In vivo efficacy of Ingavirin against pandemic A (H1N1/09)v influenza virus

Ingavirin was shown to be efficient in inhibition of the pandemic influenza virus strains A/California/04/2009 (H1N1)v, A/California/07/2009 (H1N1)v, A/Moscow/225/2009 (H1N1)v and A/Moscow/226/2009 (H1N1)v. as well as the influenza virus strain A/Aichi/2/68 (H3N2) in the lungs of the infected mice. After oral administration of Ingavirin the titers of the influenza virus strains in the lung homogenates lowered.     

甲型H1N1流感病毒在季流病毒、禽流感病毒共感染时变异可能性的验证

目的 甲型H1N1流感病毒A/California/7/2009分别与A/Brisbane/10/07和A/ShenZhen/406H/06共感染小型香猪,预测甲流病毒在与季流H3N2病毒/甲流病毒与禽流感病毒共感染时是否会发生变异.方法 分别将A/California/7/2009(CA7)与A/Brisbane/10/07(H3N2),A/California/7/2009与A/Shenzhen/406H/06(H5N1)对5～6月龄小型猪共感染,小型猪经复方氯胺酮0.1 mL/kg麻醉后进行滴鼻感染,感染后第5天安乐死动物,取动物肺组织作病毒测序分析.结果 A/California/7/2009(CA7)与A/Brisbane/10/07(H3N2)共感染后,A/California/7/2009病毒PB1基因993位G→A突变,PA基因1659位G→A突变,没有氨基酸的变异.A/California/7/2009与A/Shenzhen/406H/06(H5N1)共感染后A/California/7/2009病毒PB2基因1711位T→C突变.碱基的突变未引起氨基酸的变异.结论 A/California/7/2009(CA7)与A/Brisbane/10/07(H3N2),A/California/7/2009与A/Shenzhen/406H/06(H5N1)共感染后在猪的体内没有发生病毒重组、变异.     

Evolution trends of the 2009 pandemic influenza A (H1N1) viruses in different continents from March 2009 to April 2012

The World Health Organization (WHO) announced that the 2009 pandemic influenza A (H1N1) viruses, A/California/07/2009 (H1N1) — like virus, has gone into the post-pandemic period on August 10, 2010. People still have some concerns the virus would likely mutate and become a new pandemic virus in the future. Here, we use MUSCLE program and graphic mapping method to look into the evolutionary characteristics of the 6219 hemagglutinin and 4860 neuraminidase full-length sequences from March 2009 to April 2012. The graphic and statistical analyses showed that the novel pandemic isolates, A/California/07/2009 (H1N1) — like virus, experienced several different times. During the early-pandemic period (03/2009-08/2009), the viruses have spread globally in several clusters and deviated slightly from the recommended vaccine strain, A/California/07/2009. During the pandemic period (09/2009-08/2010), new clusters began to emerge from Asia and North America, and further deviated from the recommended vaccine strain. During the postpandemic period (09/2010-08/2011) and the recent period (09/2011-04/2012), the original cluster with the recommended vaccine isolate, A/California/07/2009, has nearly disappeared. The deviation degree between the new clusters and the vaccine isolate became larger and larger. However, the deviation degree and the deviation speed were low. The WHO did not choose a new vaccine isolate instead of the original vaccine isolate, A/California/07/2009. Even so, it is necessary to monitor continuously the 2009 pandemic influenza A (H1N1) viruses.     

Evolution of the Hemagglutinin Gene of the Influenza A(H1N1)pdm09 Virus in Morocco During Two Influenza Seasons 2009–2011

To study genetic evolution of Moroccan influenza A(H1N1)pdm09 virus strains, we conducted a molecular characterization of the hemagglutinin gene subunit 1 (HA1) of 36 influenza A(H1N1)pdm09 virus strains. The stains were collected from patients in Rabat and Casablanca during two influenza seasons 2009–2010 and 2010–2011. Nucleotide and amino acid sequences of 14 influenza A(H1N1)pdm09 virus strains from 2009 to 2010 were ~97 and 99 %, respectively, similar to the reference strain A/California/07/2009 (H1N1). Phylogenetic analysis of 22 influenza A(H1N1)pdm09 virus strains from 2010 to 2011 revealed a co-circulation of three well-described different genetic groups. Most important, none of the identified groups showed significant changes at the antigenic site of the virus HA1 subunit which may alter the efficacy of California/07/2009 (H1N1) vaccine.     

