Extensive geographical mixing of 2009 human H1N1 influenza A virus in a single university community

Despite growing interest in the molecular epidemiology of influenza virus, the pattern of viral spread within individual communities remains poorly understood. To determine the phylogeography of influenza virus in a single population, we examined the spatial diffusion of H1N1/09 influenza A virus within the student body of the University of California, San Diego (UCSD), sampling for a 1-month period between October and November 2009. Despite the highly focused nature of our study, an analysis of complete viral genome sequences revealed between 24 and 33 independent introductions of H1N1/09 into the UCSD community, comprising much of the global genetic diversity in this virus. These data were also characterized by a relatively low level of on-campus transmission as well as extensive spatial mixing, such that there was little geographical clustering by either student residence or city ZIP code. Most notably, students experiencing illness on the same day and residing in the same dorm possessed phylogenetically distinct lineages. H1N1/09 influenza A virus is therefore characterized by a remarkable spatial fluidity, which is likely to impede community-based methods for its control, including class cancellations, quarantine, and chemoprophylaxis.     

Molecular character of influenza A/H1N1 2009: Implications for spread and control

The world is experiencing a pandemic of influenza that emerged in March 2009, due to a novel strain designated influenza A/H1N1 2009. This strain is closest in molecular sequence to swine influenza viruses, but differs from all previously known influenza by a minimum of 6.1%, and from prior “seasonal” H1N1 by 27.2%, giving it great potential for widespread human infection. While spread into India was delayed for two months by an aggressive interdiction program, since 1 August 2009 most cases in India have been indigenous. H1N1 2009 has differentially struck younger patients who are naïve susceptibles to its antigenic subtype, while sparing those >60 who have crossreactive antibody from prior experience with influenza decades ago and the 1977 “swine flu” vaccine distributed in the United States. It also appears to more severely affect pregnant women. It emanated from a single source in central Mexico, but its precise geographical and circumstantial origins, from either Eurasia or the Americas, remain uncertain. While currently a mild pandemic by the standard of past pandemics, the seriousness of H1N1 2009 especially among children should not be underestimated. There is potential for the virus, which continues to adapt to humans, to change over time into a more severe etiologic agent by any of several foreseeable mutations. Mass acceptance of the novel H1N1 2009 vaccine worldwide will be essential to its control. Having spread globally in a few months, affecting millions of people, it is likely to remain circulating in the human population for a decade or more.     

Predicting the epidemic sizes of influenza A/H1N1, A/H3N2, and B: a statistical method

Background

The epidemic sizes of influenza A/H3N2, A/H1N1, and B infections vary from year to year in the United States. We use publicly available US Centers for Disease Control (CDC) influenza surveillance data between 1997 and 2009 to study the temporal dynamics of influenza over this period.

Methods and Findings

Regional outpatient surveillance data on influenza-like illness (ILI) and virologic surveillance data were combined to define a weekly proxy for the incidence of each strain in the United States. All strains exhibited a negative association between their cumulative incidence proxy (CIP) for the whole season (from calendar week 40 of each year to calendar week 20 of the next year) and the CIP of the other two strains (the complementary CIP) from the start of the season up to calendar week 2 (or 3, 4, or 5) of the next year. We introduce a method to predict a particular strain''s CIP for the whole season by following the incidence of each strain from the start of the season until either the CIP of the chosen strain or its complementary CIP exceed certain thresholds. The method yielded accurate predictions, which generally occurred within a few weeks of the peak of incidence of the chosen strain, sometimes after that peak. For the largest seasons in the data, which were dominated by A/H3N2, prediction of A/H3N2 incidence always occurred at least several weeks in advance of the peak.

Conclusion

Early circulation of one influenza strain is associated with a reduced total incidence of the other strains, consistent with the presence of interference between subtypes. Routine ILI and virologic surveillance data can be combined using this new method to predict the relative size of each influenza strain''s epidemic by following the change in incidence of a given strain in the context of the incidence of cocirculating strains. Please see later in the article for the Editors'' Summary     

