Mutation Analysis of 2009 Pandemic Influenza A(H1N1) Viruses Collected in Japan during the Peak Phase of the Pandemic

BACKGROUND: Pandemic influenza A(H1N1) virus infection quickly circulated worldwide in 2009. In Japan, the first case was reported in May 2009, one month after its outbreak in Mexico. Thereafter, A(H1N1) infection spread widely throughout the country. It is of great importance to profile and understand the situation regarding viral mutations and their circulation in Japan to accumulate a knowledge base and to prepare clinical response platforms before a second pandemic (pdm) wave emerges. METHODOLOGY: A total of 253 swab samples were collected from patients with influenza-like illness in the Osaka, Tokyo, and Chiba areas both in May 2009 and between October 2009 and January 2010. We analyzed partial sequences of the hemagglutinin (HA) and neuraminidase (NA) genes of the 2009 pdm influenza virus in the collected clinical samples. By phylogenetic analysis, we identified major variants of the 2009 pdm influenza virus and critical mutations associated with severe cases, including drug-resistance mutations. RESULTS AND CONCLUSIONS: Our sequence analysis has revealed that both HA-S220T and NA-N248D are major non-synonymous mutations that clearly discriminate the 2009 pdm influenza viruses identified in the very early phase (May 2009) from those found in the peak phase (October 2009 to January 2010) in Japan. By phylogenetic analysis, we found 14 micro-clades within the viruses collected during the peak phase. Among them, 12 were new micro-clades, while two were previously reported. Oseltamivir resistance-related mutations, i.e., NA-H275Y and NA-N295S, were also detected in sporadic cases in Osaka and Tokyo

Whole-Genome Analysis of Human Influenza A Virus Reveals Multiple Persistent Lineages and Reassortment among Recent H3N2 Viruses

Understanding the evolution of influenza A viruses in humans is important for surveillance and vaccine strain selection. We performed a phylogenetic analysis of 156 complete genomes of human H3N2 influenza A viruses collected between 1999 and 2004 from New York State, United States, and observed multiple co-circulating clades with different population frequencies. Strikingly, phylogenies inferred for individual gene segments revealed that multiple reassortment events had occurred among these clades, such that one clade of H3N2 viruses present at least since 2000 had provided the hemagglutinin gene for all those H3N2 viruses sampled after the 2002–2003 influenza season. This reassortment event was the likely progenitor of the antigenically variant influenza strains that caused the A/Fujian/411/2002-like epidemic of the 2003–2004 influenza season. However, despite sharing the same hemagglutinin, these phylogenetically distinct lineages of viruses continue to co-circulate in the same population. These data, derived from the first large-scale analysis of H3N2 viruses, convincingly demonstrate that multiple lineages can co-circulate, persist, and reassort in epidemiologically significant ways, and underscore the importance of genomic analyses for future influenza surveillance

Polymerase basic protein 1 (PB1) as a molecular determinant of fitness and adaptation in influenza a virus