Determination of the Binding Affinity of Virions of Pandemic Influenza A/California/7/2009 (H1N1pdm09) Virus for Fetuin and Mucin by the Competitive and Kinetic Methods

Biophysics - Abstract—The affinity of binding of pandemic influenza A/California/7/2009 (H1N1pdm09) virus for fetuin and mucin was studied. Three methods based on the enzyme-linked...     

Improved antigen yield in pandemic H1N1 (2009) candidate vaccine viruses with chimeric hemagglutinin molecules

The candidate pandemic H1N1 vaccine virus NIBRG-121 was derived by reverse genetics and comprises the hemagglutinin (HA) and neuraminidase (NA) genes from A/California/7/2009 (CAL) on an A/Puerto Rico/8/34 (PR8) backbone. NIBRG-121 was found to grow poorly in eggs, compared to seasonal H1N1 candidate vaccine viruses. Based on our previous study with H5N1 candidate vaccine viruses, we generated two new viruses with chimeric PR8/CAL HA genes. Here we show that these new viruses have considerably improved growth in eggs and are therefore better candidate vaccine viruses for use in production of pandemic H1N1 (2009) vaccine.     

A single base-pair change in 2009 H1N1 hemagglutinin increases human receptor affinity and leads to efficient airborne viral transmission in ferrets

The 2009 H1N1 influenza A virus continues to circulate among the human population as the predominant H1N1 subtype. Epidemiological studies and airborne transmission studies using the ferret model have shown that the transmission efficiency of 2009 H1N1 viruses is lower than that of previous seasonal strains and the 1918 pandemic H1N1 strain. We recently correlated this reduced transmission efficiency to the lower binding affinity of the 2009 H1N1 hemagglutinin (HA) to α2→6 sialylated glycan receptors (human receptors). Here we report that a single point mutation (Ile219→Lys; a base pair change) in the glycan receptor-binding site (RBS) of a representative 2009 H1N1 influenza A virus, A/California/04/09 or CA04/09, quantitatively increases its human receptor-binding affinity. The increased human receptor-affinity is in the same range as that of the HA from highly transmissible seasonal and 1918 pandemic H1N1 viruses. Moreover, a 2009 H1N1 virus carrying this mutation in the RBS (generated using reverse genetics) transmits efficiently in ferrets by respiratory droplets thereby reestablishing our previously observed correlation between human receptor-binding affinity and transmission efficiency. These findings are significant in the context of monitoring the evolution of the currently circulating 2009 H1N1 viruses.     

Oseltamivir boosts 2009 H1N1 virus infectivity in vitro

The neuraminidase inhibitor oseltamivir has been identified to have significant anti-influenza activity in clinical practice. However, its efficacy has not been verified in enough subtypes of influenza A virus, particularly, the current pandemic virus, H1N1. In vitro, using our influenza pseudotyped particle system, oseltamivir displayed significant inhibitory effects on viral NA activity and pp release. Conversely, a boosting effect on viral infection was observed, particularly with the 2009 H1N1 pp at oseltamivir concentrations above 0.025 μM. Further testing on two wild 2009 H1N1 virus strains, A/California/07/09 and A/Sichuan/1/09, as well as a seasonal flu virus, A/Baoan/51/2008, confirmed these findings.     

Detection of Extensive Cross-Neutralization between Pandemic and Seasonal A/H1N1 Influenza Viruses Using a Pseudotype Neutralization Assay

Background

Cross-immunity between seasonal and pandemic A/H1N1 influenza viruses remains uncertain. In particular, the extent that previous infection or vaccination by seasonal A/H1N1 viruses can elicit protective immunity against pandemic A/H1N1 is unclear.