甲型H1N1流感病毒裂解疫苗的免疫原性和安全性

目的评价甲型H1N1流感病毒裂解疫苗(简称甲型H1N1流感疫苗)的免疫原性和安全性。方法按照随机、双盲、安慰剂对照的原则,采用0、21天免疫程序,选择3岁及3岁以上健康者1 202人。分组为3~11岁、12~17岁、≥60岁组,按照人数基本为1∶1的比例随机分别接种7.5μg和15.0μg甲型H1N1流感疫苗;18~59岁组按照人数基本为1∶1∶1的比例随机分别接种7.5μg、15.0μg甲型H1N1流感疫苗和安慰剂对照。观察各组接种后的不良反应率以及免疫前后血凝抑制(HI)抗体阳转率、保护率、GMT水平和平均增长倍数。结果受试对象的安全性结果显示7.5μg和15.0μg组不良反应发生率分别为8.74%(48/549)和13.88%(74/533),其中Ⅱ级反应率分别为0.36%(2/549)和1.13%(6/533),未观察到Ⅲ级及以上不良反应和其他异常反应及严重不良事件。2剂接种未见不良反应叠加现象。7.5μg或15.0μg试验疫苗首剂免疫后,血清抗体阳性率分别为85.13%(395/464)和90.77%(413/455),保护率分别为85.56%(397/464)和91.43%(416/455),抗体GMT较免疫前分别增长36.1倍和52.6倍。2剂免疫后,血清抗体阳性率分别是97.84%(454/464)和99.12%(451/455),保护率分别是98.06%(455/464)和9 9.56%(453/455),抗体GMT较免疫前分别增长63.3倍和96.0倍。4个年龄组(3~11岁、12~17岁、18~59岁及≥60岁年龄组)7.5μg和15.0μg组HI抗体阳性率和保护率均大于70%,GMT较免疫前均增长2.5倍以上,结果显示7.5μg和15.0μg甲型H1N1流感疫苗接种1剂后抗体水平已达到研究方案中设定的预期标准,免疫2剂后抗体阳性率和抗体水平明显提高。结论临床试验表明甲型H1N1流感疫苗具有良好的安全性和免疫原性,且接种1剂15.0μg甲型H1N1流感疫苗,即可在3岁和3岁以上人群中产生良好的免疫效果。     

Susceptibility of pandemic influenza virus A 2009 H1N1 and highly pathogenic avian influenza virus A H5N1 to antiinfluenza agents in cell culture

The data on cytotoxicity and antiviral activity of commercial antivirals, such as Remantadine, Oseltamivir, Arbidol and Ribavirin in the MDCK cell culture infected with highly pathogenic (H5N1) and pandemic 2009 (H1N1) influenza A viruses are presented. The study of the antiviral activity of antivirals in the MDCK cells culture demonstrated that Arbidol, Rimantadine and Ribavirin efficiently inhibited reproduction of the highly pathogenic H5N1 influenza viruses isolated from sick birds. Arbidol and Oseltamivir carboxylate selectively inhibited reproduction of the pandemic 2009 H1N1 influenza A viruses with changed specificity to the cell receptors, causing severe influenza in men, while remantadine had no effect on their reproduction.     

Comparative analyses of pandemic H1N1 and seasonal H1N1, H3N2, and influenza B infections depict distinct clinical pictures in ferrets

Influenza A and B infections are a worldwide health concern to both humans and animals. High genetic evolution rates of the influenza virus allow the constant emergence of new strains and cause illness variation. Since human influenza infections are often complicated by secondary factors such as age and underlying medical conditions, strain or subtype specific clinical features are difficult to assess. Here we infected ferrets with 13 currently circulating influenza strains (including strains of pandemic 2009 H1N1 [H1N1pdm] and seasonal A/H1N1, A/H3N2, and B viruses). The clinical parameters were measured daily for 14 days in stable environmental conditions to compare clinical characteristics. We found that H1N1pdm strains had a more severe physiological impact than all season strains where pandemic A/California/07/2009 was the most clinically pathogenic pandemic strain. The most serious illness among seasonal A/H1N1 and A/H3N2 groups was caused by A/Solomon Islands/03/2006 and A/Perth/16/2009, respectively. Among the 13 studied strains, B/Hubei-Wujiagang/158/2009 presented the mildest clinical symptoms. We have also discovered that disease severity (by clinical illness and histopathology) correlated with influenza specific antibody response but not viral replication in the upper respiratory tract. H1N1pdm induced the highest and most rapid antibody response followed by seasonal A/H3N2, seasonal A/H1N1 and seasonal influenza B (with B/Hubei-Wujiagang/158/2009 inducing the weakest response). Our study is the first to compare the clinical features of multiple circulating influenza strains in ferrets. These findings will help to characterize the clinical pictures of specific influenza strains as well as give insights into the development and administration of appropriate influenza therapeutics.     

