PA residues in the 2009 H1N1 pandemic influenza virus enhance avian influenza virus polymerase activity in mammalian cells

The 2009 pandemic influenza virus (pH1N1) is a swine-origin reassortant containing human, avian, and swine influenza genes. We have previously shown that the polymerase complex of the pH1N1 strain A/California/04/2009 (Cal) is highly active in mammalian 293T cells, despite the avian origin of both its PA and PB2. In this study, we analyzed the polymerase residues that are responsible for high pH1N1 polymerase activity in the mammalian host. Characterization of polymerase complexes containing various combinations of Cal and avian influenza virus A/chicken/Nanchang/3-120/01 (H3N2) (Nan) by reporter gene assay indicates that Cal PA, but not PB2, is a major contributing factor to high Cal polymerase activity in 293T cells. In particular, Cal PA significantly activates the otherwise inactive Nan polymerase at 37 and 39°C but not at the lower temperature of 34°C. Further analysis using site-directed mutagenesis showed that the Cal PA residues 85I, 186S, and 336M contribute to enhanced activity of the Cal polymerase. Recombinant A/WSN/33 (H1N1) (WSN) viruses containing Nan NP and polymerase (PA, PB1, PB2) genes with individual mutations in PA at residues 85, 186, and 336 produced higher levels of viral protein than the virus containing wild-type (WT) Nan PA. Interestingly, compared to the WT, the virus containing the 85I mutation grew faster in human A549 cells and the 336M mutation most significantly enhanced pathogenicity in a mouse model, among the three PA mutations tested. Our results suggest that multiple mutations in PA, which were rarely present in previous influenza isolates, are involved in mammalian adaptation and pathogenicity of the 2009 pH1N1.     

Diagnosis of influenza viruses with special reference to novel H1N1 2009 influenza virus

On 15 April and 17 April 2009, novel swineorigin influenza A (H1N1) virus was identifi ed in specimens obtained from two epidemiologically unlinked patients in the United States. The ongoing outbreak of novel H1N1 2009 influenza (swine influenza) has caused more than 3,99,232 laboratory confi rmed cases of pandemic influenza H1N1 and over 4735 deaths globally. This novel 2009 influenza virus designated as H1N1 A/swine/California/04/2009 virus is not zoonotic swine flu and is transmitted from person to person and has higher transmissibility then that of seasonal influenza viruses. In India the novel H1N1 virus infection has been reported from all over the country. A total of 68,919 samples from clinically suspected persons have been tested for influenza A H1N1 across the country and 13,330 (18.9%) of them have been found positive with 427 deaths. At the All India Institute of Medical Sciences, New Delhi India, we tested 1096 clinical samples for the presence of novel H1N1 influenza virus and seasonal influenza viruses. Of these 1096 samples, 194 samples (17.7%) were positive for novel H1N1 influenza virus and 197 samples (18%) were positive for seasonal influenza viruses. During outbreaks of emerging infectious diseases accurate and rapid diagnosis is critical for minimizing further spread through timely implementation of appropriate vaccines and antiviral treatment. Since the symptoms of novel H1N1 influenza infection are not specifi c, laboratory confi rmation of suspected cases is of prime importance.     

No evidence for intra-segment recombination of 2009 H1N1 influenza virus in swine

Hao (2011) reported that the PB2 genes of three swine influenza A viruses were likely generated through homologous recombination between two closely related parental strains. However, we show that Hao's observation is an artifact of incorrect taxon sampling arising through the lack of an appropriate evolutionary context. Through rigorous phylogenetic analyses we explain the evolutionary origins of these stains and confirm the lack of any statistical support for intra-segmental recombination.     

The seroprevalence of pandemic influenza H1N1 (2009) virus in China

Background

Mainland China experienced pandemic influenza H1N1 (2009) virus (pH1N1) with peak activity during November-December 2009. To understand the geographic extent, risk factors, and attack rate of pH1N1 infection in China we conducted a nationwide serological survey to determine the prevalence of antibodies to pH1N1.

