Reassortment Patterns in Swine Influenza Viruses

Three human influenza pandemics occurred in the twentieth century, in 1918, 1957, and 1968. Influenza pandemic strains are the results of emerging viruses from non-human reservoirs to which humans have little or no immunity. At least two of these pandemic strains, in 1957 and in 1968, were the results of reassortments between human and avian viruses. Also, many cases of swine influenza viruses have reportedly infected humans, in particular, the recent H1N1 influenza virus of swine origin, isolated in Mexico and the United States. Pigs are documented to allow productive replication of human, avian, and swine influenza viruses. Thus it has been conjectured that pigs are the “mixing vessel” that create the avian-human reassortant strains, causing the human pandemics. Hence, studying the process and patterns of viral reassortment, especially in pigs, is a key to better understanding of human influenza pandemics. In the last few years, databases containing sequences of influenza A viruses, including swine viruses, collected since 1918 from diverse geographical locations, have been developed and made publicly available. In this paper, we study an ensemble of swine influenza viruses to analyze the reassortment phenomena through several statistical techniques. The reassortment patterns in swine viruses prove to be similar to the previous results found in human viruses, both in vitro and in vivo, that the surface glycoprotein coding segments reassort most often. Moreover, we find that one of the polymerase segments (PB1), reassorted in the strains responsible for the last two human pandemics, also reassorts frequently.     

Prospective of Genomics in Revealing Transmission, Reassortment and Evolution of Wildlife-Borne Avian Influenza A (H5N1) Viruses

The outbreak of highly pathogenic avian influenza (HPAI) H5N1 disease has led to significant loss of poultry and wild life and case fatality rates in humans of 60%. Wild birds are natural hosts for all avian influenza virus subtypes and over120 bird species have been reported with evidence of H5N1 infection. Influenza A viruses possess a segmented RNA genome and are characterized by frequently occurring genetic reassortment events, which play a very important role in virus evolution and the spread of novel gene constellations in immunologically naïve human and animal populations. Phylogenetic analysis of whole genome or sub-genomic sequences is a standard means for delineating genetic variation, novel reassortment events, and surveillance to trace the global transmission pathways. In this paper, special emphasis is given to the transmission and circulation of H5N1 among wild life populations, and to the reassortment events that are associated with inter-host transmission of the H5N1 viruses when they infect different hosts, such as birds, pigs and humans. In addition, we review the inter-subtype reassortment of the viral segments encoding inner proteins between the H5N1 viruses and viruses of other subtypes, such as H9N2 and H6N1. Finally, we highlight the usefulness of genomic sequences in molecular epidemiological analysis of HPAI H5N1 and the technical limitations in existing analytical methods that hinder them from playing a greater role in virological research.     

Alternative Reassortment Events Leading to Transmissible H9N1 Influenza Viruses in the Ferret Model

Influenza A H9N2 viruses are common poultry pathogens that occasionally infect swine and humans. It has been shown previously with H9N2 viruses that reassortment can generate novel viruses with increased transmissibility. Here, we demonstrate the modeling power of a novel transfection-based inoculation system to select reassortant viruses under in vivo selective pressure. Plasmids containing the genes from an H9N2 virus and a pandemic H1N1 (pH1N1) virus were transfected into HEK 293T cells to potentially generate the full panel of possible H9 reassortants. These cells were then used to inoculate ferrets, and the population dynamics were studied. Two respiratory-droplet-transmissible H9N1 viruses were selected by this method, indicating a selective pressure in ferrets for the novel combination of surface genes. These results show that a transfection-based inoculation system is a fast and efficient method to model reassortment and highlight the risk of reassortment between H9N2 and pH1N1 viruses.     

