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Influenza is a highly contagious viral infection associated with excessive hospitalizations and deaths throughout the world. Continuous antigenic shift and drift is not only responsible for this devastating effect of influenza but also causes ineffectiveness of antiviral drugs and vaccines. In this study, we investigated the effectiveness of ribavirin, oseltamivir, and amantadine drugs in vitro against nine influenza A isolates collected during June 2012–August 2013 from different slums in Dhaka city. The effectiveness of these drugs was determined by measuring the inhibition of virus-induced cytopathic effect on MDCK cells through MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Our data showed that all nine influenza isolates (6 H1N1 pdm09 and 3 H3N2 subtypes) were completely susceptible to ribavirin (The 50% effective concentrations, EC50 3.0 µg/ml) and oseltamivir (EC50 0.35 µg/ml). When influenza A infection was challenged with amantadine drug, eight out of nine isolates (88%) demonstrated susceptibility to amantadine drug (EC50 0.30 µg/ml) while one H1N1 pdm09 isolate exhibited higher EC50 value (>10 µg/ml) beyond the cell tolerance level of drug (>5 µg/ml). Genetic analysis of transmembrane matrix protein 2 (M2), which is a target for the amantadine drug and vital for viral replication, showed a substitution of amino acid at position 31(S31 N) of that amantadine-resistant isolate indicating the possible reason of amantadine drug resistance.

Antigenic changes in the HA1 domain of the influenza A/H3N2 hemagglutinin (HA) present a challenge in the design of the annual influenza vaccine. We examined the genetic variability in the nucleotide and amino acid of encoding HA1 sequences of the influenza A/H3N2 virus during the 2015 influenza season in Thailand. Toward this, the HA genes of 45 influenza A/H3N2 strains were amplified and sequenced. Although a clade 3C.3a strain (A/Switzerland/9715293/2013) was chosen for the 2015 vaccine, phylogenetic analysis demonstrated that strains belonging to clade 3C.2a (96 %) instead of clade 3C.3a (4 %) were circulating that year. Sequence analysis showed that seven codons were under positive selection, five of which were located inside the antigenic epitopes. The percentages of the perfect match vaccine efficacy (VE) estimated by the P epitope model against circulating strains suggested antigenic drift of the dominant epitopes A and B, which contributed to reduced VE of the 2015 vaccine. However, the 2016 vaccine strain (A/Hong Kong/4801/2014) was closely related and well matched against the circulating strain (mean of VE = 79.3 %). These findings provide data on the antigenic drift of the influenza A/H3N2 virus circulating in Thailand and further support continual monitoring and surveillance of the antigenic changes on HA1.

The current circulating influenza B viruses can be divided into two major phylogenetic lineages: the Victoria and Yamagata lineages. We conducted a survey of influenza B viruses in Hubei and Zhejiang provinces during 2009-2010. Out of 341 throat swabs, 18 influenza B viruses were isolated. Five isolates were selected for genetic and phylogenetic analysis. The molecular analyses revealed that all the isolates had similar antigenic characteristics to B/Brisbane/60/2008. However, in the three viruses isolated from Zhejiang, a single asparagine to aspartic acid substitution in position 197 was observed, thereby eliminating the glycosylation at that site and possibly causing an antigenic change. None of the viruses had amino acid mutations at positions 116, 149, 152, 198, 222, 250, 291, and 402 of the neuraminidase (NA) gene, predicting that the viruses would still be sensitive to NA inhibitors. Phylogenetic analyses revealed that all five isolates were closely related to B/Brisbane/60/2008-the 2010 vaccine strain-and contained Victoria-like hemagglutinin and Yamagata-like NA genes, suggesting that reassortment may had occurred. In addition, similar phylogenetic patterns among the acidic polymerase, nucleoprotein and matrix protein genes, as well as between the basic polymerase 1 and basic polymerase 2 genes, were observed, suggesting possible functional interactions among these proteins. All the results highlighted the importance of molecular monitoring of influenza B viruses for reassortment and antigenic drift.

