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.     

Seroprevalence of Antibodies to Highly Pathogenic Avian Influenza A (H5N1) Virus among Close Contacts Exposed to H5N1 Cases,China, 2005–2008

To assess the extent of highly pathogenic avian influenza (HPAI) A (H5N1) virus transmission, we conducted sero-epidemiologic studies among close contacts exposed to H5N1 cases in mainland China during 2005–2008. Blood specimens were collected from 87 household members and 332 social contacts of 23 H5N1 index cases for HPAI H5N1 serological testing by modified horse red-blood-cell hemagglutinin inhibition and microneutralization assays. All participants were interviewed with a standardized questionnaire to collect information about the use of personal protective equipment, illness symptoms, exposure to an H5N1 case during the infectious period, and poultry exposures. Two (2.3%) household contacts tested positive for HPAI H5N1 virus antibody, and all social contacts tested negative. Both seropositive cases had prolonged, unprotected, close contact with a different H5N1 index case, including days of bed-care or sleeping together during the index case’s infectious period, and did not develop any illness. None of the 419 close contacts used appropriate personal protective equipment including 17% who reported providing bedside care or having physical contact with an H5N1 case for at least 12 hours. Our findings suggest that HPAI H5N1 viruses that circulated among poultry in mainland China from 2005–2008 were not easily transmitted to close contacts of H5N1 cases.     

Influenza virus A (H5N1): a pandemic risk?

Influenza A subtype H5N1 has represented a growing alarm since its recent identification in Asia. Previously thought to infect only wild birds and poultry, H5N1 has now infected humans, cats, pigs and other mammals in an ongoing outbreak, often with a fatal outcome. In order to evaluate the risk factors for human infection with influenza virus H5N1, here we summarize 53 case patients confirmed with H5N1 infection during 2006. The review also compares the mortality rate among human cases from late 2003 until 15 June 2006 in different countries. Neither how these viruses are transmitted to humans nor the most effective way to reduce the risk for infection is fully understood. The association between household contact with diseased poultry in human infection has been demonstrated. This association could possibly operate by 2 mechanisms. First, transmission may be by inhalation or conjunctival deposition of large infectious droplets which may travel only in short distances. Second, having infected poultry in the home and preparation of infected poultry for consumption may result in exposure to higher virus concentrations than other types of exposure. There is so far no significant evidence for repeated human to human transmission, yet some cases of human to human transmission among the family relatives in Indonesia, Azerbaijan, Iraq and Turkey have been described. Recent outbreaks of highly pathogenic avian influenza A virus (H5N1 subtype) infections in poultry and humans (through direct contact with infected birds) have raised concerns that a new influenza pandemic might occur in the near future.     

Chances and Limitations of Wild Bird Monitoring for the Avian Influenza Virus H5N1 — Detection of Pathogens Highly Mobile in Time and Space

Highly pathogenic influenza virus (HPAIV) H5N1 proved to be remarkably mobile in migratory bird populations where it has led to extensive outbreaks for which the true number of affected birds usually cannot be determined. For the evaluation of avian influenza monitoring and HPAIV early warning systems, we propose a time-series analysis that includes the estimation of confidence intervals for (i) the prevalence in outbreak situations or (ii) in the apparent absence of disease in time intervals for specified regional units. For the German outbreak regions in 2006 and 2007, the upper 95% confidence limit allowed the detection of prevalences below 1% only for certain time intervals. Although more than 25,000 birds were sampled in Germany per year, the upper 95% confidence limit did not fall below 5% in the outbreak regions for most of the time. The proposed analysis can be used to monitor water bodies and high risk areas, also as part of an early-warning system. Chances for an improved targeting of the monitoring system as part of a risk-based approach are discussed with the perspective of reducing sample sizes.     

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

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

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

Background

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

Objective

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

Methods

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

Results

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

Conclusion

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

Improving the Clinical Diagnosis of Influenza—a Comparative Analysis of New Influenza A (H1N1) Cases

Background

The presentation of new influenza A(H1N1) is broad and evolving as it continues to affect different geographic locations and populations. To improve the accuracy of predicting influenza infection in an outpatient setting, we undertook a comparative analysis of H1N1(2009), seasonal influenza, and persons with acute respiratory illness (ARI) in an outpatient setting.