Tese de doutoramento, Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2017The World Health Organization and the National Institute of Allergy and Infectious Diseases reported growth deficits of influenza A(H1N1)pdm09 reverse genetic pandemic vaccine virus seeds. These have compromised the effective and timely distribution of vaccines in the 2009 pandemics and accentuated the need to improve the process of vaccine production. In pre-pandemic A(H5N1) research, seed viruses produced by reverse genetics have also been reported to present growth deficits. These deficits have been attributed to a putative sub-optimal protein interaction. The dynamics of the genetic evolution of influenza A viruses appears to suggest a gene segregation pattern between the Polymerase Basic protein 1 (PB1) and antigenic proteins Hemagglutinin (HA) and Neuraminidase (NA). In the reassortment events that lead to the emergence of the 1957 e 1968 pandemic viruses, the contemporary seasonal viruses acquired PB1 genomic segment together with antigenic glycoproteins originating from avian viruses. A similar pattern was identified in 1947, where a reassortment event between seasonal viruses, involving PB1 and antigenic proteins, has altered the epidemiology of the infection to a near-pandemic geographic dispersion. In both situations, viral fitness appears to have benefitted from acquiring a PB1 genomic segment homologous to antigenic proteins. Also, in retrospective studies on the genomic composition of high yield seasonal vaccine seeds produced by classical reassortment, PB1 is frequently co-incorporated with antigenic proteins HA and NA, further suggesting that the interaction between these proteins could have an impact in viral fitness. In this context, we proposed to address the question of PB1 genomic segment being a molecular determinant of fitness and adaptation in influenza A virus and, particularly, of the functional compatibility between PB1 and antigenic proteins being a driver of the overall viral fitness and putatively exploitable to improve seed virus production. The A(H1N1)pdm09 virus was used a model for this research because it is a product of viral reassortment with an unprecedented genomic composition of segments originating from avian, swine and human seasonal viruses. Additionally, the 2009 pandemic vaccine virus presented severe growth deficits and, since the A(H1N1)pdm09 persists in circulation with a seasonal epidemiologic profile, the demand for high yield A(H1N1)pdm09 vaccine seeds will be continuous and the need to adequate the immunogenic strain to the circulating viruses will be recurrent because of antigenic drifts. The objectives of this research were defined as 1) to evaluate the genetic evolution of PB1 in the zoonotic transmission of swine influenza virus and infer its putative contribution towards viral fitness and adaptation, and 2) to determine if the functional or structural compatibility between PB1 and antigenic proteins is a molecular determinant of the overall virus fitness in the reverse genetics A(H1N1)pdm09 vaccine seed model. The approach followed to accomplish objective 1 was to select a study sample of PB1 nucleotide sequences from swine virus that have infected the human host, to analyze phylogeny and mutation trends and to search for putative markers for viral adaptation on the basis of viral molecular epidemiology, genomic location of the polymorphisms and amino-acid properties. Our major findings were that the evolutionary history of PB1 is traceable in terms of lineage and host origin. Specific genomic markers in PB1 appear to putatively relate to the viral adaptation to mammalian hosts, 336I, 361R, 468K and 584Q, and to the viral adaptation to new genomic backgrounds possibly in the sequence of reassortment events, such as 638D and 618D. Residues 298I, 386K and 517V have been found to putatively relate to an enhanced compatibility between PB1 and HA of the H1 subtype, in the mammalian host. A subsequent in vitro investigation of the phenotypic impact of mutations L298I, R386K and I517V acquired by the A(H1N1)pdm09 during its evolutionary history, was performed by generating an A(H1N1)pdm09 recombinant virus and an A(H1N1)pdm09 reassortant in which the specific mutations have been reverted, by reverse genetics. This approach has resulted in two major findings. Acquiring these mutations has been found to putatively promote conformational changes in PB1 and enhance the span of complementary nucleotides possibly involved in PB1 interaction with HA at the RNA level and, on the other hand, has proven detrimental to viral growth kinetics in vitro. These findings have lead us to suggest that the interaction between genomic segments at the RNA level could be a determinant of co-segregation, concordant with a selective packaging model proposed by other authors, but that the mechanisms that drive this process are probably not dependent on a replicative advantage. Our approach to accomplishing objective 2) to determine if the functional or structural compatibility between PB1 and antigenic proteins is a molecular determinant of the overall virus fitness in the reverse genetic A(H1N1)pdm09 vaccine seed model, was to determine the genetic profile of A(H1N1)pdm09 strains circulating in Portugal during the pandemic period and select a prototype immunogenic strain, to generate reassortant viruses with the genomic composition of A(H1N1)pdm09 seed viruses prototypes bearing PB1 homologous and heterologous to antigenic proteins, and to evaluate viral growth and antigen yield in vitro. A sample of specimens collected from the pandemic period in Portugal were evaluated for genetic and phenotypic features and a strain similar to the consensus was selected as a prototype strain. Vaccine seed prototypes of the selected A(H1N1)pdm09 strain in an A/PuertoRico/08/34 backbone were generated by reverse genetics to present the genomic compositions of the 6:2 classical vaccine seed (PR8:HA,NA A(H1N1)pdm09) and a 5:3 seed prototype in which the PB1 segment from the immunogenic strain is co-incorporated with the antigenic proteins (PR8:HA,NA,PB1 A(H1N1)pdm09). Our major findings were that the presence of PB1 homologous to antigenic protein significantly increased viral replication, hemagglutination capacity and Neuraminidase activity. We have establishing proof of concept that, in the PR8:A(H1N1)pdm09 seed virus model, viral growth and antigen yield can be significantly improved by the inclusion of PB1 from the immunogenic strain when compared to the classical seed virus prototype. We consider that, additionally to the role of PB1 protein in viral replication, PB1 genomic segment may be a molecular determinant of the overall virus fitness and a determinant factor in the molecular epidemiology of the viruses by establishing interactions with other segments at the RNA level and by, apparently, being able to genetically change and adapt to improve these interactions. Further research is necessary to clarify the mechanisms of viral genome packaging, the role of interactions at the RNA level in establishing the co-segregation patterns and the specificities of this interactions at the subtype level. However, it becomes clear that the functional compatibility between PB1 and antigenic proteins is a driver of the overall viral fitness in the A(H1N1)pdm09 and is putatively exploitable to improve seed virus production. We also consider that exploring the concept of the compatibility between gene segments or proteins being a determinant factor in the overall viral fitness, can result in major improvements in the production of reverse genetics seed viruses of different influenza subtypes. Also, being aware of the fact that the genomic composition of influenza viruses can have a major phenotypic impact, and that consequently is a determinant of virulence even though the mechanisms that drive the selective packaging remain unclear, we consider that its inclusion in the risk assessment of influenza strains would be extremely relevant for seasonal and pandemic preparedness