Spreading patterns of the influenza A (H1N1) pandemic

We investigate the dynamics of the 2009 influenza A (H1N1/S-OIV) pandemic by analyzing data obtained from World Health Organization containing the total number of laboratory-confirmed cases of infections--by country--in a period of 69 days, from 26 April to 3 July, 2009. Specifically, we find evidence of exponential growth in the total number of confirmed cases and linear growth in the number of countries with confirmed cases. We also find that, i) at early stages, the cumulative distribution of cases among countries exhibits linear behavior on log-log scale, being well approximated by a power law decay; ii) for larger times, the cumulative distribution presents a systematic curvature on log-log scale, indicating a gradual change to lognormal behavior. Finally, we compare these empirical findings with the predictions of a simple stochastic model. Our results could help to select more realistic models of the dynamics of influenza-type pandemics.     

Seroprevalence and severity of 2009 pandemic influenza A H1N1 in Taiwan

Background

This study is to determine the seroprevalence of the pandemic influenza A H1N1 virus (pH1N1) in Taiwan before and after the 2009 pandemic, and to estimate the relative severity of pH1N1 infections among different age groups.

Methodology/Principal Findings

A total of 1544 and 1558 random serum samples were collected from the general population in Taiwan in 2007 and 2010, respectively. Seropositivity was defined by a hemagglutination inhibition titer to pH1N1 (A/Taiwan/126/09) ≥1:40. The seropositivity rate of pH1N1 among the unvaccinated subjects and national surveillance data were used to compare the proportion of infections that led to severe diseases and fatalities among different age groups. The overall seroprevalence of pH1N1 was 0.91% (95% confidence interval [CI] 0.43–1.38) in 2007 and significantly increased to 29.9% (95% CI 27.6–32.2) in 2010 (p<0.0001), with the peak attack rate (55.4%) in 10–17 year-old adolescents, the lowest in elderly ≥65 years (14.1%). The overall attack rates were 20.6% (188/912) in unvaccinated subjects. Among the unvaccinated but infected populations, the estimated attack rates of severe cases per 100,000 infections were significantly higher in children aged 0–5 years (54.9 cases, odds ratio [OR] 4.23, 95% CI 3.04–5.90) and elderly ≥ 65years (22.4 cases, OR 2.76, 95% CI 1.99–3.83) compared to adolescents aged 10–17 years (13.0 cases). The overall case-fatality rate was 0.98 per 100,000 infections without a significant difference in different age groups.

Conclusions/Significance

Pre-existing immunity against pH1N1 was rarely identified in Taiwanese at any age in 2007. Young children and elderly – the two most lower seroprotection groups showed the greatest vulnerability to clinical severity after the pH1N1 infections. These results imply that both age groups should have higher priority for immunization in the coming flu season.     

Characterization of neuraminidases from the highly pathogenic avian H5N1 and 2009 pandemic H1N1 influenza A viruses

To study the precise role of the neuraminidase (NA), and its stalk region in particular, in the assembly, release, and entry of influenza virus, we deleted the 20-aa stalk segment from 2009 pandemic H1N1 NA (09N1) and inserted this segment, now designated 09s60, into the stalk region of a highly pathogenic avian influenza (HPAI) virus H5N1 NA (AH N1). The biological characterization of these wild-type and mutant NAs was analyzed by pseudotyped particles (pseudoparticles) system. Compared with the wild-type AH N1, the wild-type 09N1 exhibited higher NA activity and released more pseudoparticles. Deletion/insertion of the 09s60 segment did not alter this relationship. The infectivity of pseudoparticles harboring NA in combination with the hemagglutinin from HPAI H5N1 (AH H5) was decreased by insertion of 09s60 into AH N1 and was increased by deletion of 09s60 from 09N1. When isolated from the wild-type 2009H1N1 virus, 09N1 existed in the forms (in order of abundance) dimer>tetramer>monomer, but when isolated from pseudoparticles, 09N1 existed in the forms dimer>monomer>tetramer. After deletion of 09s60, 09N1 existed in the forms monomer>dimer. AH N1 from pseudoparticles existed in the forms monomer>dimer, but after insertion of 09s60, it existed in the forms dimer>monomer. Deletion/insertion of 09s60 did not alter the NA glycosylation pattern of 09N1 or AH N1. The 09N1 was more sensitive than the AH N1 to the NA inhibitor oseltamivir, suggesting that the infectivity-enhancing effect of oseltamivir correlates with robust NA activity.     