Methodology/Principal Findings

Neutralizing titers against seasonal A/H1N1 (A/Brisbane/59/2007) and against pandemic A/H1N1 (A/California/04/2009) were measured using an HIV-1-based pseudovirus neutralization assay. Using this highly sensitive assay, we found that a large fraction of subjects who had never been exposed to pandemic A/H1N1 express high levels of pandemic A/H1N1 neutralizing titers. A significant correlation was seen between neutralization of pandemic A/H1N1 and neutralization of a standard seasonal A/H1N1 strain. Significantly higher pandemic A/H1N1 neutralizing titers were measured in subjects who had received vaccination against seasonal influenza in 2008–2009. Higher pandemic neutralizing titers were also measured in subjects over 60 years of age.

Conclusions/Significance

Our findings reveal that the extent of protective cross-immunity between seasonal and pandemic A/H1N1 influenza viruses may be more important than previously estimated. This cross-immunity could provide a possible explanation of the relatively mild profile of the recent influenza pandemic.     

Naturally Occurring Mutations in the PA Gene Are Key Contributors to Increased Virulence of Pandemic H1N1/09 Influenza Virus in Mice

We examined the molecular basis of virulence of pandemic H1N1/09 influenza viruses by reverse genetics based on two H1N1/09 virus isolates (A/California/04/2009 [CA04] and A/swine/Shandong/731/2009 [SD731]) with contrasting pathogenicities in mice. We found that four amino acid mutations (P224S in the PA protein [PA-P224S], PB2-T588I, NA-V106I, and NS1-I123V) contributed to the lethal phenotype of SD731. In particular, the PA-P224S mutation when combined with PA-A70V in CA04 drastically reduced the virus''s 50% mouse lethal dose (LD₅₀), by almost 1,000-fold.     

甲型H1N1流感疫苗生产用工作毒种的质量控制

根据中国药典2005年版三部和WHO"人用大流行流感疫苗制备的指导原则"相关要求,以及各企业的申报规程,对全国10家甲型H1N1流感疫苗生产企业工作毒种A/Californ ia/07/2009 NYMC X-179A进行毒种检定,结果均符合中国药典2005年版三部和各企业申报规程的要求。     

Characterization of the 2009 Pandemic A/Beijing/501/2009 H1N1 Influenza Strain in Human Airway Epithelial Cells and Ferrets

Background

A novel 2009 swine-origin influenza A H1N1 virus (S-OIV H1N1) has been transmitted among humans worldwide. However, the pathogenesis of this virus in human airway epithelial cells and mammals is not well understood.

Methodology/Principal Finding

In this study, we showed that a 2009 A (H1N1) influenza virus strain, A/Beijing/501/2009, isolated from a human patient, caused typical influenza-like symptoms including weight loss, fluctuations in body temperature, and pulmonary pathological changes in ferrets. We demonstrated that the human lung adenocarcinoma epithelial cell line A549 was susceptible to infection and that the infected cells underwent apoptosis at 24 h post-infection. In contrast to the seasonal H1N1 influenza virus, the 2009 A (H1N1) influenza virus strain A/Beijing/501/2009 induced more cell death involving caspase-3-dependent apoptosis in A549 cells. Additionally, ferrets infected with the A/Beijing/501/2009 H1N1 virus strain exhibited increased body temperature, greater weight loss, and higher viral titers in the lungs. Therefore, the A/Beijing/501/2009 H1N1 isolate successfully infected the lungs of ferrets and caused more pathological lesions than the seasonal influenza virus. Our findings demonstrate that the difference in virulence of the 2009 pandemic H1N1 influenza virus and the seasonal H1N1 influenza virus in vitro and in vivo may have been mediated by different mechanisms.

Conclusion/Significance

Our understanding of the pathogenesis of the 2009 A (H1N1) influenza virus infection in both humans and animals is broadened by our findings that apoptotic cell death is involved in the cytopathic effect observed in vitro and that the pathological alterations in the lungs of S-OIV H1N1-infected ferrets are much more severe.     