Microarray Multiplex Assay for the Simultaneous Detection and Discrimination of Influenza A and Influenza B Viruses

In this study, we present a microarray approach for the typing of influenza A and B viruses, and the subtyping of H1 and H3 subtypes. We designed four pairs of specific multiplex RT-PCR primers and eight specific oligonucleotide probes and prepared microarrays to identify the specific subtype of influenza virus. Through amplification and fluorescent marking of the multiplex RT-PCR products on the M gene of influenza A and B viruses and the HA gene of subtypes H1 and H3, the PCR products were hybridized with the microarray, and the results were analyzed using a microarray scanner. The results demonstrate that the chip developed by our research institute can detect influenza A and B viruses specifically and identify the subtypes H1 and H3 at a minimum concentration of 1 × 10² copies/μL of viral RNA. We tested 35 clinical samples and our results were identical to other fluorescent methods. The microarray approach developed in this study provides a reliable method for the monitoring and testing of seasonal influenza.     

Estimating the Lineage Dynamics of Human Influenza B Viruses

The prediction of the lineage dynamics of influenza B viruses for the next season is one of the largest obstacles for constructing an appropriate influenza trivalent vaccine. Seasonal fluctuation of transmissibility and epidemiological interference between the two major influenza B lineages make the lineage dynamics complicated. Here we construct a parsimonious model describing the lineage dynamics while taking into account seasonal fluctuation of transmissibility and epidemiological interference. Using this model we estimated the epidemiological and evolutional parameters with the time-series data of the lineage specific isolates in Japan from the 2010–2011 season to the 2014–2015 season. The basic reproduction number is similar between Victoria and Yamagata, with a minimum value during one year as 0.82 (95% highest posterior density (HPD): 0.77–0.87) for the Yamagata and 0.83 (95% HPD: 0.74–0.92) for Victoria, the amplitude of seasonal variation of the basic reproduction number is 0.77 (95% HPD:0.66–0.87) for Yamagata and 1.05 (95% HPD: 0.89–1.02) for Victoria. The duration for which the acquired immunity is effective against infection by the Yamagata lineage is shorter than the acquired immunity for Victoria, 424.1days (95% HPD:317.4–561.5days). The reduction rate of susceptibility due to immune cross-reaction is 0.51 (95% HPD: 0.084–0.92) for the immunity obtained from the infection with Yamagata against the infection with Victoria and 0.62 (95% HPD: 0.42–0.80) for the immunity obtained from the infection with Victoria against the infection with Yamagata. Using estimated parameters, we predicted the dominant lineage in 2015–2016 season. The accuracy of this prediction is 68.8% if the emergence timings of the two lineages are known and 61.4% if the emergence timings are unknown. Estimated seasonal variation of the lineage specific reproduction number can narrow down the range of emergence timing, with an accuracy of 64.6% if the emergence times are assumed to be the time at which the estimated reproduction number exceeds one.     

Isolation of Influenza A and B Viruses in HeLa Cells

The HeLa cell line which is one of the most popular cell lines was shown to be suitable for isolation of types A (H₃N₂) and B influenza viruses from throat washings of patients. Sixty-nine and 67 out of 147 throat washings taken from patients during the period from January to April, 1994, were positive for influenza A virus in HeLa cells and MDCK cells, respectively. Seven out of 10 throat washings taken between January and March, 1993, were positive for influenza B virus in MDCK. Of these 7, 4 were also positive for HeLa cells.     

The Recent Establishment of North American H10 Lineage Influenza Viruses in Australian Wild Waterfowl and the Evolution of Australian Avian Influenza Viruses

