）. The clinical features of the disease were closely observed and documented. Oseltamivir was given to all patients within 48 hours after the onset of disease and maintained for 5 days. Results The mean age of the 72 patients was 21 years old. Forty of them were men and 32 were women. The median incubation of the virus was 2 days （1 to 7 days）. The most common symptoms were fever （97.2%） and cough （77.8%）. The rate of gastrointestinal symptoms including nausea, vomiting, and diarrhea was 2.8%. Fever typically lasted for 3 days （1 to 9 days）. The median time from the onset to positive results of real-time RT-PCR was 6 days （3 to 13 days）. After treatment with oseltamivir, most patients became afebrile within 48 hours. Only one aged patient with a history of glaucoma and hypothyroidism was found to have lung infiltration on chest X-ray. Conclusions The initial cases of pandemic influenza A （H1N1） virus infection in Macao SAR showed that most of the infected persons had a mild course. The virus could be detected by real-time RT-PCR within a median of 6 days from the onset. Oseltamivir was effective.

Sixty-two severe and critical patients with 2009 influenza A （H1N1） in Shanghai, China

Background Pandemic influenza A （H1N1） emerged rapidly in China in May 2009. Preliminary comparisons with seasonal influenza suggest that pandemic 2009 influenza A （HIN1） disproportionately affects younger ages and causes generally mild disease. To characterize disease progress, comorbidities, and treatment outcomes among consecutive severe and critically ill patients in a hospital served as a reference center for the care of patients with HIN1 in Shanghai, China.Methods A retrospective study on 62 severe and critically ill patients with 2009 influenza A （H1N1） was conducted in Shanghai Public Health Clinical Center. Demographic data, symptoms, comorbidities, disease progression, treatments, and clinical outcomes were collected for analysis.

Pathogenesis of pandemic H1N1 2009 influenza virusinfection and the implication on management

The pandemic H1N12009 influenza virus has caused thefirst influenza pandemic of the 21st century,leading to disproportionate fatalities in the low-risk population despite the generally mild nature of the illness.Advances in science and technology have allowed very detailed study on the pathogenesis of this novel virus,and many have already been published in less than a year after the start of the pandemic.Information generated from cell lines,animal models,and clinical data analysis has provided us with greater understanding of the behavior of this virus and the associated host response.The new knowledge will allow us to formulate scientifically sound and evidence-based management plans.

Effect of Rorrico, extracted from group of Chinese medicines, on influenza A and H1N1 infections

OBJECTIVE: To investigate the therapeutic and preventive effects of Rorrico on influenza, especially influenza A viral infection, including swine flu(H1N1)in humans.METHODS: Eighty-nine subjects were recruited in Hong Kong and Macao, and divided into treatment group(TG) and prevention group(PG) based on their influenza A and swine flu symptoms. All subjects were prescribed Rorrico or placebo, and monitored by a Chinese medicine practitioner. Blood samples were collected before and after 7-day Rorrico or placebo treatment for laboratory investigations.RESULTS: After treatment, there were some full recoveries and obvious relief of onset symptoms in the TG. Blood test results showed that Rorrico produced(a) no adverse effects on subjects' renal andliver functions, muscle enzyme and hematological status,(b) no up-regulation of pro-inflammatory cytokines tumour necrosis factor-a and interleukin-18 in both TG and PG,(c) mild yet statistically significant elevation of plasma mannose-binding lectin(MBL) in PG.CONCLUSION: Rorrico has no up-regulating effect on the participants' immune response, or, equally likely, the immuno-modulatory effects of Rorrico do not non-specifically or unnecessarily promote inflammation when not required. It is possible that oral administration of Rorrico can promote hepatic synthesis of MBL in healthy PG subjects, thereby conferring increased protection against infection.

Anti-bacterial and anti-viral effects of Fengreqing oral liquid(风热清口服液) in vitro and in vivo

OBJECTIVE:To investigate the anti-bacterial and anti-viral effects of Fengreqing oral liquid(风热清口服液,FRQ)in vitro and in vivo.METHODS:The minimum inhibitory concentrations of Fengreqing Oral Liquid against six gram-positive bacteria(Staphylococcus aureus,Streptococcus mutans,Peptostreptococcus anaerobius,Hemolytic streptococcus,Streptococcus pneumoniae,Klebsiella pneumoniae),seven gram-negative bacteria(Escherichia coli,Actinobacillus actinomycetemcomitans,Porphyromonas gingivalis,Haemophilus influenzae,Helicobacter pylori,Pseudomonas aeruginosa,Gardnerella vaginalis)and Candida albicans were detected by the paper disc diffusion method.The inhibition rate of A/Puerto Rico/8/34(H1N1)(PR8)influenza virus in different concentrations of Fengreqing oral solution was detected by chicken embryo method.CCK8 method was used to detect the half-cell infection of RSV,VSV and CVB3.The effect of FRQ on the survival curve of mice was detected by using co-infection model of Streptococcus pneumoniae and influenza virus.RESULTS:In vitro,FRQ can inhibit Actinobacillus actinomycetemcomitans,Helicobacter pylori,Gardnerella vaginalis,Staphylococcus aureus,Streptococcus mutans and Streptococcus pneumoniae and has an antiviral effect on the envelope virus H1N1.In vivo,Fengreqing oral solution had therapeutic effect on influenza-Streptococcus pneumoniae co-infection in mice,significantly improving the survival rate of mice.The medium dose and low dose FRQ significantly prolonged the survival time of mice.CONCLUSION:FRQ has good anti-bacterial and anti-viral effects in vivo and in vitro.

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.