Methodology/Principal Findings

Stored serum samples (n = 2,379) collected during 2006-2008 were used to estimate baseline serum reactogenicity to pH1N1. In January 2010, we used a multistage-stratified random sampling method to select 50,111 subjects who met eligibility criteria and collected serum samples and administered a standardized questionnaire. Antibody response to pH1N1 was measured using haemagglutination inhibition (HI) assay and the weighted seroprevalence was calculated using the Taylor series linearization method. Multivariable logistic regression analyses were used to examine risk factors for pH1N1 seropositivity. Baseline seroprevalence of pH1N1 antibody (HI titer ≥40) was 1.2%. The weighted seroprevalence of pH1N1 among the Chinese population was 21.5%(vaccinated: 62.0%; unvaccinated: 17.1%). Among unvaccinated participants, those aged 6-15 years (32.9%) and 16-24 years (30.3%) had higher seroprevalence compared with participants aged 25–59 years (10.7%) and ≥60 years (9.9%, P<0.0001). Children in kindergarten and students had higher odds of seropositivity than children in family care (OR: 1.36 and 2.05, respectively). We estimated that 207.7 million individuals (15.9%) experienced pH1N1 infection in China.

Conclusions/Significance

The Chinese population had low pre-existing immunity to pH1N1 and experienced a relatively high attack rate in 2009 of this virus. We recommend routine control measures such as vaccination to reduce transmission and spread of seasonal and pandemic influenza viruses.     

上海2009甲型H1N1流行性感冒病毒全基因组分析

2009年全球暴发2009甲型H1N1流行性感冒(简称流感)疫情,上海于2009年5月出现第1例输入型病例。为了解上海地区输入型2009甲型H1N1流感病毒的生物学特征,以上海较早发现的2例输入型甲型H1N1流感患者作为研究对象,分离出A/Shanghai/37T/2009和A/Shanghai/71T/2009病毒,利用实时定量荧光反转录-聚合酶链反应(RT-PCR)鉴定病毒,通过扫描透射电子显微镜观察、免疫荧光检测、全基因组测序和生物信息软件分析,对这2株流感病毒形态、结构、耐药性、基因特点和病毒型别等进行研究。结果显示,病毒呈现正黏病毒颗粒形态特征;犬肾(MDCK)细胞内的病毒能与患者恢复期血清反应。此2株病毒的全基因核酸序列和氨基酸序列与美国参考株A/California/04/2009(H1N1)有较高同源性,其中第31位氨基酸残基发生改变。对金刚烷胺耐药,而对奥司他韦敏感。基于全基因组的系统发育分析,确认此2株病毒属2009甲型H1N1流感病毒。     

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.     

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.     

Large-scale evolutionary surveillance of the 2009 H1N1 influenza A virus using resequencing arrays

In April 2009, a new influenza A (H1N1 2009) virus emerged that rapidly spread around the world. While current variants of this virus have caused widespread disease, particularly in vulnerable groups, there remains the possibility that future variants may cause increased virulence, drug resistance or vaccine escape. Early detection of these virus variants may offer the chance for increased containment and potentially prevention of the virus spread. We have developed and field-tested a resequencing kit that is capable of interrogating all eight segments of the 2009 influenza A(H1N1) virus genome and its variants, with added focus on critical regions such as drug-binding sites, structural components and mutation hotspots. The accompanying base-calling software (EvolSTAR) introduces novel methods that utilize neighbourhood hybridization intensity profiles and substitution bias of probes on the microarray for mutation confirmation and recovery of ambiguous base queries. Our results demonstrate that EvolSTAR is highly accurate and has a much improved call rate. The high throughput and short turn-around time from sample to sequence and analysis results (30 h for 24 samples) makes this kit an efficient large-scale evolutionary biosurveillance tool.     

Susceptibility of antiviral drugs against 2009 influenza A (H1N1) virus

The recent outbreak of the novel strain of influenza A (H1N1) virus has raised a global concern of the future risk of a pandemic. To understand at the molecular level how this new H1N1 virus can be inhibited by the current anti-influenza drugs and which of these drugs it is likely to already be resistant to, homology modeling and MD simulations have been applied on the H1N1 neuraminidase complexed with oseltamivir, and the M2-channel with adamantanes bound. The H1N1 virus was predicted to be susceptible to oseltamivir, with all important interactions with the binding residues being well conserved. In contrast, adamantanes are not predicted to be able to inhibit the M2 function and have completely lost their binding with the M2 residues. This is mainly due to the fact that the M2 transmembrane of the new H1N1 strain contains the S31N mutation which is known to confer resistance to adamantanes.     