Pandemic outbreaks of H1N1 swine influenza virus have been reported since 2009. Reassortant H1N2 viruses that contain genes from the pandemic H1N1 virus have been isolated in Italy and the United States. However, there is limited information regarding the molecular characteristics of reassortant H1N2 swine influenza viruses in eastern China. Active influenza surveillance programs in Zhejiang Province identified a novel H1N2 influenza virus isolated from pigs displaying clinical signs of influenza virus infection. Whole-genome sequencing was performed and this strain was compared with other influenza viruses available in GenBank. Phylogenetic analysis suggested that the novel strain contained genes from the 2009 pandemic human H1N1 and swine H3N2 viruses. BALB/c mice were infected with the isolated virus to assess its virulence in mice. While the novel H1N2 isolate replicated well in mice, it was found to be less virulent. These results provide additional evidence that swine serve as intermediate hosts or ‘mixing vessels’ for novel influenza viruses. They also emphasize the importance of surveillance in the swine population for use as an early warning system for influenza outbreaks in swine and human populations.

There are two different mechanisms by which influenza viruses might evolve: (1) Because the RNA genome of influenza viruses is segmented, new strains can suddenly be produced by reassortment, as happens, for example, during antigenic shift, creating new pandemic strains. (2) New viruses evolve relatively slowly by stepwise mutation and selection, for example, during antigenic or genetic drift. Influenza A viruses were found in various vertebrate species, where they form reservoirs that do not easily mix. While human influenza A viruses do not spread in birds and vice versa, the species barrier to pigs is relatively low, so that pigs might function as "mixing vessels" for the creation of new pandemic reassortants in Southeast Asia, where the probability is greatest for double infection of pigs by human and avian influenza viruses. Phylogenetic studies revealed that about 100 years ago, an avian influenza A virus had crossed the species barrier, presumably first to pigs, and from there to humans, forming the new stable human and classical swine lineages. In 1979, again, an avian virus showed up in the North European swine population, forming another stable swine lineage. The North European swine isolates from 1979 until about 1985 were genetically extremely unstable. A hypothesis is put forward stating that a mutator mutation is necessary to enable influenza virus to cross the species barrier by providing the new host with sufficient variants from which it can select the best fitting ones. As long as the mutator mutation is still present, such a virus should be able to cross the species barrier a second time, as happened about 100 years ago. Although the most recent swine isolates from northern Germany are again genetically stable, we nevertheless should be on the lookout to see if a North European swine virus shows up in the human population in the near future.

During recent years, a conceptual shift took place with respect to the genetic dynamics of influenza A viruses. In difference of the widely accepted approach that avian viral strains have the capacity to infect man only after undergoing genetic reassortment within pigs, it is now contended that direct transfection of man by intact avian-harbored viral genotypes is an actual, recurrent move, which may bring bout the generation of a new pandemic strain. This cardinal conceptual shift has been propelled by the appearance in 1997 of the zoonotic avian influenza H5N1 virus––a virulent, not yet contagious strain for humans––and ostensibly followed a genuine, unprecedented path within the evolutionary paradigm of Influenza A virus. This paper suggests that direct avian-human genetic interface is a pristine fundamental within the natural history of this protean pathogen, points at earlier as well as corroborative findings leading to such postulation, and regards the course of the H5N1 virus (and alike), as a readily detectable and traceable one, presently, rather then a novel development It further examines the general feasibility of various components of that interface at large, such that give rise––whether gradually or abruptly––to pandemic genotypes, in terms of infectivity, pathogenicity and contagiousness. Within that context, the anticipated involvement of certain human-adapted antigenic subtypes is referred to, extrapolatively. Connectedly, the significance of natural ice as plausible regenerator of influenza A viruses, and its possible contribution to the emergence and reemergence of pandemic strains are accentuated.