Concurrent Acquisition of a Single Nucleotide Polymorphism in Diverse Influenza H5N1 Clade 2.2 Sub-clades

Highly pathogenic Influenza A H5N1 was first identified in Guangdong Province in 1996, followed by human cases in Hong Kong in 1997. The number of confirmed human cases now exceeds 300, and the associated Case Fatality Rate exceeds 60%. The genetic diversity of the serotype continues to increase. Four distinct clades or sub-clades have been linked to human cases. The gradual genetic changes identified in the sub-clades have been attributed to copy errors by viral encoded polymerases that lack an editing function, thereby resulting in antigenic drift. We report here the concurrent acquisition of the same polymorphism by multiple, genetically distinct, clade 2.2 sub-clades in Egypt, Russia, and Ghana. These changes are not easily explained by the current theory of “random mutation” through copy error, and are more easily explained by recombination with a common source. This conclusion is supported by additional polymorphisms shared by clade 2.2 isolates in Egypt and Germany

Aggregation of Single Nucleotide Polymorphisms in a Human H5N1 Clade 2.2 Hemagglutinin

The evolution of H5N1 has attracted significant interest 1-4 due to linkages with avian 5,6 and human infections 7,8. The basic tenets of influenza genetics 9 attribute genetic drift to replication errors caused by a polymerase complex that lacks a proof reading function. However, recent analysis 10 of swine influenza genes identifies regions copied with absolute fidelity for more than 25 years. In addition, polymorphism tracing of clade 2.2 H5N1 single nucleotide polymorphisms identify concurrent acquisition 11 of the same polymorphism onto multiple genetic backgrounds in widely dispersed geographical locations. Here we show the aggregation of regional clade 2.2 polymorphisms from Germany, Egypt, and sub-Sahara Africa onto a human Nigerian H5N1 hemagglutinin (HA), implicating recombination in the dispersal and aggregation of single nucleotide polymorphisms from closely related genomes

Aggregation of Single Nucleotide Polymorphisms in a Human H5N1 Clade 2.2 Hemagglutinin

The rapid evolution of the H5N1 serotype of avian influenza has been explained by a mechanism involving the selection of single nucleotide polymorphisms generated by copy errors. The recent emergence of H5N1 Clade 2.2 in fifty countries, offered a unique opportunity to view the acquisition of new polymorphism in these evolving genomes. We analyzed the H5N1 hemagglutinin gene from a fatal human case from Nigeria in 2007. The newly emerged polymorphisms were present in diverse H5N1 isolates from the previous year. The aggregation of these polymorphisms from clade 2.2 sub-clades was not supported by recent random mutations, and was most easily explained by recombination between closely related sequences

Pandemic potential of highly pathogenic avian influenza clade 2.3.4.4 A(H5) viruses

The panzootic caused by A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) A(H5) viruses has occurred in multiple waves since 1996. From 2013 onwards, clade 2.3.4.4 viruses of subtypes A(H5N2), A(H5N6), and A(H5N8) emerged to cause panzootic waves of unprecedented magnitude among avian species accompanied by severe losses to the poultry industry around the world. Clade 2.3.4.4 A(H5) viruses have expanded in distinct geographical and evolutionary pathways likely via long distance migratory bird dispersal onto several continents and by poultry trade among neighboring countries. Coupled with regional circulation, the viruses have evolved further by reassorting with local viruses. As of February 2019, there have been 23 cases of humans infected with clade 2.3.4.4 H5N6 viruses, 16 (70%) of which had fatal outcomes. To date, no HPAI A(H5) virus has caused sustainable human-to-human transmission. However, due to the lack of population immunity in humans and ongoing evolution of the virus, there is a continuing risk that clade 2.3.4.4 A(H5) viruses could cause an influenza pandemic if the ability to transmit efficiently among humans was gained. Therefore, multisectoral collaborations among the animal, environmental, and public health sectors are essential to conduct risk assessments and develop countermeasures to prevent disease and to control spread. In this article, we describe an assessment of the likelihood of clade 2.3.4.4 A(H5) viruses gaining human-to-human transmissibility and impact on human health should such human-to-human transmission occur. This structured analysis assessed properties of the virus, attributes of the human population, and ecology and epidemiology of these viruses in animal hosts

Single Assay for Simultaneous Detection and Differential Identification of Human and Avian Influenza Virus Types, Subtypes, and Emergent Variants

For more than four decades the cause of most type A influenza virus infections of humans has been attributed to only two viral subtypes, A/H1N1 or A/H3N2. In contrast, avian and other vertebrate species are a reservoir of type A influenza virus genome diversity, hosting strains representing at least 120 of 144 combinations of 16 viral hemagglutinin and 9 viral neuraminidase subtypes. Viral genome segment reassortments and mutations emerging within this reservoir may spawn new influenza virus strains as imminent epidemic or pandemic threats to human health and poultry production. Traditional methods to detect and differentiate influenza virus subtypes are either time-consuming and labor-intensive (culture-based) or remarkably insensitive (antibody-based). Molecular diagnostic assays based upon reverse transcriptase-polymerase chain reaction (RT-PCR) have short assay cycle time, and high analytical sensitivity and specificity. However, none of these diagnostic tests determine viral gene nucleotide sequences to distinguish strains and variants of a detected pathogen from one specimen to the next. Decision-quality, strain- and variant-specific pathogen gene sequence information may be critical for public health, infection control, surveillance, epidemiology, or medical/veterinary treatment planning. The Resequencing Pathogen Microarray (RPM-Flu) is a robust, highly multiplexed and target gene sequencing-based alternative to both traditional culture- or biomarker-based diagnostic tests. RPM-Flu is a single, simultaneous differential diagnostic assay for all subtype combinations of type A influenza viruses and for 30 other viral and bacterial pathogens that may cause influenza-like illness. These other pathogen targets of RPM-Flu may co-infect and compound the morbidity and/or mortality of patients with influenza. The informative specificity of a single RPM-Flu test represents specimen-specific viral gene sequences as determinants of virus type, A/HN subtype, virulence, host-range, and resistance to antiviral agents

Genome Analysis Linking Recent European and African Influenza (H5N1) Viruses

Although linked, these viruses are distinct from earlier outbreak strains

Global Surveillance of Emerging Influenza Virus Genotypes by Mass Spectrometry

Effective influenza surveillance requires new methods capable of rapid and inexpensive genomic analysis of evolving viral species for pandemic preparedness, to understand the evolution of circulating viral species, and for vaccine strain selection. We have developed one such approach based on previously described broad-range reverse transcription PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology.Analysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide sub-species identification and infer influenza virus H and N subtypes. Using this approach, we detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, direct analysis of 656 human clinical respiratory specimens collected over a seven-year period (1999-2006) showed correct identification of the viral species and subtypes with >97% sensitivity and specificity. Base composition derived clusters inferred from this analysis showed 100% concordance to previously established clades. Ongoing surveillance of samples from the recent influenza virus seasons (2005-2006) showed evidence for emergence and establishment of new genotypes of circulating H3N2 strains worldwide. Mixed viral quasispecies were found in approximately 1% of these recent samples providing a view into viral evolution.Thus, rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio