H5N1 avian influenza in China

H5N1 highly pathogenic avian influenza virus was first detected in a goose in Guangdong Province of China in 1996. Multiple genotypes of H5N1 viruses have been identified from apparently healthy waterfowl since 1999. In the years 2004–2008, over 100 outbreaks in domestic poultry occurred in 23 provinces and caused severe economic damage to the poultry industry in China. Beginning from 2004, a culling plus vaccination strategy has been implemented for the control of epidemics. Since then, over 35420000 poultry have been depopulated, and over 55 billion doses of the different vaccines have been used to control the outbreaks. Although it is logistically impossible to vaccinate every single bird in China due to the large poultry population and the complicated rearing styles, there is no doubt that the increased vaccination coverage has resulted in decreased disease epidemic and environmental virus loading. The experience in China suggests that vaccination has played an important role in the protection of poultry from H5N1 virus infection, the reduction of virus load in the environment, and the prevention of H5N1 virus transmission from poultry to humans. Supported by the Key Animal Infectious Disease Control Program of the Ministry of Agriculture, the Chinese National S&T Plan(Grant No. 2004BA519A-57), National Key Basic Research and Development Program of China (Grant Nos: 2005CB523005, 2005CB523200).     

Comparison of the evolution of recent and late phase of old influenza A (H1N1) viruses

A comparison of the evolutionary tree of new influenza A (H1N1) viruses to that of old H1N1 viruses which disappeared in 1957 was performed. The evolutionary trees of the hemagglutinin (HA) molecule based on amino acid sequences of the HA1 polypeptide were constructed with old and new H1N1 viruses isolated from 1947 to 1957 and 1986 to 2000, respectively. The evolutionary history of recent H1N1 viruses was similar to that of old H1N1 viruses just before the disappearance in two respects. Firstly, both viruses did not originate from the viruses of the previous H1N1 epidemic season but originated from the viruses branched off at the same point on the mainstream stem as the viruses of two H1N1 epidemic seasons earlier. Secondly, recent H1N1 viruses mainly circulating in Japan have a deletion at amino acid residue 134, located close to residue 131, which was deleted in old H1N1 viruses at the time of the disappearance. However, different from the evolutionary history of old H1N1 viruses, in the 1999/2000 H1N1 epidemic season, the H1N1 viruses which were located on the same lineage as the previous epidemic viruses were also isolated sporadically in Japan.     

Receptor recognition mechanism of human influenza A H1N1 (1918), avian influenza A H5N1 (2004), and pandemic H1N1 (2009) neuraminidase

Influenza A neuraminidase (NA) is a target for anti-influenza drugs. The function of this enzyme is to cleave a glycosidic linkage of a host cell receptor that links sialic acid (Sia) to galactose (Gal), to allow the virus to leave an infected cell and propagate. The receptor is an oligosaccharide on the host cell surface. There are two types of oligosaccharide receptor; the first, which is found mainly on avian epithelial cell surfaces, links Sia with Gal by an α2,3 glycosidic linkage; in the second, found mainly on human epithelial cell surfaces, linkage is via an α2,6 linkage. Some researchers believe that NAs from different viruses show selectivity for each type of linkage, but there is limited information available to confirm this hypothesis. To see if the linkage type is more specific to any particular NA, a number of NA-receptor complexes of human influenza A H1N1 (1918), avian influenza A H5N1 (2004), and a pandemic strain of H1N1 (2009) were constructed using homology modeling and molecular dynamics simulation. The results show that the two types of receptor analogues bound to NAs use different mechanisms. Moreover, it was found that a residue unique to avian virus NA is responsible for the recognition of the Siaα2,3Gal receptor, and a residue unique to human virus NA is responsible for the recognition of Siaα2,6Gal. We believe that this finding could explain how NAs of different virus origins always possess some unique residues.     