Methodology/Principal Findings

Comparative analyses of one hundred non-matched cases each of PCR confirmed H1N1(2009), seasonal influenza, and ARI cases. Multivariate analysis was performed to look for predictors of influenza infection. Receiver operating characteristic curves were constructed for various combinations of clinical and laboratory case definitions. The initial clinical and laboratory features of H1N1(2009) and seasonal influenza were similar. Among ARI cases, fever, cough, headache, rhinorrhea, the absence of leukocytosis, and a normal chest radiograph positively predict for both PCR-confirmed H1N1-2009 and seasonal influenza infection. The sensitivity and specificity of current WHO and CDC influenza-like illness (ILI) criteria were modest in predicting influenza infection. However, the combination of WHO ILI criteria with the absence of leukocytosis greatly improved the accuracy of diagnosing H1N1(2009) and seasonal influenza (positive LR of 7.8 (95%CI 3.5–17.5) and 9.2 (95%CI 4.1–20.3) respectively).

Conclusions/Significance

The clinical presentation of H1N1(2009) infection is largely indistinguishable from that of seasonal influenza. Among patients with acute respiratory illness, features such as a temperature greater than 38°C, rhinorrhea, a normal chest radiograph, and the absence of leukocytosis or significant gastrointestinal symptoms were all positively associated with H1N1(2009) and seasonal influenza infection. An enhanced ILI criteria that combines both a symptom complex with the absence of leukocytosis on testing can improve the accuracy of predicting both seasonal and H1N1-2009 influenza infection.     

Anomalies in the Influenza Virus Genome Database: New Biology or Laboratory Errors?

A search of the influenza virus genome database reveals anomalies associated with a nonnegligible number of submitted sequences. There are many pairs of viral segments that are very close to each other in nucleotide sequence but relatively far apart in reported time of isolation, resulting in an abnormally low evolutionary rate. Also, some sequences show clear evidence of apparent homologous recombination, a process normally assumed to be extremely rare or nonexistent in this virus. These findings may point to surprising new biology but are perhaps more readily explained by stock contamination or other errors in the sequencing laboratories.     

The Cold European Winter of 2005–2006 Assisted the Spread and Persistence of H5N1 Influenza Virus in Wild Birds

In January 2006, a major cold spell affected Europe, coinciding with an increase of H5N1 influenza virus detected in wild birds, mostly dead mute swans, starting along the River Danube and the Mediterranean coast line. Subsequently H5N1 detections in wild birds were concentrated in central and western parts of Europe, reaching a peak in mid February. We tested the hypothesis that the geographic distribution of these H5N1 infections was modulated by the long-term wintering line, the 0°C isotherm marking the limit beyond which areas are largely unsuitable for wintering waterfowl. Given the particularly cold 2005–2006 European winter, we also considered the satellite-derived contemporary frost conditions. This brought us to select the long-term maximum rather than the mean January 0°C isotherm as the best approximation for the 2005–2006 wintering line. Our analysis shows that H5N1 detection sites were closer to the wintering line than would be expected by chance, even when the geographic distribution of water bird wintering sites was accounted for. We argue that partial frost conditions in water bodies are conducive to bird congregation, and this may have enhanced H5N1 transmission and local spread. Because the environmental virus load also would build up in these hot spots, H5N1 virus may have readily persisted during the spring, at least in cooler areas. We conclude that H5N1 introduction, spread, and persistence in Europe may have been enhanced by the cold 2005–2006 winter.     

Spatial and Temporal Association of Outbreaks of H5N1 Influenza Virus Infection in Wild Birds with the 0°C Isotherm

