唐山市甲型H1N1流感病毒血凝素基因序列分析

目的 分析2010—2016年唐山市甲型H1N1流感病毒血凝素(hemagglutinin,HA)基因序列进化特征.方法 选取唐山市3家哨点医院流感样病例分离到的24株甲型H1N1病毒,通过RT-PCR和测序方法获得HA基因的全长序列,运用分子生物学软件和统计学软件对序列进行拼接、比对和分析.结果 同源进化分析显示,24株甲型H1N1流感病毒HA基因与疫苗株A/California/7/2009的核苷酸和氨基酸的同源性分别为97.0%~99.0%和97.0%~98.5%.进化分析显示,2010—2016年唐山地区流行的甲型H1N1流感病毒属于1、7、6三个基因分支,其中6分支毒株分为6C、6B、6B.1和6B.2亚支.氨基酸位点分析显示,不同毒株与疫苗株比较存在8~16处氨基酸位点改变,其中7个变异涉及3个抗原表位:H138Q/Y和S203T突变位于Ca区,N125S、K153E、S162N、K163T/Q突变位于Sa区,S185T突变位于Sb区同时也位于受体结合部位;2015—2016流行季6B.1分支毒株抗原位点S162N突变增加了新的潜在糖基化位点.结论 与疫苗株比较,随着时间推移唐山地区甲型H1N1流感病毒发生了抗原漂变,未来仍应关注6B分支流行株的变化.     

Pandemic influenza A(H1N1)pdm09: risk of infection in primary healthcare workers

Background

Healthcare workers in primary care are at risk of infection during an influenza pandemic. The 2009 influenza pandemic provided an opportunity to assess this risk.

Aim

To measure the prevalence of seropositivity to influenza A(H1N1)pdm09 among primary healthcare workers in Canterbury, New Zealand, following the 2009 influenza pandemic, and to examine associations between seropositivity and participants’ sociodemographic characteristics, professional roles, work patterns, and seasonal influenza vaccination status.

Design and setting

An observational study involving a questionnaire and testing for influenza A(H1N1)pdm09 seropositivity in all primary healthcare workers in Canterbury, New Zealand between December 2009 and February 2010.

Method

Participants completed a questionnaire that recorded sociodemographic and professional data, symptoms of influenza-like illness, history of seasonal influenza vaccination, and work patterns. Serum samples were collected and haemagglutination inhibition antibody titres to influenza A(H1N1)pdm09 measured.

Results

Questionnaires and serum samples were received from 1027 participants, from a workforce of 1476 (response rate 70%). Seropositivity was detected in 224 participants (22%). Receipt of seasonal influenza vaccine (odds ratio [OR] = 2.0, 95% confidence interval [CI] = 1.2 to 3.3), recall of influenza (OR = 1.9, 95% CI = 1.3 to 2.8), and age ≤45 years (OR = 1.4, 95% CI = 1.0 to 1.9) were associated with seropositivity.

Conclusion

A total of 22% of primary care healthcare workers were seropositive. Younger participants, those who recalled having influenza, and those who had been vaccinated against seasonal influenza were more likely to be seropositive. Working in a dedicated influenza centre was not associated with an increased risk of seropositivity.     

Hemagglutinin variants of influenza A(H1N1)pdm09 virus with reduced affinity for sialic acid receptors

In influenza A H1 virus, amino acids at position 190 and 225 of HA affect replication and transmission. In this study, we show that the mutation D190Y in the HA of influenza AH1N1pdm09 virus reduces the affinity of the virus for sialic acid receptors expressed at the surface of red blood cells from different species without affecting virus replication in MDCK cells.     

Complete Sequences of the Neuraminidase Genes of Swine Influenza Viruses (H1N1) Associated with the Respiratory Disease in Pigs

The complete nucleotide sequences of neuraminidase (NA) of two swine influenza viruses (H1N1) are presented. A/Sw/Quebec/5393/91 (SwQc91) virus, associated with the chronic respiratory disease and A/Sw/Quebec/192/81 (SwQc81) virus, associated with the acute respiratory disease, were used. The deduced amino acid sequences of NA of SwQc91 and SwQc81 viruses showed a high degree (>95%) of similarity. The NA gene of both viruses was a single open reading frame of 1459 nucleotides coding for 469 aa with a 5′ noncoding region of 21 nucleotides and a 3′ noncoding region of 28 nucleotides. The comparison of two sequences showed that there were 23 differences recorded for SwQc91 strain, of which 5, 6, and 12 differences were recorded in the hydrophobic, stalk and head regions, respectively. A potential antigenic determinant changed from Ala to Thr at position 453 and there was a new potential glycosylation site present at position 88 for SwQc91 strain whereas it was absent at position 50 when compared with SwQc81 strain. Estimates of genetic distance and phylogenic tree analysis showed that SwQc91 and SwQc81 viruses were closely related with each other and with the American strain, A/Sw/Wisconsin/4754/94. However, the swine viruses represented a distinct group that was considerably divergent from the group of human viruses. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of oseltamivir‐resistant influenza A(H1N1)pdm09 viruses in Taiwan in 2009–2011

