Influenza A/H1N1 2009 Pandemic and Respiratory Virus Infections,Beijing, 2009–2010

To determine the role of the pandemic influenza A/H1N1 2009 (A/H1N1 2009pdm) in acute respiratory tract infections (ARTIs) and its impact on the epidemic of seasonal influenza viruses and other common respiratory viruses, nasal and throat swabs taken from 7,776 patients with suspected viral ARTIs from 2006 through 2010 in Beijing, China were screened by real-time PCR for influenza virus typing and subtyping and by multiplex or single PCR tests for other common respiratory viruses. We observed a distinctive dual peak pattern of influenza epidemic during the A/H1N1 2009pdm in Beijing, China, which was formed by the A/H1N1 2009pdm, and a subsequent influenza B epidemic in year 2009/2010. Our analysis also shows a small peak formed by a seasonal H3N2 epidemic prior to the A/H1N1 2009pdm peak. Parallel detection of multiple respiratory viruses shows that the epidemic of common respiratory viruses, except human rhinovirus, was delayed during the pandemic of the A/H1N1 2009pdm. The H1N1 2009pdm mainly caused upper respiratory tract infections in the sampled patients; patients infected with H1N1 2009pdm had a higher percentage of cough than those infected with seasonal influenza or other respiratory viruses. Our findings indicate that A/H1N1 2009pdm and other respiratory viruses except human rhinovirus could interfere with each other during their transmission between human beings. Understanding the mechanisms and effects of such interference is needed for effective control of future influenza epidemics.     

Guillain-Barré Syndrome and Adjuvanted Pandemic Influenza A (H1N1) 2009 Vaccines: A Multinational Self-Controlled Case Series in Europe

Background

The risk of Guillain-Barré syndrome (GBS) following the United States'' 1976 swine flu vaccination campaign in the USA led to enhanced active surveillance during the pandemic influenza (A(H1N1)pdm09) immunization campaign. This study aimed to estimate the risk of GBS following influenza A(H1N1)pdm09 vaccination.

Methods

A self-controlled case series (SCCS) analysis was performed in Denmark, Finland, France, Netherlands, Norway, Sweden, and the United Kingdom. Information was collected according to a common protocol and standardised procedures. Cases classified at levels 1–4a of the Brighton Collaboration case definition were included. The risk window was 42 days starting the day after vaccination. Conditional Poisson regression and pooled random effects models estimated adjusted relative incidences (RI). Pseudo likelihood and vaccinated-only methods addressed the potential contraindication for vaccination following GBS.

Results

Three hundred and three (303) GBS and Miller Fisher syndrome cases were included. Ninety-nine (99) were exposed to A(H1N1)pdm09 vaccination, which was most frequently adjuvanted (Pandemrix and Focetria). The unadjusted pooled RI for A(H1N1)pdm09 vaccination and GBS was 3.5 (95% Confidence Interval (CI): 2.2–5.5), based on all countries. This lowered to 2.0 (95% CI: 1.2–3.1) after adjustment for calendartime and to 1.9 (95% CI: 1.1–3.2) when we accounted for contra-indications. In a subset (Netherlands, Norway, and United Kingdom) we further adjusted for other confounders and there the RI decreased from 1.7 (adjusted for calendar month) to 1.4 (95% CI: 0.7–2.8), which is the main finding.

Conclusion

This study illustrates the potential of conducting European collaborative vaccine safety studies. The main, fully adjusted analysis, showed that the RI of GBS was not significantly elevated after influenza A(H1N1)pdm09 vaccination (RI = 1.4 (95% CI: 0.7–2.8). Based on the upper limits of the pooled estimate we can rule out with 95% certainty that the number of excess GBS cases after influenza A(H1N1)pdm09 vaccination would be more than 3 per million vaccinated.     