Inside the outbreak of the 2009 influenza A (H1N1)v virus in Mexico

Background

Influenza viruses pose a threat to human health because of their potential to cause global disease. Between mid March and mid April a pandemic influenza A virus emerged in Mexico. This report details 202 cases of infection of humans with the 2009 influenza A virus (H1N1)v which occurred in Mexico City as well as the spread of the virus throughout the entire country.

Methodology and Findings

From May 1st to May 5th nasopharyngeal swabs, derived from 751 patients, were collected at 220 outpatient clinics and 28 hospitals distributed throughout Mexico City. Analysis of samples using real time RT-PCR revealed that 202 patients out of the 751 subjects (26.9%) were confirmed to be infected with the new virus. All confirmed cases of human infection with the strain influenza (H1N1)v suffered respiratory symptoms. The greatest number of confirmed cases during the outbreak of the 2009 influenza A (H1N1)v were seen in neighbourhoods on the northeast side of Mexico City including Iztapalapa, Gustavo A. Madero, Iztacalco, and Tlahuac which are the most populated areas in Mexico City. Using these data, together with data reported by the Mexican Secretariat of Health (MSH) to date, we plot the course of influenza (H1N1)v activity throughout Mexico.

Conclusions

Our data, which is backed up by MSH data, show that the greatest numbers of the 2009 influenza A (H1N1) cases were seen in the most populated areas. We speculate on conditions in Mexico which may have sparked this flu pandemic, the first in 41 years. We accept the hypothesis that high population density and a mass gathering which took in Iztapalapa contributed to the rapid spread of the disease which developed in three peaks of activity throughout the Country.     

Mutations in polymerase genes enhanced the virulence of 2009 pandemic H1N1 influenza virus in mice

Influenza A virus can infect a wide variety of animal species with illness ranging from mild to severe, and is a continual cause for concern. Genetic mutations that occur either naturally or during viral adaptation in a poorly susceptible host are key mechanisms underlying the evolution and virulence of influenza A virus. Here, the variants containing PA-A36T or PB2-H357N observed in the mouse-adapted descendants of 2009 pandemic H1N1 virus (pH1N1), A/Sichuan/1/2009 (SC), were characterized. Both mutations enhanced polymerase activity in mammalian cells. These effects were confirmed using recombinant SC virus containing polymerase genes with wild type (WT) or mutant PA or PB2. The PA-A36T mutant showed enhanced growth property compared to the WT in both human A549 cells and porcine PK15 cells in vitro, without significant effect on viral propagation in murine LA-4 cells and pathogenicity in mice; however, it did enhance the lung virus titer. PB2-H357N variant demonstrated growth ability comparable to the WT in A549 cells, but replicated well in PK15, LA-4 cells and in mice with an enhanced pathogenic phenotype. Despite such mutations are rare in nature, they could be observed in avian H5 and H7 subtype viruses which were currently recognized to pose potential threat to human. Our findings indicated that pH1N1 may adapt well in mammals when acquiring these mutations. Therefore, future molecular epidemiological surveillance should include scrutiny of both markers because of their potential impact on pathogenesis.     

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。     

Replication and pathogenesis of the pandemic (H1N1) 2009 influenza virus in mammalian models

This study aimed to characterize the replication and pathogenic properties of a Korean pandemic (H1N1) 2009 influenza virus isolate in ferrets and mice. Ferrets infected with A/Korea/01/2009 (H1N1) virus showed mild clinical signs. The virus replicated well in lungs and slightly in brains with no replication in any other organs. Severe bronchopneumonia and thickening of alveolar walls were detected in the lungs. Viral antigens were detected in the bronchiolar epithelial cells, in peribronchial glands with severe peribronchitis and in cells present in the alveoli. A/Korea/01/2009 (H1N1) virus-infected mice showed weight loss and pathological lung lesions including perivascular cuffing, interstitial pneumonia and alveolitis. The virus replicated highly in the lungs and slightly in the nasal tissues. Viral antigens were detected in bronchiolar epithelial cells, pneumocytes and interstitial macrophages. However, seasonal H1N1 influenza virus did not replicate in the lungs of ferrets, and viral antigens were not detected. Thus, this Korean pandemic (H1N1) 2009 isolate infected the lungs of ferrets and mice successfully and caused more pathological lesions than did the seasonal influenza virus.     