The early isolated swine‐origin influenza A(H1N1)pdm09 viruses were susceptible to oseltamivir; however, there is a concern about whether oseltamivir‐resistant influenza A(H1N1)pdm09 viruses will spread worldwide as did the oseltamivir‐resistant seasonal influenza A(H1N1) viruses in 2007–2008. In this study, the frequency of oseltamivir resistance in influenza A(H1N1)pdm09 viruses was determined in Taiwan. From May 2009 to April 2011, 1,335 A(H1N1)pdm09‐positive cases in Taiwan were tested for the H275Y mutation in the neuraminidase (NA) gene that confers resistance to oseltamivir. Among these, 15 patients (1.1%) were found to be infected with H275Y virus. All the resistant viruses were detected after the patients have received the oseltamivir. The overall monthly ratio of H275Y‐harboring viruses ranged between 0% and 2.88%, and the peak was correlated with influenza epidemics. The genetic analysis revealed that the oseltamivir‐resistant A(H1N1)pdm09 viruses can emerged from different variants with a great diversity under drug pressure. The ratio of NA/HA activities in different clades of oseltamivir‐resistant viruses was reduced compared to those in the wild‐type viruses, indicating that the balance of NA/HA in the current oseltamivir‐resistant influenza A(H1N1)pdm09 viruses was interfered. It is possible that H275Y‐bearing A(H1N1)pdm09 virus has not yet spread globally because it lacks the essential permissive mutations that can compensate for the negative impact on fitness by the H275Y amino acid substitution in NA. Continuous monitoring the evolution patterns of sensitive and resistant viruses is required to respond to possible emergence of resistant viruses with permissive genetic background which enable the wide spread of resistance. J. Med. Virol. 85:379–387, 2013. © 2012 Wiley Periodicals, Inc.     

Letter to the Editor: Drift in the Nucleoprotein Gene of Swine Influenza Virus (H1N1) Causing Respiratory Disease in Pigs

The nucleoprotein (NP) gene of swine influenza H1N1 variant, A/Sw/Quebec/5393/91 (SwQc91) was sequenced. When compared with other H1N1 strains, 12 amino acid (aa) replacements were observed in the 101–484 aa region of the NP protein including two aas, 345 and 430, representing the unique lineage of swine viruses. Phylogenetic analysis showed a drift in the NP gene.     

流感及H5N1亚型禽流感病毒液相检测芯片的制备

目的 建立利用液相芯片技术检测甲、乙型流感和H5N1亚型高致病性禽流感病毒的方法,并对该方法进行评价。方法 对GenBank中甲型流感病毒的NP、乙型流感病毒的HA以及高致病性禽流感病毒（H5N1）的H5、N1基因片段序列进行同源性比对,根据保守序列,设计针对各基因的简并引物和寡核苷酸探针,制备探针偶联微球,将样本核酸多重PCR扩增产物与微球进行杂交,以Bio-Plex液相芯片检测系统进行芯片检测。结果 该方法可以对甲型流感病毒的NP基因、乙型流感病毒的HA基因以及高致病性禽流感病毒（H5N1）的H5、N1基因同时进行检测,病毒核酸的最低检出量为1pg,检测特异性高。结论 成功构建了甲、乙型流感病毒和H5N1亚型高致病性禽流感病毒液相芯片检测系统,为流感、禽流感的快速检测、诊断奠定了基础。     

Co-infection with influenza A/H1N1 and A/H3N2 viruses in a patient with influenza-like illness during the winter/spring of 2008 in Shanghai, China

Co-infection with different influenza viruses occurs naturally and plays an important role in epidemiology and pathogenicity. To monitor the prevalence of influenza viruses in humans during seasonal influenza epidemics in Shanghai, China, and to analyze the genetic characteristics of the viruses, 365 nasopharyngeal swabs collected from patients with influenza-like illness between January and April 2008, were tested by a colloidal gold assay, viral isolation in Madin-Darby canine kidney (MDCK) cells, direct immunofluorescence assay and multiplex RT-PCR. The genetic characteristics of the viruses were analyzed by full-length PCR amplification of the HA segment. One case of co-infection with influenza A/H1N1 and A/H3N2 viruses was detected among the 7 cases of A/H1N1, 84 cases of A/H3N2 and 48 cases of influenza B virus during the winter/spring of 2008. All influenza A/H1N1 and A/H3N2 isolates were similar, including the co-infecting isolates. The present study demonstrates the possibility of natural co-infection with different types of influenza viruses in humans, which could provide the opportunity for the occurrence of viral genetic reassortment within the human respiratory tract.     

