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After more than 2 years of pandemic caused by SARS-CoV-2, COVID-19 is still a national concern in many countries worldwide. Coupled with the PHI test of recombination, the close-to-wide pairing distances of linkage disequilibrium suggested that the genomes of SARS-CoV-2 had little to no recombination influence. [Extracted from the article]

Abstract Background The emergence and co-circulation of two different clades (clade 1 and 2) of H5N1 influenza viruses in Vietnam necessitates the availability of a diagnostic assay that can detect both variants. Results We developed a single real-time RT-PCR assay for detection of both clades of H5N1 viruses, directly from clinical specimens, using locked nucleic acid TaqMan probes. Primers and probe used in this assay were designed based on a highly conserved region in the HA gene of H5N1 viruses. The analytical sensitivity of the assay was < 0.5 PFU and 10 - 100 ssDNA plasmid copies. A total of 106 clinical samples (58 from patients infected with clade 1, 2.1 or 2.3 H5N1 viruses and 48 from uninfected or seasonal influenza A virus-infected individuals) were tested by the assay. The assay showed 97% concordance with initial diagnostics for H5 influenza virus infection with a specificity of 100%. Conclusions This assay is a useful tool for diagnosis of H5N1 virus infections in regions where different genetic clades are co-circulating.

Background Since their emergence in Indonesia in 2005, 200 human infections with clade 2.1 highly pathogenic avian influenza A/H5N1 virus have been reported, associated with exceptionally high mortality (84%) compared to regions affected by other genetic clades of this virus. To provide potential clues towards understanding this high mortality, detailed clinical virological analyses were performed in specimens from 180 H5N1 patients, representing 90% of all Indonesian patients and 20% of reported H5N1-infected patients globally. Methods H5N1 RNA was quantified in available upper- and lower-respiratory tract specimens as well as fecal and blood samples from 180 patients with confirmed infection between 2005 and 2017. Mutations in the neuraminidase and M2 genes that confer resistance to oseltamivir and adamantanes were assessed. Fatal and nonfatal cases were compared. Results High viral RNA loads in nasal and pharyngeal specimens were associated with fatal outcome. Mortality increased over time during the study period, which correlated with increasing viral RNA loads on admission. Furthermore, the prevalence of amantadine resistance–conferring M2 mutations increased over time, and viral loads were higher in patients infected with viruses that harbored these mutations. Compared to observations from other regions, viral RNA was detected more frequently in feces (80%) and particularly in blood (85%), and antiviral responses to oseltamivir appeared less pronounced. Conclusions These observations confirm the association of viral load with outcome of human H5N1 infections and suggest potential differences in virulence and antiviral responses to oseltamivir that may explain the exceptionally high mortality related to clade 2.1 H5N1 infections in Indonesia. [ABSTRACT FROM AUTHOR]

Background: The emergence and co-circulation of two different clades (clade 1 and 2) of H5N1 influenza viruses in Vietnam necessitates the availability of a diagnostic assay that can detect both variants. Results: We developed a single real-time RT-PCR assay for detection of both clades of H5N1 viruses, directly from clinical specimens, using locked nucleic acid TaqMan probes. Primers and probe used in this assay were designed based on a highly conserved region in the HA gene of H5N1 viruses. The analytical sensitivity of the assay was < 0.5 PFU and 10 - 100 ssDNA plasmid copies. A total of 106 clinical samples (58 from patients infected with clade 1, 2.1 or 2.3 H5N1 viruses and 48 from uninfected or seasonal influenza A virus-infected individuals) were tested by the assay. The assay showed 97% concordance with initial diagnostics for H5 influenza virus infection with a specificity of 100%. Conclusions: This assay is a useful tool for diagnosis of H5N1 virus infections in regions where different genetic clades are co-circulating. [ABSTRACT FROM AUTHOR]

Highly pathogenic avian influenza (HPAI) viruses are enzootic in wild birds and poultry and continue to cause human infections with high mortality. To date, more than 850 confirmed human cases of H5N1 virus infection have been reported, of which ~60% were fatal. Global concern persists that these or similar avian influenza viruses will evolve into viruses that can transmit efficiently between humans, causing a severe influenza pandemic. It was shown previously that a change in receptor specificity is a hallmark for adaptation to humans and evolution towards a transmittable virus. Substantial genetic diversity was detected within the receptor binding site of hemagglutinin of HPAI A/H5N1 viruses, evolved during human infection, as detected by next generation sequencing. Here we investigated the functional impact of substitutions that were detected during these human infections. Upon rescue of 21 mutant viruses, most substitutions in the RBS resulted in viable virus, but virus replication, entry and stability were often impeded. None of the tested substitutions individually resulted in a clear switch in receptor preference as measured with modified red blood cells and glycan arrays. Although several combinations of the substitutions can lead to human-type receptor specificity, accumulation of multiple amino acid substitutions within a single hemagglutinin during human infection is rare, thus reducing the risk of virus adaptation to humans. Importance H5 viruses continue to be a threat for public health. Because these viruses are immunologically novel to humans, they could spark a pandemic when adapted to transmit between humans. Avian influenza viruses need several adaptive mutations to bind to human- type receptors, increase HA stability, and replicate in human cells. However, knowledge on adaptive mutations during human infections is limited. A previous study showed substantial diversity within the receptor binding site of H5N1 during human infection. We therefore analyzed the observed amino acid changes phenotypically in a diverse set of assays, including virus replication, stability and receptor specificity. None of the tested substitutions resulted in a clear step towards a human adapted virus capable of aerosol transmission. It is notable that acquiring human-type receptor specificity needs multiple amino acid mutations, and that variability at key position 226 is not tolerated, reducing the risk of them being acquired naturally. [ABSTRACT FROM AUTHOR]