人H7N9禽流感病毒与H1N1流感病毒感染小鼠病理学损伤的比较

目的比较分析H7N9病毒与H1N1病毒感染小鼠病理学损伤特点,初步探讨两种病毒感染致小鼠急性肺损伤的致病机制。方法 H7N9病毒与H1N1病毒分别感染小鼠,观察不同病毒感染后小鼠生存率,并于不同时间点取心、肝、脾、肺、肾、脑、肠等组织,伊红-苏木素染色并进行组织病理学分析,免疫组化检测病毒抗原分布及中性粒细胞浸润。综合分析肺组织病理损伤与病毒复制、宿主免疫反应之间的关系。结果 H7N9病毒感染小鼠肺及脾脏损伤较轻,存活率较高。H1N1病毒感染的小鼠肺及脾脏损伤较重,感染后9 d全部死亡;两种病毒抗原主要分布于支气管上皮细胞、少量间质细胞和肺泡上皮细胞,病毒复制水平无明显差异。但H1N1病毒感染后肺及脾脏中均有大量中性粒细胞浸润,小鼠机体炎症反应明显强于H7N9病毒感染后小鼠炎症反应。结论 H7N9病毒与H1N1病毒感染后小鼠病理学损伤特点及程度均不同,病毒复制是小鼠肺损伤的诱发因素但并非决定因素,宿主针对病毒感染产生的免疫反应程度与急性肺损伤密切相关。     

Protection against divergent influenza H1N1 virus by a centralized influenza hemagglutinin

Influenza poses a persistent worldwide threat to the human population. As evidenced by the 2009 H1N1 pandemic, current vaccine technologies are unable to respond rapidly to this constantly diverging pathogen. We tested the utility of adenovirus (Ad) vaccines expressing centralized consensus influenza antigens. Ad vaccines were produced within 2 months and protected against influenza in mice within 3 days of vaccination. Ad vaccines were able to protect at doses as low as 10(7) virus particles/kg indicating that approximately 1,000 human doses could be rapidly generated from standard Ad preparations. To generate broadly cross-reactive immune responses, centralized consensus antigens were constructed against H1 influenza and against H1 through H5 influenza. Twenty full-length H1 HA sequences representing the main branches of the H1 HA phylogenetic tree were used to create a synthetic centralized gene, HA1-con. HA1-con minimizes the degree of sequence dissimilarity between the vaccine and existing circulating viruses. The centralized H1 gene, HA1-con, induced stronger immune responses and better protection against mismatched virus challenges as compared to two wildtype H1 genes. HA1-con protected against three genetically diverse lethal influenza challenges. When mice were challenged with 1934 influenza A/PR/8/34, HA1-con protected 100% of mice while vaccine generated from 2009 A/TX/05/09 only protected 40%. Vaccination with 1934 A/PR/8/34 and 2009 A/TX/05/09 protected 60% and 20% against 1947 influenza A/FM/1/47, respectively, whereas 80% of mice vaccinated with HA1-con were protected. Notably, 80% of mice challenged with 2009 swine flu isolate A/California/4/09 were protected by HA1-con vaccination. These data show that HA1-con in Ad has potential as a rapid and universal vaccine for H1N1 influenza viruses.     

Complex patterns of human antisera reactivity to novel 2009 H1N1 and historical H1N1 influenza strains

Background

During the 2009 influenza pandemic, individuals over the age of 60 had the lowest incidence of infection with approximately 25% of these people having pre-existing, cross-reactive antibodies to novel 2009 H1N1 influenza isolates. It was proposed that older people had pre-existing antibodies induced by previous 1918-like virus infection(s) that cross-reacted to novel H1N1 strains.

Methodology/Principal Findings

Using antisera collected from a cohort of individuals collected before the second wave of novel H1N1 infections, only a minority of individuals with 1918 influenza specific antibodies also demonstrated hemagglutination-inhibition activity against the novel H1N1 influenza. In this study, we examined human antisera collected from individuals that ranged between the ages of 1 month and 90 years to determine the profile of seropositive influenza immunity to viruses representing H1N1 antigenic eras over the past 100 years. Even though HAI titers to novel 2009 H1N1 and the 1918 H1N1 influenza viruses were positively associated, the association was far from perfect, particularly for the older and younger age groups.

Conclusions/Significance

Therefore, there may be a complex set of immune responses that are retained in people infected with seasonal H1N1 that can contribute to the reduced rates of H1N1 influenza infection in older populations.     