Transmission of aerosolized seasonal H1N1 influenza A to ferrets

Influenza virus is a major cause of morbidity and mortality worldwide, yet little quantitative understanding of transmission is available to guide evidence-based public health practice. Recent studies of influenza non-contact transmission between ferrets and guinea pigs have provided insights into the relative transmission efficiencies of pandemic and seasonal strains, but the infecting dose and subsequent contagion has not been quantified for most strains. In order to measure the aerosol infectious dose for 50% (aID₅₀) of seronegative ferrets, seasonal influenza virus was nebulized into an exposure chamber with controlled airflow limiting inhalation to airborne particles less than 5 µm diameter. Airborne virus was collected by liquid impinger and Teflon filters during nebulization of varying doses of aerosolized virus. Since culturable virus was accurately captured on filters only up to 20 minutes, airborne viral RNA collected during 1-hour exposures was quantified by two assays, a high-throughput RT-PCR/mass spectrometry assay detecting 6 genome segments (Ibis T5000™ Biosensor system) and a standard real time RT-qPCR assay. Using the more sensitive T5000 assay, the aID₅₀ for A/New Caledonia/20/99 (H1N1) was approximately 4 infectious virus particles under the exposure conditions used. Although seroconversion and sustained levels of viral RNA in upper airway secretions suggested established mucosal infection, viral cultures were almost always negative. Thus after inhalation, this seasonal H1N1 virus may replicate less efficiently than H3N2 virus after mucosal deposition and exhibit less contagion after aerosol exposure.     

Avian influenza H5N1 transmission in households, Indonesia

Background

Disease transmission patterns are needed to inform public health interventions, but remain largely unknown for avian influenza H5N1 virus infections. A recent study on the 139 outbreaks detected in Indonesia between 2005 and 2009 found that the type of exposure to sources of H5N1 virus for both the index case and their household members impacted the risk of additional cases in the household. This study describes the disease transmission patterns in those outbreak households.

Methodology/Principal Findings

We compared cases (n = 177) and contacts (n = 496) in the 113 sporadic and 26 cluster outbreaks detected between July 2005 and July 2009 to estimate attack rates and disease intervals. We used final size household models to fit transmission parameters to data on household size, cases and blood-related household contacts to assess the relative contribution of zoonotic and human-to-human transmission of the virus, as well as the reproduction number for human virus transmission. The overall household attack rate was 18.3% and secondary attack rate was 5.5%. Secondary attack rate remained stable as household size increased. The mean interval between onset of subsequent cases in outbreaks was 5.6 days. The transmission model found that human transmission was very rare, with a reproduction number between 0.1 and 0.25, and the upper confidence bounds below 0.4. Transmission model fit was best when the denominator population was restricted to blood-related household contacts of index cases.

Conclusions/Significance

The study only found strong support for human transmission of the virus when a single large cluster was included in the transmission model. The reproduction number was well below the threshold for sustained transmission. This study provides baseline information on the transmission dynamics for the current zoonotic virus and can be used to detect and define signatures of a virus with increasing capacity for human-to-human transmission.     

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。     

Genetic and phylogenetic analyses of influenza A H1N1pdm virus in Buenos Aires, Argentina

An influenza pandemic caused by swine-origin influenza virus A/H1N1 (H1N1pdm) spread worldwide in 2009, with 12,080 confirmed cases and 626 deaths occurring in Argentina. A total of 330 H1N1pdm viruses were detected from May to August 2009, and phylogenetic and genetic analyses of 21 complete genome sequences from both mild and fatal cases were achieved with reference to concatenated whole genomes. In addition, the analysis of another 16 hemagglutinin (HA), neuraminidase (NA), and matrix (M) gene sequences of Argentinean isolates was performed. The microevolution timeline was assessed and resistance monitoring of an NA fragment from 228 samples throughout the 2009 pandemic peak was performed by sequencing and pyrosequencing. We also assessed the viral growth kinetics for samples with replacements at the genomic level or special clinical features. In this study, we found by Bayesian inference that the Argentinean complete genome sequences clustered with globally distributed clade 7 sequences. The HA sequences were related to samples from the northern hemisphere autumn-winter from September to December 2009. The NA of Argentinean sequences belonged to the New York group. The N-4 fragment as well as the hierarchical clustering of samples showed that a consensus sequence prevailed in time but also that different variants, including five H275Y oseltamivir-resistant strains, arose from May to August 2009. Fatal and oseltamivir-resistant isolates had impaired growth and a small plaque phenotype compared to oseltamivir-sensitive and consensus strains. Although these strains might not be fit enough to spread in the entire population, molecular surveillance proved to be essential to monitor resistance and viral dynamics in our country.     