2009甲型H1N1流感病毒研究进展

2009年3月在美国和墨西哥爆发的新型甲型H1N1流感在很短的时间内便扩散到世界多个国家,形成了流感的大流行,引起世界卫生组织和各国的高度重视。综述新型甲型H1N1流感病毒的基因组来源、目前主要的检测手段,并对预防和治疗的方法进行简单介绍。     

A/California/7/2009与A/California/4/2009病毒感染力比较

目的甲型H1N1流感病毒A/California/7/2009与A/California/4/2009病毒序列比较同源性在99%以上,本实验旨在比较两株病毒感染BALB/c小鼠研究感染力强弱。方法分别将A/California/7/2009（CA7）与A/California/4/2009（CA4）两株病毒分别连续10倍稀释后,对4~6周龄雌性BALB/c小鼠经乙醚麻醉后进行滴鼻攻毒,每个稀释度接种10只实验小鼠,测定CA7 MLD50为101.24/0.05 mL,检测小鼠感染、致病的多项指标,观察期为14 d。结果相同TCID50的CA7和CA4病毒感染小鼠,CA4感染小鼠后14 d内死亡率为20%,而CA7感染小鼠后8 d内死亡率为100%。CA7 106TCID50感染的小鼠病理表现为重度弥漫性间质性肺炎,CA4 106TCID50感染的小鼠病理表现为中度-重度间质性肺炎。结论在相同条件下,CA7感染力明显强于CA4。     

A/H6N1亚型禽流感病毒致病性评估及与2009 A/H1N1亚型流感病毒在哺乳动物体内的遗传兼容性

目的探讨人、禽流感病毒在哺乳动物体内的遗传兼容性,为下一步研究H6亚型禽流感病毒重配和致病性变异的分子机制奠定基础。方法野鸭源A/H6N1亚型禽流感病毒A/Mallard/SanJiang/275/2007以101EID50～106EID50的攻毒剂量经鼻内途径感染小鼠,通过临床症状观察、病毒滴定和病理切片观察进行病毒学和组织学两方面检测对小鼠的致病性;同时,将此病毒与2009年A/H1N1流感病毒A/Changchun/01/2009(H1N1)混合感染豚鼠,分析两株病毒在哺乳动物体内的遗传兼容性。每天采集豚鼠鼻洗液并用噬斑纯化技术获得重配病毒,对获得的重配病毒进行全基因组序列的测定。结果 H6N1亚型禽流感病毒能直接感染小鼠,但对小鼠不致死。106EID50的攻毒剂量可有效感染小鼠,攻毒后第5天,小鼠表现出被毛较粗乱、活动减少、体重下降、呼吸急促的临床症状,但至攻毒后第10天开始康复,而对照组(MOCK)小鼠在14 d的观察期内无明显临床症状。病毒滴定结果表明,该病毒主要在小鼠肺脏和鼻甲骨中复制,病毒滴度可达104.5EID50/mL。病理学观察发现感染小鼠肺泡壁增厚,有大量炎性细胞浸润,纤维蛋白渗出并伴有轻微出血;在A/H6N1和A/H1N1混合感染豚鼠的重配实验中,经过三轮噬斑纯化从豚鼠鼻洗液中分离到6株重配病毒,说明A/H6N1亚型禽流感病毒与A/H1N1亚型流感病毒具有很好的遗传兼容性,能在豚鼠体内能发生重配。结论野鸭源A/H6N1亚型流感病毒无需适应就能够感染哺乳动物;该病毒与A/H1N1流感病毒具有很好的遗传兼容性,在哺乳动物体内能够发生基因重配,产生新的重配病毒,其公共卫生意义应引起高度关注。     

A Two-Year Surveillance of 2009 Pandemic Influenza A (H1N1) in Guangzhou,China: From Pandemic to Seasonal Influenza?

In this two-years surveillance of 2009 pandemic influenza A (H1N1) (pH1N1) in Guangzhou, China, we reported here that the scale and duration of pH1N1 outbreaks, severe disease and fatality rates of pH1N1 patients were significantly lower or shorter in the second epidemic year (May 2010-April 2011) than those in the first epidemic year (May 2009-April 2010) (P<0.05), but similar to those of seasonal influenza (P>0.05). Similar to seasonal influenza, pre-existing chronic pulmonary diseases was a risk factor associated with fatal cases of pH1N1 influenza. Different from seasonal influenza, which occurred in spring/summer seasons annually, pH1N1 influenza mainly occurred in autumn/winter seasons in the first epidemic year, but prolonged to winter/spring season in the second epidemic year. The information suggests a tendency that the epidemics of pH1N1 influenza may probably further shift to spring/summer seasons and become a predominant subtype of seasonal influenza in coming years in Guangzhou, China.     

