Biology subjects

Wentworth, D. E.

Publications and source records attributed to Wentworth, D. E..

3 recordsLinked to original sources

bioRxiv2018

Complete genome direct RNA sequencing of influenza A virus

For the first time, a complete genome of an RNA virus has been sequenced in its original form. Previously, RNA was sequenced by the chemical degradation of radiolabelled RNA, a difficult method that produced only short sequences. Instead, RNA has usually been sequenced indirectly by copying it into cDNA, which is often amplified to dsDNA by PCR and subsequently analyzed using a variety of DNA sequencing methods. We designed an adapter to short highly conserved termini of the influenza virus genome to target the (-) sense RNA into a protein nanopore on the Oxford Nanopore MinION sequencing platform. Utilizing this method and total RNA extracted from the allantoic fluid of infected chicken eggs, we demonstrate successful sequencing of the complete influenza virus genome with 100% nucleotide coverage, 99% consensus identity, and 99% of reads mapped to influenza. By utilizing the same methodology we can redesign the adapter in order to expand the targets to include viral mRNA and (+) sense cRNA, which are essential to the viral life cycle. This has the potential to identify and quantify splice variants and base modifications, which are not practically measurable with current methods.

genomics↗

bioRxiv2018

Avian influenza viruses in wild birds: virus evolution in a multi-host ecosystem

Wild ducks and gulls are the major reservoirs for avian influenza A viruses (AIVs). The mechanisms that drive AIV evolution are complex at sites where various duck and gull species from multiple flyways breed, winter or stage. The Republic of Georgia is located at the intersection of three migratory flyways: Central Asian Flyway, East Asian/East African Flyway and Black Sea/Mediterranean Flyway. For six consecutive years (2010-2016), we collected AIV samples from various duck and gull species that breed, migrate and overwinter in Georgia. We found substantial subtype diversity of viruses that varied in prevalence from year to year. Low pathogenic (LP)AIV subtypes included H1N1, H2N3, H2N5, H2N7, H3N8, H4N2, H6N2, H7N3, H7N7, H9N1, H9N3, H10N4, H10N7, H11N1, H13N2, H13N6, H13N8, H16N3, plus two H5N5 and H5N8 highly pathogenic (HP)AIVs belonging to clade 2.3.4.4. Whole genome phylogenetic trees showed significant host species lineage restriction for nearly all gene segments and significant differences for LPAIVs among different host species in observed reassortment rates, as defined by quantification of phylogenetic incongruence, and in nucleotide diversity. Hemagglutinin clade 2.3.4.4 H5N8 viruses, circulated in Eurasia during 2014-2015 did not reassort, but analysis after its subsequent dissemination during 2016-2017 revealed reassortment in all gene segments except NP and NS. Some virus lineages appeared to be unrelated to AIVs in wild bird populations in other regions with maintenance of local AIV viruses in Georgia, whereas other lineages showed considerable genetic inter-relationship with viruses circulating in other parts of Eurasia and Africa, despite relative under-sampling in the area.\n\nImportanceWaterbirds (e.g., gulls/ducks) are natural reservoirs of avian influenza viruses (AIVs) and have been shown to mediate dispersal of AIV at inter-continental scales during seasonal migration. The segmented genome of influenza viruses enables viral RNA from different lineages to mix or re-assort when two viruses infect the same host. Such reassortant viruses have been identified in most major human influenza pandemics and several poultry outbreaks. Despite their importance, we have only recently begun to understand AIV evolution and reassortment in their natural host reservoirs. This comprehensive study illustrates of AIV evolutionary dynamics within a multi-host ecosystem at a stop-over site where three major migratory flyways intersect. Our analysis of this ecosystem over a six-year period provides a snapshot of how these viruses are linked to global AIV populations. Understanding the evolution of AIVs in the natural host is imperative to both mitigating the risk of incursion into domestic poultry and potential risk to mammalian hosts including humans.

microbiology↗

bioRxiv2017

Despite egg-adaptive mutations, the 2012-13 H3N2 influenza vaccine induced comparable antibody titers to the intended strain

BackgroundInfluenza vaccination aims to prevent infection by influenza virus and reduce associated morbidity and mortality; however, vaccine effectiveness (VE) can be modest, especially for subtype A/H3N2. Failure to achieve consistently high VE has been attributed both to mismatches between the vaccine and circulating influenza strains and to the vaccine's elicitation of protective immunity in only a subset of the population. The low H3N2 VE in 2012-13 was attributed to egg-adaptive mutations that created antigenic mismatch between the intended (A/Victoria/361/2011) and actual vaccine strain (IVR-165).\n\nMethodsWe investigate the basis of the low VE in 2012-2013 by evaluating whether vaccinated and unvaccinated individuals were infected by different viral strains and assessing the serologic responses to A/Victoria/361/2011 and the IVR-165 vaccine strain in an adult cohort before and after vaccination.\n\nResultsWe found no significant genetic differences between the strains that infected vaccinated and unvaccinated individuals. Vaccination increased titers to A/Victoria/361/2011 as much as to IVR-165. These results are consistent with the hypothesis that vaccination served merely to boost preexisting cross-reactive immune responses, which provided limited protection against infection with the circulating influenza strains.\n\nConclusionsIn contrast to suggestive analyses based on ferret antisera, low H3N2 VE in 2012-13 does not appear to be due to the failure of the egg-adapted strain to induce a response to the intended vaccine strain. Instead, low VE might have been caused by the emergence of anti-genically novel influenza strains and low vaccine immunogenicity in a subset of the population.

epidemiology↗