Swine-origin pandemic H1N1 influenza virus-like particles produced in insect cells induce hemagglutination inhibiting antibodies in BALB/c mice

Recent outbreaks of influenza A highlight the importance of rapid and sufficient supply for pandemic and inter-pandemic vaccines. Classical manufacturing methods for influenza vaccines fail to satisfy this demand. Alternatively, cell culture-based production systems and virus-like particle (VLP)-based technologies have been established. We developed swine-origin pandemic H1N1 influenza VLPs consisting of hemagglutinin (A/California/04/2009) and matrix protein. Hemagglutinin and matrix protein were co-expressed in insect cells by the baculovirus expression system. VLPs were harvested from infection supernatants, purified and used for intraperitoneal immunization of BALB/c mice. Immunization induced high serum antibody titers against A/California/04/2009 as well as hemagglutination inhibiting antibodies. Additionally, we compared VLP production in two different insect cell lines, Sf9 and BTI-TN5B1-4 (High Five™). Taken together VLPs represent a potential strategy for the fight against new pandemic influenza viruses.     

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.     

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.     

Minor changes in the hemagglutinin of influenza A(H1N1)2009 virus alter its antigenic properties

Background

The influenza A(H1N1)2009 virus has been the dominant type of influenza A virus in Finland during the 2009–2010 and 2010–2011 epidemic seasons. We analyzed the antigenic characteristics of several influenza A(H1N1)2009 viruses isolated during the two influenza seasons by analyzing the amino acid sequences of the hemagglutinin (HA), modeling the amino acid changes in the HA structure and measuring antibody responses induced by natural infection or influenza vaccination.

Methods/Results

Based on the HA sequences of influenza A(H1N1)2009 viruses we selected 13 different strains for antigenic characterization. The analysis included the vaccine virus, A/California/07/2009 and multiple California-like isolates from 2009–2010 and 2010–2011 epidemic seasons. These viruses had two to five amino acid changes in their HA1 molecule. The mutation(s) were located in antigenic sites Sa, Ca1, Ca2 and Cb region. Analysis of the antibody levels by hemagglutination inhibition test (HI) indicated that vaccinated individuals and people who had experienced a natural influenza A(H1N1)2009 virus infection showed good immune responses against the vaccine virus and most of the wild-type viruses. However, one to two amino acid changes in the antigenic site Sa dramatically affected the ability of antibodies to recognize these viruses. In contrast, the tested viruses were indistinguishable in regard to antibody recognition by the sera from elderly individuals who had been exposed to the Spanish influenza or its descendant viruses during the early 20^th century.

Conclusions

According to our results, one to two amino acid changes (N125D and/or N156K) in the major antigenic sites of the hemagglutinin of influenza A(H1N1)2009 virus may lead to significant reduction in the ability of patient and vaccine sera to recognize A(H1N1)2009 viruses.     

The M segment of the 2009 new pandemic H1N1 influenza virus is critical for its high transmission efficiency in the guinea pig model

A remarkable feature of the 2009 pandemic H1N1 influenza virus is its efficient transmissibility in humans compared to that of precursor strains from the triple-reassortant swine influenza virus lineage, which cause only sporadic infections in humans. The viral components essential for this phenotype have not been fully elucidated. In this study, we aimed to determine the viral factors critical for aerosol transmission of the 2009 pandemic virus. Single or multiple segment reassortments were made between the pandemic A/California/04/09 (H1N1) (Cal/09) virus and another H1N1 strain, A/Puerto Rico/8/34 (H1N1) (PR8). These viruses were then tested in the guinea pig model to understand which segment of Cal/09 virus conferred transmissibility to the poorly transmissible PR8 virus. We confirmed our findings by generating recombinant A/swine/Texas/1998 (H3N2) (sw/Tx/98) virus, a representative triple-reassortant swine virus, containing segments of the Cal/09 virus. The data showed that the M segment of the Cal/09 virus promoted aerosol transmissibility to recombinant viruses with PR8 and sw/Tx/98 virus backgrounds, suggesting that the M segment is a critical factor supporting the transmission of the 2009 pandemic virus.     