Did Media Attention of the 2009 A(H1N1) Influenza Epidemic Increase Outpatient Antibiotic Use in France?: A Time-Series Analysis

Background

In France, the 2009 A(H1N1) influenza epidemic occurred between September 2009 and January 2010. Sparking widespread controversy, it was intensely reported in the media. Despite therapeutic inefficacy, antibiotic consumption and viral respiratory infections are positively correlated, particularly in France, where antibiotic overconsumption is well-known. We first determined the period when media coverage was high, and then compared, during this period, observed outpatient antibiotic consumption to estimated outpatient antibiotic consumption “without media attention”.

Materials and Methods

To evaluate media coverage, two online databases were consulted: Factiva and Europresse. To quantify outpatient antibiotic consumption, we used data on reimbursements of outpatient systemic antibiotics from the computerized databases of the two main National Health Insurance agencies. Influenza-like syndromes data came from the French GPs Sentinelles Network. Weekly time-series of antibiotic consumption were modeled by autoregressive moving-average models with exogenous inputs and interventions. Analyses were computed for the entire series and by age group (0–5, 6–15, 16–60, and >60 years).

Results

Media coverage was intense between April 2009 and January 2010. No effect on total outpatient antibiotic consumption was observed during the whole mediatic period. However, during the epidemic in France (September 2009-January 2010), we found an antibiotic underconsumption for the entire series, 0–5 and >60 years. Additionally, at the beginning of the pandemic, when cases were still outside France (June 2009-August 2009), we found an antibiotic overconsumption for patients >16 years.

Conclusion

The early period of A(H1N1) virus circulation compared with seasonal influenza or an overdeclaration of ILS cases might explain the antibiotic underconsumption observed during the period of active A(H1N1) virus transmission in France. At the pandemic onset, when uncertainty was high, the overconsumption observed for individuals >16 years might have been caused by alarmist media reporting. Additional analyses are needed to understand the determinants of antibiotic consumption during this period.     

Twin Peaks: A/H1N1 Pandemic Influenza Virus Infection and Vaccination in Norway, 2009–2010

Background

Vaccination campaigns against A/H1N1 2009 pandemic influenza virus (A/H1N1p) began in autumn 2009 in Europe, after the declaration of the pandemic at a global level. This study aimed to estimate the proportion of individuals vaccinated against A/H1N1p in Norway who were already infected (asymptomatically or symptomatically) by A/H1N1p before vaccination, using a mathematical model.

Methods

A dynamic, mechanistic, mathematical model of A/H1N1p transmission was developed for the Norwegian population. The model parameters were estimated by calibrating the model-projected number of symptomatic A/H1N1p cases to the number of laboratory-confirmed A/H1N1p cases reported to the surveillance system, accounting for potential under-reporting. It was assumed in the base case that the likelihood of vaccination was independent of infection/disease state. A sensitivity analysis explored the effects of four scenarios in which current or previous symptomatic A/H1N1p infection would influence the likelihood of being vaccinated.

Results

The number of model-projected symptomatic A/H1N1p cases by week during the epidemic, accounting for under-reporting and timing, closely matched that of the laboratory-confirmed A/H1N1p cases reported to the surveillance system. The model-projected incidence of symptomatic A/H1N1p infection was 27% overall, 55% in people <10 years old and 41% in people 10–20 years old. The model-projected percentage of individuals vaccinated against A/H1N1p who were already infected with A/H1N1p before being vaccinated was 56% overall, 62% in people <10 years old and 66% in people 10–20 years old. The results were sensitive to assumptions about the independence of vaccination and infection; however, even when current or previous symptomatic A/H1N1p infection was assumed to reduce the likelihood of vaccination, the estimated percentage of individuals who were infected before vaccination remained at least 32% in all age groups.

