To better understand the ecology and epidemiology of the highly pathogenic avian infl uenza virus in its transcontinental spread, we sequenced and analyzed the complete genomes of 36 recent infl uenza A (H5N1) viruses collected from birds in Europe, northern Africa, and southeastern Asia. These sequences, among the fi rst complete genomes of infl uenza (H5N1) viruses outside Asia, clearly depict the lineages now infecting wild and domestic birds in Europe and Africa and show the relationships among these isolates and other strains affecting both birds and humans. The isolates fall into 3 distinct lineages, 1 of which contains all known non-Asian isolates. This new Euro-African lineage, which was the cause of several recent (2006) fatal human infections in Egypt and Iraq, has been introduced at least 3 times into the European-African region and has split into 3 distinct, independently evolving sublineages. One isolate provides evidence that 2 of these sublineages have recently reassorted.

BackgroundEffective 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.Methods and Principal FindingsAnalysis 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.Conclusion/SignificanceThus, 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.

Three hundred eighteen European cows and 115 buffaloes were vaccinated with locally prepared Smithburn vaccine, of which, 100 cows and 20 buffaloes were pregnant. Twenty-eight cows aborted within 72 days post-vaccination, buffaloes did not abort. Blood samples collected 77 days post-vaccination from aborted cows, 17 pregnant cows, 5 pregnant buffaloes, and 32 non-pregnant cows. Sera were tested by ELISA for anti-RVF IgM and IgG. All aborted cows were strongly positive for IgG. Five of 17 cows and two of five buffaloes that did not abort were IgG positive. The percentage of IgM positives in aborted cows was 25% and 0% in non-aborted cows. The percentage of IgG positives in pregnant non-aborted cows was lower than in non-pregnant cows. The percentage of IgG positives of non-pregnant cows was lower than pregnant aborted cows. Virus was isolated from one aborted fetus. The nucleotide sequence of fetus virus was compared to Smithburn of Onderstepoort, local Smithburn and virus isolates from 1993 to 1994 and 1977 RVF outbreaks. The nucleotide sequences of Onderstepoort and Egyptian Smithburn vaccines were almost identical. The sequences of 1993-1994 isolates were identical to 1977 outbreak virus. Virus from the fetus had two mutations; it is apparently a variant that is genetically distant from local Smithburn and Onderstepoort vaccines. Fetus virus was genetically distant from virus of 1993/1994 and 1977 outbreaks. In conclusion, antibody response to vaccination with local Smithburn had occurred in some, but not all the cows and buffaloes. Virus isolation from the fetus suggests in utero transmission of used vaccine virus, which resulted in high abortions in European cows.

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Summary 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.