Pathogenesis and transmission of triple-reassortant swine H1N1 influenza viruses isolated before the 2009 H1N1 pandemic

The 2009 H1N1 pandemic influenza virus represents the greatest incidence of human infection with an influenza virus of swine origin to date. Moreover, triple-reassortant swine (TRS) H1N1 viruses, which share similar host and lineage origins with 2009 H1N1 viruses, have been responsible for sporadic human cases since 2005. Similar to 2009 H1N1 viruses, TRS viruses are capable of causing severe disease in previously healthy individuals and frequently manifest with gastrointestinal symptoms; however, their ability to cause severe disease has not been extensively studied. Here, we evaluated the pathogenicity and transmissibility of two TRS viruses associated with disease in humans in the ferret model. TRS and 2009 H1N1 viruses exhibited comparable viral titers and histopathologies following virus infection and were similarly unable to transmit efficiently via respiratory droplets in the ferret model. Utilizing TRS and 2009 H1N1 viruses, we conducted extensive hematologic and blood serum analyses on infected ferrets to identify lymphohematopoietic parameters associated with mild to severe influenza virus infection. Following H1N1 or H5N1 influenza virus infection, ferrets were found to recapitulate several laboratory abnormalities previously documented with human disease, furthering the utility of the ferret model for the assessment of influenza virus pathogenicity.     

Age-matched comparison of children hospitalized for 2009 pandemic H1N1 influenza with those hospitalized for seasonal H1N1 and H3N2

BACKGROUND: A wide spectrum of clinical manifestation ranging from deaths to a mild course of disease has been reported in children infected with the 2009 pandemic H1N1 (pH1N1) influenza. METHODOLOGY/MAJOR FINDINGS: We conducted an age-matched control study comparing children hospitalized for pH1N1 with historic controls infected with seasonal H1N1 and H3N2 influenza to correct for the effect of age on disease susceptibility and clinical manifestations. We also compared children with pH1N1 to children concurrently admitted for seasonal influenza during the pandemic period to adjust for differences in health-seeking behavior during the pandemic or other potential bias associated with historic controls. There was no death or intensive care admission. Children with pH1N1 were more likely to have at least one risk condition for influenza, an underlying chronic pulmonary condition, more likely to have asthma exacerbation and to be treated with oseltamivir. There was no difference in other aspects of the clinical course or outcome. CONCLUSION: Disease manifestation of children hospitalized for pH1N1 infection was mild in our patient population.     

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。     

Antibody dynamics of 2009 influenza A (H1N1) virus in infected patients and vaccinated people in China

Background

To evaluate the risk of the recurrence and the efficiency of the vaccination, we followed-up antibody responses in patients with the 2009 pandemic H1N1 influenza and persons who received the pandemic H1N1 vaccine in Guangzhou China.

Methods

We collected serum samples from 129 patients and 86 vaccinated persons at day 0, 15, 30, 180 after the disease onset or the vaccination, respectively. Antibody titers in these serum samples were determined by haemagglutination inhibition (HI) assay using a local isolated virus strain A/Guangdong Liwan/SWL1538/2009(H1N1).

Results

HI antibody positive rate of the patients increased significantly from 0% to 60% at day 15 (χ² = 78, P<0.001) and 100% at day 30 (χ² = 23, P<0.001), but decreased significantly to 52% at day 180 (χ² = 38, P<0.001), while that of vaccinated subjects increased from 0% to 78% at day 15 (χ² = 110, P<0.001) and 81% at day 30 (χ² = 0.32, P = 0.57), but decreased significantly to 34% at day 180 (χ² = 39, P<0.001). Geometric mean titers (GMT) of HI antibodies in positive samples from the patients did not change significantly between day 15 and day 30 (T = 0.92, P = 0.36), but it decreased significantly from 80 at day 30 to 52 at day 180 (T = 4.5, P<0.001). GMT of vaccinated persons increased significantly from 100 at day 15 to 193 at day 30 (T = 4.5, P<0.001), but deceased significantly to 74 at day 180 (T = 5.1, P<0.001). Compared to the patients, the vaccinated subjects showed lower seroconversion rate (χ² = 11, P<0.001; χ² = 5.9, P = 0.015), but higher GMT (T = 6.0, P<0.001; T = 3.6, P = 0.001) at day 30 and day 180, respectively.