Conclusion

This analysis suggests that over half the people vaccinated against A/H1N1p in Norway during the 2009 pandemic may already have been infected by A/H1N1p before being vaccinated.     

High Rate of A(H1N1)pdm09 Infections among Rural Thai Villagers, 2009–2010

Background

Pandemic influenza A(H1N1)pdm09 emerged in Thailand in 2009. A prospective longitudinal adult cohort and household transmission study of influenza-like illness (ILI) was ongoing in rural Thailand at the time of emergence. Symptomatic and subclinical A(H1N1)pdm09 infection rates in the cohort and among household members were evaluated.

Methods

A cohort of 800 Thai adults underwent active community-based surveillance for ILI from 2008–2010. Acute respiratory samples from ILI episodes were tested for A(H1N1)pdm09 by qRT-PCR; acute and 60-day convalescent blood samples were tested by A(H1N1)pdm09 hemagglutination inhibition assay (HI). Enrollment, 12-month and 24-month follow-up blood samples were tested for A(H1N1)pdm09 seroconversion by HI. Household members of influenza A-infected cohort subjects with ILI were enrolled in household transmission investigations in which day 0 and 60 blood samples and acute respiratory samples were tested by either qRT-PCR or HI for A(H1N1)pdm09. Seroconversion between annual blood samples without A(H1N1)pdm09-positive ILI was considered as subclinical infection.

Results

The 2-yr cumulative incidence of A(H1N1)pdm09 infection in the cohort in 2009/2010 was 10.8% (84/781) with an annual incidence of 1.2% in 2009 and 9.7% in 2010; 83.3% of infections were subclinical (50% in 2009 and 85.9% in 2010). The 2-yr cumulative incidence was lowest (5%) in adults born ≤1957. The A(H1N1)pdm09 secondary attack rate among household contacts was 47.2% (17/36); 47.1% of these infections were subclinical. The highest A(H1N1)pdm09 secondary attack rate among household contacts (70.6%, 12/17) occurred among children born between 1990 and 2003.

Conclusion

Subclinical A(H1N1)pdm09 infections in Thai adults occurred frequently and accounted for a greater proportion of all A(H1N1)pdm09 infections than previously estimated. The role of subclinical infections in A(H1N1)pdm09 transmission has important implications in formulating strategies to predict and prevent the spread of A(H1N1)pdm09 and other influenza virus strains.     

Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 μ1A (AP-1 mu1A)

Kidney anion exchanger 1 (kAE1) mediates chloride (Cl⁻) and bicarbonate (HCO₃⁻) exchange at the basolateral membrane of kidney α-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl⁻/HCO₃⁻ exchange at the basolateral membrane and failure of proton (H⁺) secretion at the apical membrane, causing a kidney disease - distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 μ1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXØ motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1 trafficking of kidney α-intercalated cells.     

Molecular Evolution of Human H1N1 and H3N2 Influenza A Virus in Thailand, 2006–2009

Background

Annual seasonal influenza outbreaks are associated with high morbidity and mortality.

Objective

To index and document evolutionary changes among influenza A H1N1 and H3N2 viruses isolated from Thailand during 2006–2009, using complete genome sequences.

Methods

Nasopharyngeal aspirates were collected from patients diagnosed with respiratory illness in Thailand during 2006–2009. All samples were screened for Influenza A virus. A total of 13 H1N1 and 21 H3N2 were confirmed and whole genome sequenced for the evolutionary analysis using standard phylogenetic approaches.

Results

Phylogenetic analysis of HA revealed a clear diversification of seasonal from vaccine strain lineages. H3N2 seasonal clusters were closely related to the WHO recommended vaccine strains in each season. Most H1N1 isolates could be differentiated into 3 lineages. The A/Brisbane/59/2007 lineage, a vaccine strain for H1N1 since 2008, is closely related with the H1N1 subtypes circulating in 2009. HA sequences were conserved at the receptor-binding site. Amino acid variations in the antigenic site resulted in a possible N-linked glycosylation motif. Recent H3N2 isolates had higher genetic variations compared to H1N1 isolates. Most substitutions in the NP protein were clustered in the T-cell recognition domains.