Novel Genotypes of H9N2 Influenza A Viruses Isolated from Poultry in Pakistan Containing NS Genes Similar to Highly Pathogenic H7N3 and H5N1 Viruses

The impact of avian influenza caused by H9N2 viruses in Pakistan is now significantly more severe than in previous years. Since all gene segments contribute towards the virulence of avian influenza virus, it was imperative to investigate the molecular features and genetic relationships of H9N2 viruses prevalent in this region. Analysis of the gene sequences of all eight RNA segments from 12 viruses isolated between 2005 and 2008 was undertaken. The hemagglutinin (HA) sequences of all isolates were closely related to H9N2 viruses isolated from Iran between 2004 and 2007 and contained leucine instead of glutamine at position 226 in the receptor binding pocket, a recognised marker for the recognition of sialic acids linked α2–6 to galactose. The neuraminidase (NA) of two isolates contained a unique five residue deletion in the stalk (from residues 80 to 84), a possible indication of greater adaptation of these viruses to the chicken host. The HA, NA, nucleoprotein (NP), and matrix (M) genes showed close identity with H9N2 viruses isolated during 1999 in Pakistan and clustered in the A/Quail/Hong Kong/G1/97 virus lineage. In contrast, the polymerase genes clustered with H9N2 viruses from India, Iran and Dubai. The NS gene segment showed greater genetic diversity and shared a high level of similarity with NS genes from either H5 or H7 subtypes rather than with established H9N2 Eurasian lineages. These results indicate that during recent years the H9N2 viruses have undergone extensive genetic reassortment which has led to the generation of H9N2 viruses of novel genotypes in the Indian sub-continent. The novel genotypes of H9N2 viruses may play a role in the increased problems observed by H9N2 to poultry and reinforce the continued need to monitor H9N2 infections for their zoonotic potential