Characterizing the epidemiology of the 2009 influenza A/H1N1 pandemic in Mexico

Background

Mexico''s local and national authorities initiated an intense public health response during the early stages of the 2009 A/H1N1 pandemic. In this study we analyzed the epidemiological patterns of the pandemic during April–December 2009 in Mexico and evaluated the impact of nonmedical interventions, school cycles, and demographic factors on influenza transmission.

Methods and Findings

We used influenza surveillance data compiled by the Mexican Institute for Social Security, representing 40% of the population, to study patterns in influenza-like illness (ILIs) hospitalizations, deaths, and case-fatality rate by pandemic wave and geographical region. We also estimated the reproduction number (R) on the basis of the growth rate of daily cases, and used a transmission model to evaluate the effectiveness of mitigation strategies initiated during the spring pandemic wave. A total of 117,626 ILI cases were identified during April–December 2009, of which 30.6% were tested for influenza, and 23.3% were positive for the influenza A/H1N1 pandemic virus. A three-wave pandemic profile was identified, with an initial wave in April–May (Mexico City area), a second wave in June–July (southeastern states), and a geographically widespread third wave in August–December. The median age of laboratory confirmed ILI cases was ∼18 years overall and increased to ∼31 years during autumn (p<0.0001). The case-fatality ratio among ILI cases was 1.2% overall, and highest (5.5%) among people over 60 years. The regional R estimates were 1.8–2.1, 1.6–1.9, and 1.2–1.3 for the spring, summer, and fall waves, respectively. We estimate that the 18-day period of mandatory school closures and other social distancing measures implemented in the greater Mexico City area was associated with a 29%–37% reduction in influenza transmission in spring 2009. In addition, an increase in R was observed in late May and early June in the southeast states, after mandatory school suspension resumed and before summer vacation started. State-specific fall pandemic waves began 2–5 weeks after school reopened for the fall term, coinciding with an age shift in influenza cases.

Conclusions

We documented three spatially heterogeneous waves of the 2009 A/H1N1 pandemic virus in Mexico, which were characterized by a relatively young age distribution of cases. Our study highlights the importance of school cycles on the transmission dynamics of this pandemic influenza strain and suggests that school closure and other mitigation measures could be useful to mitigate future influenza pandemics. Please see later in the article for the Editors'' Summary     

Comparative study of the hemagglutinin and neuraminidase genes of influenza A virus H3N2, H9N2, and H5N1 subtypes using bioinformatics techniques

To investigate the genomic patterns of influenza A virus subtypes, such as H3N2, H9N2, and H5N1, we collected 1842 sequences of the hemagglutinin and neuraminidase genes from the NCBI database and parsed them into 7 categories: accession number, host species, sampling year, country, subtype, gene name, and sequence. The sequences that were isolated from the human, avian, and swine populations were extracted and stored in a MySQL database for intensive analysis. The GC content and relative synonymous codon usage (RSCU) values were calculated using JAVA codes. As a result, correspondence analysis of the RSCU values yielded the unique codon usage pattern (CUP) of each subtype and revealed no extreme differences among the human, avian, and swine isolates. H5N1 subtype viruses exhibited little variation in CUPs compared with other subtypes, suggesting that the H5N1 CUP has not yet undergone significant changes within each host species. Moreover, some observations may be relevant to CUP variation that has occurred over time among the H3N2 subtype viruses isolated from humans. All the sequences were divided into 3 groups over time, and each group seemed to have preferred synonymous codon patterns for each amino acid, especially for arginine, glycine, leucine, and valine. The bioinformatics technique we introduce in this study may be useful in predicting the evolutionary patterns of pandemic viruses.