Restored PB1-F2 in the 2009 pandemic H1N1 influenza virus has minimal effects in swine

PB1-F2 is an 87- to 90-amino-acid-long protein expressed by certain influenza A viruses. Previous studies have shown that PB1-F2 contributes to virulence in the mouse model; however, its role in natural hosts-pigs, humans, or birds-remains largely unknown. Outbreaks of domestic pigs infected with the 2009 pandemic H1N1 influenza virus (pH1N1) have been detected worldwide. Unlike previous pandemic strains, pH1N1 viruses do not encode a functional PB1-F2 due to the presence of three stop codons resulting in premature truncation after codon 11. However, pH1N1s have the potential to acquire the full-length form of PB1-F2 through mutation or reassortment. In this study, we assessed whether restoring the full-length PB1-F2 open reading frame (ORF) in the pH1N1 background would have an effect on virus replication and virulence in pigs. Restoring the PB1-F2 ORF resulted in upregulation of viral polymerase activity at early time points in vitro and enhanced virus yields in porcine respiratory explants and in the lungs of infected pigs. There was an increase in the severity of pneumonia in pigs infected with isogenic virus expressing PB1-F2 compared to the wild-type (WT) pH1N1. The extent of microscopic pneumonia correlated with increased pulmonary levels of alpha interferon and interleukin-1β in pigs infected with pH1N1 encoding a functional PB1-F2 but only early in the infection. Together, our results indicate that PB1-F2 in the context of pH1N1 moderately modulates viral replication, lung histopathology, and local cytokine response in pigs.     

Effect of priming with H1N1 influenza viruses of variable antigenic distances on challenge with 2009 pandemic H1N1 virus

Compared to seasonal influenza viruses, the 2009 pandemic H1N1 (pH1N1) virus caused greater morbidity and mortality in children and young adults. People over 60 years of age showed a higher prevalence of cross-reactive pH1N1 antibodies, suggesting that they were previously exposed to an influenza virus or vaccine that was antigenically related to the pH1N1 virus. To define the basis for this cross-reactivity, ferrets were infected with H1N1 viruses of variable antigenic distance that circulated during different decades from the 1930s (Alaska/35), 1940s (Fort Monmouth/47), 1950s (Fort Warren/50), and 1990s (New Caledonia/99) and challenged with 2009 pH1N1 virus 6 weeks later. Ferrets primed with the homologous CA/09 or New Jersey/76 (NJ/76) virus served as a positive control, while the negative control was an influenza B virus that should not cross-protect against influenza A virus infection. Significant protection against challenge virus replication in the respiratory tract was observed in ferrets primed with AK/35, FM/47, and NJ/76; FW/50-primed ferrets showed reduced protection, and NC/99-primed ferrets were not protected. The hemagglutinins (HAs) of AK/35, FM/47, and FW/50 differ in the presence of glycosylation sites. We found that the loss of protective efficacy observed with FW/50 was associated with the presence of a specific glycosylation site. Our results suggest that changes in the HA occurred between 1947 and 1950, such that prior infection could no longer protect against 2009 pH1N1 infection. This provides a mechanistic understanding of the nature of serological cross-protection observed in people over 60 years of age during the 2009 H1N1 pandemic.     