Influenza A H10N7 virus with a hemagglutinin gene of North American origin was detected in Australian chickens and poultry abattoir workers in New South Wales, Australia, in 2010 and in chickens in Queensland, Australia, on a mixed chicken and domestic duck farm in 2012. We investigated their genomic origins by sequencing full and partial genomes of H10 viruses isolated from wild aquatic birds and poultry in Australia and analyzed them with all available avian influenza virus sequences from Oceania and representative viruses from North America and Eurasia. Our analysis showed that the H10N7 viruses isolated from poultry were similar to those that have been circulating since 2009 in Australian aquatic birds and that their initial transmission into Australia occurred during 2007 and 2008. The H10 viruses that appear to have developed endemicity in Australian wild aquatic birds were derived from several viruses circulating in waterfowl along various flyways. Their hemagglutinin gene was derived from aquatic birds in the western states of the United States, whereas the neuraminidase was closely related to that from viruses previously detected in waterfowl in Japan. The remaining genes were derived from Eurasian avian influenza virus lineages. Our analysis of virological data spanning 40 years in Oceania indicates that the long-term evolutionary dynamics of avian influenza viruses in Australia may be determined by climatic changes. The introduction and long-term persistence of avian influenza virus lineages were observed during periods with increased rainfall, whereas bottlenecks and extinction were observed during phases of widespread decreases in rainfall. These results extend our understanding of factors affecting the dynamics of avian influenza and provide important considerations for surveillance and disease control strategies.     

Joint Inference of Migration and Reassortment Patterns for Viruses with Segmented Genomes

The structured coalescent allows inferring migration patterns between viral subpopulations from genetic sequence data. However, these analyses typically assume that no genetic recombination process impacted the sequence evolution of pathogens. For segmented viruses, such as influenza, that can undergo reassortment this assumption is broken. Reassortment reshuffles the segments of different parent lineages upon a coinfection event, which means that the shared history of viruses has to be represented by a network instead of a tree. Therefore, full genome analyses of such viruses are complex or even impossible. Although this problem has been addressed for unstructured populations, it is still impossible to account for population structure, such as induced by different host populations, whereas also accounting for reassortment. We address this by extending the structured coalescent to account for reassortment and present a framework for investigating possible ties between reassortment and migration (host jump) events. This method can accurately estimate subpopulation dependent effective populations sizes, reassortment, and migration rates from simulated data. Additionally, we apply the new model to avian influenza A/H5N1 sequences, sampled from two avian host types, Anseriformes and Galliformes. We contrast our results with a structured coalescent without reassortment inference, which assumes independently evolving segments. This reveals that taking into account segment reassortment and using sequencing data from several viral segments for joint phylodynamic inference leads to different estimates for effective population sizes, migration, and clock rates. This new model is implemented as the Structured Coalescent with Reassortment package for BEAST 2.5 and is available at https://github.com/jugne/SCORE.     

Progress and Challenge in Computational Identification of Influenza Virus Reassortment

Virologica Sinica - Genomic reassortment is an important evolutionary mechanism for influenza viruses. In this process, the novel viruses acquire new characteristics by the exchange of the intact...     

Reassortment between Avian H5N1 and Human Influenza Viruses Is Mainly Restricted to the Matrix and Neuraminidase Gene Segments

Highly pathogenic avian influenza H5N1 viruses have devastated the poultry industry in many countries of the eastern hemisphere. Occasionally H5N1 viruses cross the species barrier and infect humans, sometimes with a severe clinical outcome. When this happens, there is a chance of reassortment between H5N1 and human influenza viruses. To assess the potential of H5N1 viruses to reassort with contemporary human influenza viruses (H1N1, H3N2 and pandemic H1N1), we used an in vitro selection method to generate reassortant viruses, that contained the H5 hemagglutinin gene, and that have a replication advantage in vitro. We found that the neuraminidase and matrix gene segments of human influenza viruses were preferentially selected by H5 viruses. However, these H5 reassortant viruses did not show a marked increase in replication in MDCK cells and human bronchial epithelial cells. In ferrets, inoculation with a mixture of H5N1-pandemic H1N1 reassortant viruses resulted in outgrowth of reassortant H5 viruses that had incorporated the neuraminidase and matrix gene segment of pandemic 2009 H1N1. This virus was not transmitted via aerosols or respiratory droplets to naïve recipient ferrets. Altogether, these data emphasize the potential of avian H5N1 viruses to reassort with contemporary human influenza viruses. The neuraminidase and matrix gene segments of human influenza viruses showed the highest genetic compatibility with HPAI H5N1 virus.     