Conclusion

Vaccination of 2009 influenza A (H1N1) was effective. However, about half or more recovered patients and vaccinated persons might have lost sufficient immunity against the recurrence of the viral infection after half a year. Vaccination or re-vaccination may be necessary for prevention of the recurrence.     

达菲药物对H1N1病毒作用研究

甲型H1N1病毒在全世界范围内爆发,引起人们广泛关注,而目前疫苗和新的药物正处于研发阶段,与此同时该病毒神经氨酸酶蛋白序列不断被报道。达菲作为治疗H1N1病毒的药物被患者广泛使用。通过同源性建模的方法比较神经氨酸酶的变异情况,从而预测达菲药物对变异前后的作用效果评价。通过AUTODOCK计算结合能,发现达菲药物与神经氨酸酶的结合能维持在2.4～4.2 kJ/mol范围内,动力学常数最高值达到18.2 mM。证明达菲药物对抑制病毒进入寄主细胞有明显效果。     

Adaption of seasonal H1N1 influenza virus in mice

The experimental infection of a mouse lung with influenza A virus has proven to be an invaluable model for studying the mechanisms of viral adaptation and virulence. The mouse adaption of human influenza A virus can result in mutations in the HA and other proteins, which is associated with increased virulence in mouse lungs. In this study, a mouse-adapted seasonal H1N1 virus was obtained through serial lung-to-lung passages and had significantly increased virulence and pathogenicity in mice. Genetic analysis indicated that the increased virulence of the mouse-adapted virus was attributed to incremental acquisition of three mutations in the HA protein (T89I, N125T, and D221G). However, the mouse adaption of influenza A virus did not change the specificity and affinity of receptor binding and the pH-dependent membrane fusion of HA, as well as the in vitro replication in MDCK cells. Notably, infection with the mouse adapted virus induced severe lymphopenia and modulated cytokine and chemokine responses in mice. Apparently, mouse adaption of human influenza A virus may change the ability to replicate in mouse lungs, which induces strong immune responses and inflammation in mice. Therefore, our findings may provide new insights into understanding the mechanisms underlying the mouse adaption and pathogenicity of highly virulent influenza viruses.     

Increased pathogenicity of a reassortant 2009 pandemic H1N1 influenza virus containing an H5N1 hemagglutinin

A novel H1N1 influenza virus emerged in 2009 (pH1N1) to become the first influenza pandemic of the 21st century. This virus is now cocirculating with highly pathogenic H5N1 avian influenza viruses in many parts of the world, raising concerns that a reassortment event may lead to highly pathogenic influenza strains with the capacity to infect humans more readily and cause severe disease. To investigate the virulence of pH1N1-H5N1 reassortant viruses, we created pH1N1 (A/California/04/2009) viruses expressing individual genes from an avian H5N1 influenza strain (A/Hong Kong/483/1997). Using several in vitro models of virus replication, we observed increased replication for a reassortant CA/09 virus expressing the hemagglutinin (HA) gene of HK/483 (CA/09-483HA) relative to that of either parental CA/09 virus or reassortant CA/09 expressing other HK/483 genes. This increased replication correlated with enhanced pathogenicity in infected mice similar to that of the parental HK/483 strain. The serial passage of the CA/09 parental virus and the CA/09-483HA virus through primary human lung epithelial cells resulted in increased pathogenicity, suggesting that these viruses easily adapt to humans and become more virulent. In contrast, serial passage attenuated the parental HK/483 virus in vitro and resulted in slightly reduced morbidity in vivo, suggesting that sustained replication in humans attenuates H5N1 avian influenza viruses. Taken together, these data suggest that reassortment between cocirculating human pH1N1 and avian H5N1 influenza strains will result in a virus with the potential for increased pathogenicity in mammals.     

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.     

Estimated epidemiologic parameters and morbidity associated with pandemic H1N1 influenza

Background

In the face of an influenza pandemic, accurate estimates of epidemiologic parameters are required to help guide decision-making. We sought to estimate epidemiologic parameters for pandemic H1N1 influenza using data from initial reports of laboratory-confirmed cases.