Evolution of the Influenza A Virus: Some New Advances

Influenza is an RNA virus that causes mild to severe respiratory symptoms in humans and other hosts. Every year approximately half a million people around the world die from seasonal Influenza. But this number is substantially larger in the case of pandemics, with the most dramatic instance being the 1918 “Spanish flu” that killed more than 50 million people worldwide. In the last few years, thousands of Influenza genomic sequences have become publicly available, including the 1918 pandemic strain and many isolates from non-human hosts. Using these data and developing adequate bioinformatic and statistical tools, some of the major questions surrounding Influenza evolution are becoming tractable. Are the mutations and reassortments random? What are the patterns behind the virus’s evolution? What are the necessary and sufficient conditions for a virus adapted to one host to infect a different host? Why is Influenza seasonal? In this review, we summarize some of the recent progress in understanding the evolution of the virus

MOLECULAR PATHOGENESIS OF INFLUENZA IN SWINE AND ENGINEERING OF NOVEL RECOMBINANT INFLUENZA VIRUSES

Influenza A viruses (IAVs) belong to the family Orthomyxoviridae and represent major pathogens of both humans and animals. Swine influenza virus is an important pathogen that affects not only the swine industry, but also represents a constant threat to the turkey industry and is of particular concern to public health. In North America, H3N2 triple reassortant (TR) IAVs first emerged in 1998 and have since become endemic in swine populations. In the first part of this dissertation, we focused on the role of surface glycoproteins and PB1-F2 to unravel their roles in the virulence of TR IAVs in this important natural host. We found that surface glycoproteins are necessary and sufficient for the lung pathology, whereas the internal genes play a major role in the febrile response induced by TR H3N2 IAVs in swine. With respect to PB1-F2, we found that PB1-F2 exerts pleiotropic effects in the swine host, which are expressed in a strain-dependent manner. Pathogenicity studies in swine revealed that the presence of PB1-F2 leads the following effects in context of three TR strains tested: no effect in the context of sw/99 strain; increases the virulence of pH1N1; and decreases the virulence of ty/04. Next, we developed temperature-sensitive live attenuated influenza vaccines for use in swine and shown that these vaccines are safe and efficacious against aggressive intratracheal challenge with pH1N1. Lastly, we rearranged the genome of an avian H9N2 influenza virus to generate replication competent influenza virus vectors that provide a robust system for expression and delivery of foreign genes. As a proof-of-principle, we expressed the hemagglutinin from a prototypical highly pathogenic avian influenza virus (HPAIV) H5N1 and shown that this vectored H5 vaccine retained its safety properties in avian and mammalian species, and induced excellent protection against aggressive HPAIV H5N1 challenges in both mice and ferrets. Taken together, these studies have advanced our understanding of molecular basis of pathogenesis of influenza in the swine host and have contributed to the development of improved vaccines and influenza-based vectors with potential applications in both human and veterinary medicine

The Role of Genomics in Tracking the Evolution of Influenza A Virus

Influenza A virus causes annual epidemics and occasional pandemics of short-term respiratory infections associated with considerable morbidity and mortality. The pandemics occur when new human-transmissible viruses that have the major surface protein of influenza A viruses from other host species are introduced into the human population. Between such rare events, the evolution of influenza is shaped by antigenic drift: the accumulation of mutations that result in changes in exposed regions of the viral surface proteins. Antigenic drift makes the virus less susceptible to immediate neutralization by the immune system in individuals who have had a previous influenza infection or vaccination. A biannual reevaluation of the vaccine composition is essential to maintain its effectiveness due to this immune escape. The study of influenza genomes is key to this endeavor, increasing our understanding of antigenic drift and enhancing the accuracy of vaccine strain selection. Recent large-scale genome sequencing and antigenic typing has considerably improved our understanding of influenza evolution: epidemics around the globe are seeded from a reservoir in East-Southeast Asia with year-round prevalence of influenza viruses; antigenically similar strains predominate in epidemics worldwide for several years before being replaced by a new antigenic cluster of strains. Future in-depth studies of the influenza reservoir, along with large-scale data mining of genomic resources and the integration of epidemiological, genomic, and antigenic data, should enhance our understanding of antigenic drift and improve the detection and control of antigenically novel emerging strains