Replication-Competent Influenza B Reporter Viruses as Tools for Screening Antivirals and Antibodies

Influenza B virus is a human pathogen responsible for significant health and economic burden. Research into this pathogen has been limited by the lack of reporter viruses. Here we describe the development of both a replication-competent fluorescent influenza B reporter virus and bioluminescent influenza B reporter virus. Furthermore, we demonstrate these reporter viruses can be used to quickly monitor viral growth and permit the rapid screening of antiviral compounds and neutralizing antibodies.     

The Possible Impact of Vaccination for Seasonal Influenza on Emergence of Pandemic Influenza via Reassortment

Background

One pathway through which pandemic influenza strains might emerge is reassortment from coinfection of different influenza A viruses. Seasonal influenza vaccines are designed to target the circulating strains, which intuitively decreases the prevalence of coinfection and the chance of pandemic emergence due to reassortment. However, individual-based analyses on 2009 pandemic influenza show that the previous seasonal vaccination may increase the risk of pandemic A(H1N1) pdm09 infection. In view of pandemic influenza preparedness, it is essential to understand the overall effect of seasonal vaccination on pandemic emergence via reassortment.

Methods and Findings

In a previous study we applied a population dynamics approach to investigate the effect of infection-induced cross-immunity on reducing such a pandemic risk. Here the model was extended by incorporating vaccination for seasonal influenza to assess its potential role on the pandemic emergence via reassortment and its effect in protecting humans if a pandemic does emerge. The vaccination is assumed to protect against the target strains but only partially against other strains. We find that a universal seasonal vaccine that provides full-spectrum cross-immunity substantially reduces the opportunity of pandemic emergence. However, our results show that such effectiveness depends on the strength of infection-induced cross-immunity against any novel reassortant strain. If it is weak, the vaccine that induces cross-immunity strongly against non-target resident strains but weakly against novel reassortant strains, can further depress the pandemic emergence; if it is very strong, the same kind of vaccine increases the probability of pandemic emergence.

Conclusions

Two types of vaccines are available: inactivated and live attenuated, only live attenuated vaccines can induce heterosubtypic immunity. Current vaccines are effective in controlling circulating strains; they cannot always help restrain pandemic emergence because of the uncertainty of the oncoming reassortant strains, however. This urges the development of universal vaccines for prevention of pandemic influenza.     

Limited Compatibility of Polymerase Subunit Interactions in Influenza A and B Viruses

Despite their close phylogenetic relationship, natural intertypic reassortants between influenza A (FluA) and B (FluB) viruses have not been described. Inefficient polymerase assembly of the three polymerase subunits may contribute to this incompatibility, especially because the known protein-protein interaction domains, including the PA-binding domain of PB1, are highly conserved for each virus type. Here we show that substitution of the FluA PA-binding domain (PB1-A_1–25) with that of FluB (PB1-B_1–25) is accompanied by reduced polymerase activity and viral growth of FluA. Consistent with these findings, surface plasmon resonance spectroscopy measurements revealed that PA of FluA exhibits impaired affinity to biotinylated PB1-B_1–25 peptides. PA of FluB showed no detectable affinity to biotinylated PB1-A_1–25 peptides. Consequently, FluB PB1 harboring the PA-binding domain of FluA (PB1-AB) failed to assemble with PA and PB2 into an active polymerase complex. To regain functionality, we used a single amino acid substitution (T6Y) known to confer binding to PA of both virus types, which restored polymerase complex formation but surprisingly not polymerase activity for FluB. Taken together, our results demonstrate that the conserved virus type-specific PA-binding domains differ in their affinity to PA and thus might contribute to intertypic exclusion of reassortants between FluA and FluB viruses.