Conclusion

In this study we performed evolutionary genetic analysis of influenza A viruses in Thailand between 2006–2009. Although the current vaccine strain is efficient for controlling the circulating outbreak subtypes, surveillance is necessary to provide unambiguous information on emergent viruses. In summary, the findings of this study contribute the understanding of evolution in influenza A viruses in humans and is useful for routine surveillance and vaccine strain selection.     

Induction of CYP1A by benzo[k]fluoranthene in human hepatocytes: CYP1A1 or CYP1A2?

While fresh human hepatocyte cultures are widely used to model hepatic cytochrome P450 (CYP) regulation and activity, their CYP1A subfamily composition induced by, e.g., polycyclic aromatic hydrocarbons is ambiguous. CYP1A1, CYP1A2, or both have been reported to be expressed, and their varied roles in chemical carcinogenesis makes resolution of which CYPs are expressed essential. We have used an immunoblot system with Bis-Tris-HCl-buffered polyacrylamide gel, which clearly resolves human CYP1A1 and CYP1A2, and polyclonal goat anti-human CYP1A1/CYP1A2 and rabbit anti-human CYP1A2 antibodies to probe the expressed CYP1A1 and CYP1A2 composition of seven individual human hepatocyte cultures induced with 5 microM benzo[k]fluoranthene (BKF) for 24 h. In six of the cultures only CYP1A1 was detected, and in the seventh both CYPs were detected. In most vehicle-treated hepatocyte cultures, neither CYP1A1 nor CYP1A2 was detected. In three additional hepatocyte cultures treated individually with BKF and 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD), the resultant induced CYP1A1/1A2 profiles were essentially not influenced by the nature of the inducing agents. To develop an activity-based assay to differentiate between CYP1A1 and CYP1A2 expression in human hepatocytes, our previously published R warfarin assay (Drug Metab. Disp. (1995) 23, 1339-1345) was applied to TCDD (10 nM)-treated hepatocyte culture. The low concentration of TCDD did not produce inhibition of the warfarin metabolism-such inhibition could confound the results. Based on the ratios of 6- to 8-hydroxywarfarin formed in two cultures, the ratios of CYP1A1/CYP1A2 expressed in these cultures were determined and they agreed with the ratios determined by immunoblot analysis. Thus each individual human hepatocyte culture must be characterized for induced CYP1A1 and CYP1A2 expression in studies of CYP1A activity. The warfarin assay provides a means of characterizing the cultures.     

Influenza Virus A/Beijing/501/2009(H1N1) NS1 Interacts with β-Tubulin and Induces Disruption of the Microtubule Network and Apoptosis on A549 Cells

NS1 of influenza A virus is a key multifunctional protein that plays various roles in regulating viral replication mechanisms, host innate/adaptive immune responses, and cellular signalling pathways. These functions rely on its ability to participate in a multitude of protein-protein and protein-RNA interactions. To gain further insight into the role of NS1, a tandem affinity purification (TAP) method was utilized to find unknown interaction partner of NS1. The protein complexes of NS1 and its interacting partner were purified from A549 cell using TAP-tagged NS1 as bait, and co-purified cellular factors were identified by mass spectrometry (MS). We identified cellular β-tubulin as a novel interaction partner of NS1. The RNA-binding domain of NS1 interacts with β-tubulin through its RNA-binding domain, as judged by a glutathione S-transferase (GST) pull-down assay with the GST-fused functional domains of NS1. Immunofluorescence analysis further revealed that NS1 with β-tubulin co-localized in the nucleus. In addition, the disruption of the microtubule network and apoptosis were also observed on NS1-transfected A549 cells. Our findings suggest that influenza A virus may utilize its NS1 protein to interact with cellular β-tubulin to further disrupt normal cell division and induce apoptosis. Future work will illustrate whether this interaction is uniquely specific to the 2009 pandemic H1N1 virus.     