Methods

We obtained data on laboratory-confirmed cases of pandemic H1N1 influenza reported in the province of Ontario, Canada, with dates of symptom onset between Apr. 13 and June 20, 2009. Incubation periods and duration of symptoms were estimated and fit to parametric distributions. We used competing-risk models to estimate risk of hospital admission and case-fatality rates. We used a Markov Chain Monte Carlo model to simulate disease transmission.

Results

The median incubation period was 4 days and the duration of symptoms was 7 days. Recovery was faster among patients less than 18 years old than among older patients (hazard ratio 1.23, 95% confidence interval 1.06–1.44). The risk of hospital admission was 4.5% (95% CI 3.8%–5.2%) and the case-fatality rate was 0.3% (95% CI 0.1%–0.5%). The risk of hospital admission was highest among patients less than 1 year old and those 65 years or older. Adults more than 50 years old comprised 7% of cases but accounted for 7 of 10 initial deaths (odds ratio 28.6, 95% confidence interval 7.3–111.2). From the simulation models, we estimated the following values (and 95% credible intervals): a mean basic reproductive number (R₀, the number of new cases created by a single primary case in a susceptible population) of 1.31 (1.25–1.38), a mean latent period of 2.62 (2.28–3.12) days and a mean duration of infectiousness of 3.38 (2.06–4.69) days. From these values we estimated a serial interval (the average time from onset of infectiousness in a case to the onset of infectiousness in a person infected by that case) of 4–5 days.

Interpretation

The low estimates for R₀ indicate that effective mitigation strategies may reduce the final epidemic impact of pandemic H1N1 influenza.The emergence and global spread of pandemic H1N1 influenza led the World Health Organization to declare a pandemic on June 11, 2009. As the pandemic spreads, countries will need to make decisions about strategies to mitigate and control disease in the face of uncertainty.For novel infectious diseases, accurate estimates of epidemiologic parameters can help guide decision-making. A key parameter for any new disease is the basic reproductive number (R₀), defined as the average number of new cases created by a single primary case in a susceptible population. R₀ affects the growth rate of an epidemic and the final number of infected people. It also informs the optimal choice of control strategies. Other key parameters that affect use of resources, disease burden and societal costs during a pandemic are duration of illness, rate of hospital admission and case-fatality rate. Early in an epidemic, the case-fatality rate may be underestimated because of the temporal lag between onset of infection and death; the delay between initial identification of a new case and death may lead to an apparent increase in deaths several weeks into an epidemic that is an artifact of the natural history of the disease.We used data from initial reports of laboratory-confirmed pandemic H1N1 influenza to estimate epidemiologic parameters for pandemic H1N1 influenza. The parameters included R₀, incubation period and duration of illness. We also estimated risk of hospital admission and case-fatality rates, which can be used to estimate the burden of illness likely to be associated with this disease.     

Reassortment Networks and the evolution of pandemic H1N1 swine-origin influenza

Prior research developed Reassortment Networks to reconstruct the evolution of segmented viruses under both reassortment and mutation. We report their application to the swine-origin pandemic H1N1 virus (S-OIV). A database of all influenza A viruses, for which complete genome sequences were available in Genbank by October 2009, was created and dynamic programming was used to compute distances between all corresponding segments. A reassortment network was created to obtain the minimum cost evolutionary paths from all viruses to the exemplar S-OIV A/California/04/2009. This analysis took 35 hours on the Cray Extreme Multithreading (XMT) supercomputer, which has special hardware to permit efficient parallelization. Six specific H1N1/H1N2 bottleneck viruses were identified that almost always lie on minimum cost paths to S-OIV. We conjecture that these viruses are crucial to S-OIV evolution and worthy of careful study from a molecular biology viewpoint. In phylogenetics, ancestors are typically medians that have no functional constraints. In our method, ancestors are not inferred, but rather chosen from previously observed viruses along a path of mutation and reassortment leading to the target virus. This specificity and functional constraint render our results actionable for further experiments in vitro and in vivo.