Genetic variability of isolates of pandemic influenza A virus H1N1 isolated in Russia in 2009

Complete nucleotide sequence of the genome segments encoding the surface glycoproteins, hemagglutinin, and neuraminidase of influenza A virus H1N1 derived from the patients with influenza in the context of pandemic (H1N1) 2009 was determined out of 14 isolates of pandemic influenza. The philogenetic analysis of these sequences demonstrated their genetic similarity to the corresponding genes of the pandemic influenza virus A (H1N1) 2009 isolates obtained in other countries; each gene homology was greater than 99%. Neuraminidase mutations causing virus resistance to oseltamivir and other neuraminidase inhibitors, known from the literature, were not detected. The hemagglutinin gene mutation D222G was found in 4 isolates from autopsy material. In the hemagglutinin of pandemic A/Salekhard/01/2009(H1N1) isolate a mutation G155E leading to the increase in viral replication in developing chick embryos was detected. The nature and frequency of nucleotides substitutions within HA and NA genes were determined in the current research.     

Serological evidence of subclinical transmission of the 2009 pandemic H1N1 influenza virus outside of Mexico

Background

Relying on surveillance of clinical cases limits the ability to understand the full impact and severity of an epidemic, especially when subclinical cases are more likely to be present in the early stages. Little is known of the infection and transmissibility of the 2009 H1N1 pandemic influenza (pH1N1) virus outside of Mexico prior to clinical cases being reported, and of the knowledge pertaining to immunity and incidence of infection during April–June, which is essential for understanding the nature of viral transmissibility as well as for planning surveillance and intervention of future pandemics.

Methodology/Principal Findings

Starting in the fall of 2008, 306 persons from households with schoolchildren in central Taiwan were followed sequentially and serum samples were taken in three sampling periods for haemagglutination inhibition (HI) assay. Age-specific incidence rates were calculated based on seroconversion of antibodies to the pH1N1 virus with an HI titre of 1∶40 or more during two periods: April–June and September–October in 2009. The earliest time period with HI titer greater than 40, as well as a four-fold increase of the neutralization titer, was during April 26–May 3. The incidence rates during the pre-epidemic phase (April–June) and the first wave (July–October) of the pandemic were 14.1% and 29.7%, respectively. The transmissibility of the pH1N1 virus during the early phase of the epidemic, as measured by the effective reproductive number R₀, was 1.16 (95% confidence interval (CI): 0.98–1.34).

Conclusions

Approximately one in every ten persons was infected with the 2009 pH1N1 virus during the pre-epidemic phase in April–June. The lack of age-pattern in seropositivity is unexpected, perhaps highlighting the importance of children as asymptomatic transmitters of influenza in households. Although without virological confirmation, our data raise the question of whether there was substantial pH1N1 transmission in Taiwan before June, when clinical cases were first detected by the surveillance network.     

Genetical features of influenza virus A (H1N1) strain that caused the 2009 pandemic

Genetical features of the A(H1N1) influenza virus strain that caused the 2009 pandemic are analyzed in the review. Mutations typical for this strain, unique and similar to influenza viruses of swine, avian and seasonal types, and phenotypic (pathologic) features associated with them, that are experimentally confirmed, are described. A possibility of reassortation of avian and swine influenza viruses and possible epidemiologic consequences are discussed.     

Sublingual administration of bacteria-expressed influenza virus hemagglutinin 1 (HA1) induces protection against infection with 2009 pandemic H1N1 influenza virus

Influenza viruses are respiratory pathogens that continue to pose a significantly high risk of morbidity and mortality of humans worldwide. Vaccination is one of the most effective strategies for minimizing damages by influenza outbreaks. In addition, rapid development and production of efficient vaccine with convenient administration is required in case of influenza pandemic. In this study, we generated recombinant influenza virus hemagglutinin protein 1 (sHA1) of 2009 pandemic influenza virus as a vaccine candidate using a well-established bacterial expression system and administered it into mice via sublingual (s.l.) route. We found that s.l. immunization with the recombinant sHA1 plus cholera toxin (CT) induced mucosal antibodies as well as systemic antibodies including neutralizing Abs and provided complete protection against infection with pandemic influenza virus A/CA/04/09 (H1N1) in mice. Indeed, the protection efficacy was comparable with that induced by intramuscular (i.m.) immunization route utilized as general administration route of influenza vaccine. These results suggest that s.l. vaccination with the recombinant non-glycosylated HA1 protein offers an alternative strategy to control influenza outbreaks including pandemics.