A contributing role for anti-neuraminidase antibodies on immunity to pandemic H1N1 2009 influenza A virus

Background

Exposure to contemporary seasonal influenza A viruses affords partial immunity to pandemic H1N1 2009 influenza A virus (pH1N1) infection. The impact of antibodies to the neuraminidase (NA) of seasonal influenza A viruses to cross-immunity against pH1N1 infection is unknown.

Methods and Results

Antibodies to the NA of different seasonal H1N1 influenza strains were tested for cross-reactivity against A/California/04/09 (pH1N1). A panel of reverse genetic (rg) recombinant viruses was generated containing 7 genes of the H1N1 influenza strain A/Puerto Rico/08/34 (PR8) and the NA gene of either the pandemic H1N1 2009 strain (pH1N1) or one of the following contemporary seasonal H1N1 strains: A/Solomon/03/06 (rg Solomon) or A/Brisbane/59/07 (rg Brisbane). Convalescent sera collected from mice infected with recombinant viruses were measured for cross-reactive antibodies to pH1N1 via Hemagglutinin Inhibition (HI) or Enzyme-Linked Immunosorbent Assay (ELISA). The ectodomain of a recombinant NA protein from the pH1N1 strain (pNA-ecto) was expressed, purified and used in ELISA to measure cross-reactive antibodies. Analysis of sera from elderly humans immunized with trivalent split-inactivated influenza (TIV) seasonal vaccines prior to 2009 revealed considerable cross-reactivity to pNA-ecto. High titers of cross-reactive antibodies were detected in mice inoculated with either rg Solomon or rg Brisbane. Convalescent sera from mice inoculated with recombinant viruses were used to immunize naïve recipient Balb/c mice by passive transfer prior to challenge with pH1N1. Mice receiving rg California sera were better protected than animals receiving rg Solomon or rg Brisbane sera.

Conclusions

The NA of contemporary seasonal H1N1 influenza strains induces a cross-reactive antibody response to pH1N1 that correlates with reduced lethality from pH1N1 challenge, albeit less efficiently than anti-pH1N1 NA antibodies. These findings demonstrate that seasonal NA antibodies contribute to but are not sufficient for cross-reactive immunity to pH1N1.     

Effect of prior vaccination with a seasonal trivalent influenza vaccine on the antibody response to the influenza pandemic H1N1 2009 vaccine: a randomized controlled trial

Vaccination with the non-adjuvanted split-virion A/California/7/2009 influenza vaccine (pandemic H1N1 2009 vaccine) began in October 2009 in Japan. The present study was designed to assess the effect of prior vaccination with a seasonal trivalent influenza vaccine on the antibody response to the pandemic H1N1 2009 vaccine in healthy adult volunteers. One hundred and seventeen participants aged 22 to 62 were randomly assigned to two study groups. In Group 1 (the priming group), participants were first vaccinated with the seasonal trivalent influenza vaccine followed by two separate one-dose vaccinations of the pandemic H1N1 2009 vaccine, whereas in Group 2 (the non-priming group), the participants were first vaccinated with one dose of the pandemic H1N1 2009 vaccine, followed by simultaneous vaccination of the seasonal trivalent vaccine and the second dose of the pandemic H1N1 2009 vaccine. The participants in Group 2 had a seroprotection rate (SPR) of 79.7% and a seroconversion rate (SCR) of 79.7% in the hemagglutination-inhibition test after the first dose of the pandemic H1N1 2009 vaccine, indicating that the pandemic H1N1 2009 vaccine is sufficiently immunogenic. On the other hand, the participants of Group 1 had a significantly weaker antibody response, with a SPR of 60.8% and a SCR of 58.5%. These results indicate that prior vaccination with the seasonal trivalent influenza vaccine inhibits the antibody response to the pandemic H1N1 2009 vaccine. Therefore, the pandemic H1N1 2009 vaccine should be administered prior to vaccination with the seasonal trivalent influenza vaccine.