Wild bird movements and aggregations following spells of cold weather may have resulted in the spread of highly pathogenic avian influenza virus (HPAIV) H5N1 in Europe during the winter of 2005–2006. Waterbirds are constrained in winter to areas where bodies of water remain unfrozen in order to feed. On the one hand, waterbirds may choose to winter as close as possible to their breeding grounds in order to conserve energy for subsequent reproduction, and may be displaced by cold fronts. On the other hand, waterbirds may choose to winter in regions where adverse weather conditions are rare, and may be slowed by cold fronts upon their journey back to the breeding grounds, which typically starts before the end of winter. Waterbirds will thus tend to aggregate along cold fronts close to the 0°C isotherm during winter, creating conditions that favour HPAIV H5N1 transmission and spread. We determined that the occurrence of outbreaks of HPAIV H5N1 infection in waterbirds in Europe during the winter of 2005–2006 was associated with temperatures close to 0°C. The analysis suggests a significant spatial and temporal association of outbreaks caused by HPAIV H5N1 in wild birds with maximum surface air temperatures of 0°C–2°C on the day of the outbreaks and the two preceding days. At locations where waterbird census data have been collected since 1990, maximum mallard counts occurred when average and maximum surface air temperatures were 0°C and 3°C, respectively. Overall, the abundance of mallards (Anas platyrhynchos) and common pochards (Aythya ferina) was highest when surface air temperatures were lower than the mean temperatures of the region investigated. The analysis implies that waterbird movements associated with cold weather, and congregation of waterbirds along the 0°C isotherm likely contributed to the spread and geographical distribution of outbreaks of HPAIV H5N1 infection in wild birds in Europe during the winter of 2005–2006.     

Wild Bird’s-eye View of Influenza Virus A(H1N1) Phylogenetic Evolution

Wild bird fecal samples collected and characterized by the USDA as part of a national surveillance effort were sequenced to study the genetic relatedness of avian, swine, and human H1 and N1 subtypes. Our results find that the 2009 H1N1 human outbreak is closely related to swine virus, but falls into different clades in the H1 and N1 trees. Further, there is evidence of multiple viral genetic exchanges between birds and swine. Ongoing research across host species contributes to an understanding of the circulation of influenza viruses.     

Comparison of Influenza Outbreaks in the Republic of Kazakhstan and Russia Induced by 2009 Yearly New Variant of А(H1N1) Influenza Virus

The aim of the work is the comparison of the epidemiology of influenza and acute respiratory virus infections (ARVI) in the Republic of Kazakhstan with the corresponding influenza epidemic in Russia induced by influenza pandemic virus A/California/07/2009 in 2009.Data on influenza and ARVI from the Republic of Kazakhstan and Federal Center of influenza was collected and investigated over the course of several weeks from hospitalized patients with the same diagnosis among all population and in age groups on ...     

Seasonality of Influenza A(H7N9) Virus in China—Fitting Simple Epidemic Models to Human Cases

Background

Three epidemic waves of influenza A(H7N9) (hereafter ‘H7N9’) human cases have occurred between March 2013 and July 2015 in China. However, the underlying transmission mechanism remains unclear. Our main objective is to use mathematical models to study how seasonality, secular changes and environmental transmission play a role in the spread of H7N9 in China.

Methods

Data on human cases and chicken cases of H7N9 infection were downloaded from the EMPRES-i Global Animal Disease Information System. We modelled on chicken-to-chicken transmission, assuming a constant ratio of 10⁻⁶ human case per chicken case, and compared the model fit with the observed human cases. We developed three different modified Susceptible-Exposed-Infectious-Recovered-Susceptible models: (i) a non-periodic transmission rate model with an environmental class, (ii) a non-periodic transmission rate model without an environmental class, and (iii) a periodic transmission rate model with an environmental class. We then estimated the key epidemiological parameters and compared the model fit using Akaike Information Criterion and Bayesian Information Criterion.

Results

Our results showed that a non-periodic transmission rate model with an environmental class provided the best model fit to the observed human cases in China during the study period. The estimated parameter values were within biologically plausible ranges.

Conclusions

This study highlighted the importance of considering secular changes and environmental transmission in the modelling of human H7N9 cases. Secular changes were most likely due to control measures such as Live Poultry Markets closures that were implemented during the initial phase of the outbreaks in China. Our results suggested that environmental transmission via viral shedding of infected chickens had contributed to the spread of H7N9 human cases in China.     

A Variable Gene in a Conserved Region of the Helicobacter pylori Genome: Isotopic Gene Replacement or Rapid Evolution?