Pregnant women infected with pandemic influenza A(H1N1)pdm09 virus showed differential immune response correlated with disease severity

BackgroundDuring pregnancy, immunological and hormonal alterations place women at increased risk for influenza-related severe illnesses including hospitalization and death. Although A(H1N1) pdm09 infection resulted in increased disease severity in pregnant women, the precise mechanisms responsible for this risk have yet to be established.ObjectivesThe present study was aimed to investigate the role of host chemokines and cytokine profiles in A(H1N1) pdm09 infection regarding disease severity in pregnant women.Study designThis retrospective survey examined 41 pregnant women with confirmed A(H1N1) pdm09 infection. Of them, 12 died (D), 29 survived (S), and 17 remained uninfected and served as controls (C). Antiviral response was evaluated for IFN-β expression and gene expression profiles of cytokines (TNF-α, IL-6, IL-12, TGF-β) and chemokines (IL-8, RANTES, MCP-1, IP-10), and the viral Matrix (M1) gene was quantified and normalized using the housekeeping gene product β-actin mRNA.ResultsHigher IL-8 and TNF-α mRNA expression were found in D and S compared with C, while IL-6 showed higher expression in D. Interestingly, these results were associated with a decrease in the anti-inflammatory response of TGF-β mRNA and IFN-β. These alterations would lead to an imbalance in the immune response of those patients.ConclusionsPregnancy-related reductions in IFN-β and TGF-β expression levels and elevated levels of pro-inflammatory cytokines could explain the increased severity of infection and death of pregnant women. These findings may help improve the understanding of the high susceptibility and disease severity to influenza virus infection during pregnancy.     

The response of medical virology laboratories to the influenza A(H1N1)pdm09 outbreak in Paris Île-de-France region

The outbreak of influenza A(H1N1)pdm09 was a challenge for the laboratories of Paris Île-de-France region in charge of virological diagnosis. In order to evaluate the quality of their response to this challenge, a retrospective survey based on a self-administered standardized questionnaire was undertaken among the 18 hospital laboratories involved in A(H1N1)pdm09 virus detection over a period of 10 months from April 2009 to January 2010. All concerned laboratories responded to the survey. Due to imposed initial biosafety constraints and indications, virological diagnosis was performed in only two laboratories at the start of the studied period. Step by step, it was further settled in the other laboratories starting from June to November 2009. From the beginning, A(H1N1)pdm09-specific RT-PCR was considered the reference method while the use of rapid influenza detection tests remained temporary and concerned a minority of these laboratories. Among the overall 21,656 specimens received, a positive diagnosis of influenza A(H1N1)pdm09 was obtained in 5,390 cases (25%), the positivity range being significantly higher among women as compared to men (P < 0.0001) and subjects below 45 years of age as compared to those over 65 years (P < 0.0001). Two peaks in positivity frequency were observed at weeks 24 (30%, 8–12 June 2009) and 44 (50%, 26–30 October 2009) respectively, the latter one occurring 2 weeks earlier than the peak of epidemic at the national level. In contrast, a low positivity rate was detected at weeks 38–40 in relationship with other respiratory virus infections which were clinically misinterpreted as a peak of influenza epidemic. These data demonstrate the ability of medical virology laboratories of Paris Île-de-France region to provide in real time a valuable diagnosis of A(H1N1)pdm09 virus infection and a relevant view of outbreak evolution, suggesting they will be a crucial component in the management of future influenza epidemics.     

Detection of haemagglutinin D222 polymorphisms in influenza A(H1N1)pdm09-infected patients by ultra-deep pyrosequencing

This study was aimed at establishing the genetic heterogeneity of influenza virus haemagglutinin (HA) gene quasi-species and the polymorphisms at codon 222, by application of ultra-deep pyrosequencing (UDPS) to respiratory samples from patients hospitalized for influenza A(H1N1)pdm09 infection, presenting with severe or moderate–mild disease. HA diversity was significantly higher in samples collected from patients with severe manifestations than in those from patients with moderate–mild manifestations (p 0.02). D222 polymorphism was detected in 40.7% of patients by UDPS, and in only 7.1% by Sanger sequencing. D222E, D222G, D222N and D222A were observed in 37.0%, 11.1%, 7.4% and 3.7% of patients, respectively; 10.7% of samples harboured more than two variants. The relative frequency of each single variant showed a wide range of intrapatient variation. D222G/N/A were detected, as either minor or predominant variants, only in severe cases, whereas D222E was equally represented in severe and moderate–mild infections. Other amino acid variants were observed at different positions within the analysed HA fragment. Consistent with higher heterogeneity, non-D222 variants were more frequently detected in severe cases than in moderate–mild cases. In addition, seven non-D222 mutations carried by minority variants, not previously described, were observed.     

Evidence for Subclinical Influenza A(H1N1)pdm09 Virus Infection among Dogs in Guangdong Province,China

During 2012, we identified sampled dogs with elevated levels of antibodies (≥1:40) against A(H1N1)pdm09 virus by using a hemagglutination inhibition (HI) assay (seroprevalence, 24.7%) and a microneutralization (MN) assay (seroprevalence, 10.8%). These high seroprevalences of A(H1N1)pdm09 among dogs without clinical signs of influenza support the premise that dogs may play a role in the human influenza ecology in China.