Anti-viral activity of (−)- and (+)-usnic acids and their derivatives against influenza virus A(H1N1)2009

Influenza is a widespread respiratory infection. Every year it causes epidemics, quickly spreading from country to country, or even pandemics, involving a significant part of the human population of the earth. Being a highly variable infection, influenza easy accumulates the resistance mutations to many antivirals.Usnic acid, a dibenzofuran originally isolated from lichens belongs to the secondary metabolites and has a broad spectrum of biological activity. In humans, it can act as an anti-inflammatory, antimitotic, antineoplasic, antibacterial, and antimycotic agent. In this work we studied for the first time the antiviral activity of usnic acid and its derivatives against the pandemic influenza virus A(H1N1)pdm09. A total of 26 compounds representing (+) and (?) isomers of usnic acid and their derivates were tested for cytotoxicity and anti-viral activity in MDCK cells by microtetrazolium test and virus yield assay, respectively. Based on the results obtained, 50% cytotoxic dose (CTD₅₀) and 50% effective dose (ED₅₀) and selectivity index (SI) were calculated for each compound. Eleven of them were found to have SI higher than 10 (highest value 37.3). Absolute configuration was shown to have critical significance for the anti-viral activity. With minor exceptions, in the pair of enantiomers, (?)-usnic acid was more active comparing to (+)-isomer, but its biological activity was reversed after the usnic acid was chemically modified. Based on the obtained results, derivatives of usnic acid should be considered as prospective compounds for further optimization as anti-influenza substances.     

NS1A在A型流感病毒感染中的作用

NS1A是A型流感病毒(influenza A virus,IAV)的重要毒力因子,在IAV感染过程中具有多种调节活性,如促进病毒复制、调节感染细胞凋亡和下调宿主抗病毒免疫.深入理解NSlA在病毒感染中的作用有助于揭示IAV感染的致病机理和发现抗IAV药物的靶标.     

NS1A在A型流感病毒感染中的作用

NS1A是A型流感病毒（influenza A virus,IAV）的重要毒力因子,在IAV感染过程中具有多种调节活性,如促进病毒复制、调节感染细胞凋亡和下调宿主抗病毒免疫。深入理解NS1A在病毒感染中的作用有助于揭示IAV感染的致病机理和发现抗IAV药物的靶标。     

Supply of Neuraminidase Inhibitors Related to Reduced Influenza A (H1N1) Mortality during the 2009–2010 H1N1 Pandemic: An Ecological Study

Background

The influenza A (H1N1) pandemic swept across the globe from April 2009 to August 2010 affecting millions. Many WHO Member States relied on antiviral drugs, specifically neuraminidase inhibitors (NAIs) oseltamivir and zanamivir, to treat influenza patients in critical condition. Such drugs have been found to be effective in reducing severity and duration of influenza illness, and likely reduced morbidity during the pandemic. However, it is less clear whether NAIs used during the pandemic reduced H1N1 mortality.

Methods

Country-level data on supply of oseltamivir and zanamivir were used to predict H1N1 mortality (per 100,000 people) from July 2009 to August 2010 in forty-two WHO Member States. Poisson regression was used to model the association between NAI supply and H1N1 mortality, with adjustment for economic, demographic, and health-related confounders.

Results

After adjustment for potential confounders, each 10% increase in kilograms of oseltamivir, per 100,000 people, was associated with a 1.6% reduction in H1N1 mortality over the pandemic period (relative rate (RR) = 0.84 per log increase in oseltamivir supply). While the supply of zanamivir was considerably less than that of oseltamivir in each Member State, each 10% increase in kilogram of active zanamivir, per 100,000, was associated with a 0.3% reduction in H1N1 mortality (RR = 0.97 per log increase).

Conclusion

While there are limitations to the ecologic nature of these data, this analysis offers evidence of a protective relationship between antiviral drug supply and influenza mortality and supports a role for influenza antiviral use in future pandemics.