The present study concerns the identification of a novel coding sequence in a region of the Helicobacter pylori genome, located between JHP1069/HP1141 and JHP1071/HP1143 according to the numbering of the J99 and 26695 reference strains, respectively, and spanning three different coding DNA sequences (CDSs). The CDSs located at the centre of this locus were highly polymorphic, as determined by the analysis of 24 European isolates, 3 Asian, and 3 African isolates. Phylogenetic and molecular evolutionary analyses showed that the CDSs were not restricted to the geographical origin of the strains. Despite a very high variability observed in the deduced protein sequences, significant similarity was observed, always with the same protein families, i.e. ATPase and bacteriophage receptor/invasion proteins. Although this variability could be explained by isotopic gene replacement via horizontal transfer of a gene with the same function but coming from a variety of sources, it seems more likely that the very high sequence variation observed at this locus is the result of a strong selection pressure exerted on the corresponding gene product. The CDSs identified in the present study could be used as strain specific markers.     

Cocirculation of Avian H9N2 and Contemporary “Human” H3N2 Influenza A Viruses in Pigs in Southeastern China: Potential for Genetic Reassortment?

Pigs are permissive to both human and avian influenza viruses and have been proposed to be an intermediate host for the genesis of pandemic influenza viruses through reassortment or adaptation of avian viruses. Prospective virological surveillance carried out between March 1998 and June 2000 in Hong Kong, Special Administrative Region, People's Republic of China, on pigs imported from southeastern China, provides the first evidence of interspecies transmission of avian H9N2 viruses to pigs and documents their cocirculation with contemporary human H3N2 (A/Sydney/5/97-like, Sydney97-like) viruses. All gene segments of the porcine H9N2 viruses were closely related to viruses similar to chicken/Beijing/1/94 (H9N2), duck/Hong Kong/Y280/97 (H9N2), and the descendants of the latter virus lineage. Phylogenetic analysis suggested that repeated interspecies transmission events had occurred from the avian host to pigs. The Sydney97-like (H3N2) viruses isolated from pigs were related closely to contemporary human H3N2 viruses in all gene segments and had not undergone genetic reassortment. Cocirculation of avian H9N2 and human H3N2 viruses in pigs provides an opportunity for genetic reassortment leading to the emergence of viruses with pandemic potential.     

Preclinical Evaluation of a Replication-Deficient Intranasal ΔNS1 H5N1 Influenza Vaccine

Background

We developed a novel intranasal influenza vaccine approach that is based on the construction of replication-deficient vaccine viruses that lack the entire NS1 gene (ΔNS1 virus). We previously showed that these viruses undergo abortive replication in the respiratory tract of animals. The local release of type I interferons and other cytokines and chemokines in the upper respiratory tract may have a “self-adjuvant effect”, in turn increasing vaccine immunogenicity. As a result, ΔNS1 viruses elicit strong B- and T- cell mediated immune responses.

Methodology/Principal Findings

We applied this technology to the development of a pandemic H5N1 vaccine candidate. The vaccine virus was constructed by reverse genetics in Vero cells, as a 5∶3 reassortant, encoding four proteins HA, NA, M1, and M2 of the A/Vietnam/1203/04 virus while the remaining genes were derived from IVR-116. The HA cleavage site was modified in a trypsin dependent manner, serving as the second attenuation factor in addition to the deleted NS1 gene. The vaccine candidate was able to grow in the Vero cells that were cultivated in a serum free medium to titers exceeding 8 log₁₀ TCID_50/ml. The vaccine virus was replication deficient in interferon competent cells and did not lead to viral shedding in the vaccinated animals. The studies performed in three animal models confirmed the safety and immunogenicity of the vaccine. Intranasal immunization protected ferrets and mice from being infected with influenza H5 viruses of different clades. In a primate model (Macaca mulatta), one dose of vaccine delivered intranasally was sufficient for the induction of antibodies against homologous A/Vietnam/1203/04 and heterologous A/Indonesia/5/05 H5N1 strains.

Conclusion/Significance

Our findings show that intranasal immunization with the replication deficient H5N1 ΔNS1 vaccine candidate is sufficient to induce a protective immune response against H5N1 viruses. This approach might be attractive as an alternative to conventional influenza vaccines. Clinical evaluation of ΔNS1 pandemic and seasonal influenza vaccine candidates are currently in progress.