Investigating the Genetic Diversity of H5 Avian Influenza Viruses in the United Kingdom from 2020–2022

Publication history: Accepted - 27 April 2023; Published - 26 June 2023.Since 2020, the United Kingdom and Europe have experienced annual epizootics of high-pathogenicity avian influenza virus (HPAIV). The first epizootic, during the autumn/winter of 2020–2021, involved six H5Nx subtypes, although H5N8 HPAIV dominated in the United Kingdom. While genetic assessments of the H5N8 HPAIVs within the United Kingdom demonstrated relative homogeneity, there was a background of other genotypes circulating at a lower degree with different neuraminidase and internal genes. Following a small number of detections of H5N1 in wild birds over the summer of 2021, the autumn/winter of 2021–2022 saw another European H5 HPAIV epizootic that dwarfed the prior epizootic. This second epizootic was dominated almost exclusively by H5N1 HPAIV, although six distinct genotypes were defined. We have used genetic analysis to evaluate the emergence of different genotypes and proposed reassortment events that have been observed. The existing data suggest that the H5N1 viruses circulating in Europe during late 2020 continued to circulate in wild birds throughout 2021, with minimal adaptation, but then went on to reassort with AIVs in the wild bird population. We have undertaken an in-depth genetic assessment of H5 HPAIVs detected in the United Kingdom over two winter seasons and demonstrate the utility of in-depth genetic analyses in defining the diversity of H5 HPAIVs circulating in avian species, the potential for zoonotic risk, and whether incidents of lateral spread can be defined over independent incursions of infections from wild birds. This provides key supporting data for mitigation activities.This work was funded by the Department for Environment, Food, and Rural Affairs (Defra) (United Kingdom) and the Devolved Administrations of Scotland and Wales through the following programs of work: SV3400, SV3032, SV3006, and SE2213. Funding for diagnostic testing in Northern Ireland was provided by the Department for Agriculture, Environment, and Rural Affairs (DAERA). The writing and data analysis for the manuscript were also supported in part by the DELTA-FLU project funded by the European Union’s Horizon 2020 research and innovation program under grant agreement no. 727922. A.C.B., J.J., and I.H.B. were also partly funded by the BBSRC/Defra-funded research initiative FluMAP (BB/X006204/1)

Multi-Gene Detection and Identification of Mosquito-Borne RNA Viruses Using an Oligonucleotide Microarray

<div><p>Background</p><p>Arthropod-borne viruses are important emerging pathogens world-wide. Viruses transmitted by mosquitoes, such as dengue, yellow fever, and Japanese encephalitis viruses, infect hundreds of millions of people and animals each year. Global surveillance of these viruses in mosquito vectors using molecular based assays is critical for prevention and control of the associated diseases. Here, we report an oligonucleotide DNA microarray design, termed ArboChip5.1, for multi-gene detection and identification of mosquito-borne RNA viruses from the genera <i>Flavivirus</i> (family <i>Flaviviridae</i>), <i>Alphavirus</i> (<i>Togaviridae</i>), <i>Orthobunyavirus</i> (<i>Bunyaviridae</i>), and <i>Phlebovirus</i> (<i>Bunyaviridae</i>).</p><p>Methodology/Principal Findings</p><p>The assay utilizes targeted PCR amplification of three genes from each virus genus for electrochemical detection on a portable, field-tested microarray platform. Fifty-two viruses propagated in cell-culture were used to evaluate the specificity of the PCR primer sets and the ArboChip5.1 microarray capture probes. The microarray detected all of the tested viruses and differentiated between many closely related viruses such as members of the dengue, Japanese encephalitis, and Semliki Forest virus clades. Laboratory infected mosquitoes were used to simulate field samples and to determine the limits of detection. Additionally, we identified dengue virus type 3, Japanese encephalitis virus, Tembusu virus, <i>Culex</i> flavivirus, and a Quang Binh-like virus from mosquitoes collected in Thailand in 2011 and 2012.</p><p>Conclusions/Significance</p><p>We demonstrated that the described assay can be utilized in a comprehensive field surveillance program by the broad-range amplification and specific identification of arboviruses from infected mosquitoes. Furthermore, the microarray platform can be deployed in the field and viral RNA extraction to data analysis can occur in as little as 12 h. The information derived from the ArboChip5.1 microarray can help to establish public health priorities, detect disease outbreaks, and evaluate control programs.</p></div

PCR amplification and microarray detection of phleboviruses.

<p>Viral RNA derived from cell culture or infected mosquitoes were PCR amplified using GSPs. Amplicons were analyzed using the ArboChip5.1 microarray. POS, positive PCR amplification; NEG, negative PCR amplification; yes, detected by microarray; no, not detected by microarray; n.t., not tested.</p>a<p>Viruses propagated in cell culture and identified by microarray to species unless otherwise noted.</p>b<p>Virus-infected mosquito used for microarray evaluations, microarray detected at least one target for each virus.</p>c<p>ArboChip5.1 does not include probes specific to the target.</p>d<p>Microarray detection with genus-level probes only.</p>e<p>Not a target virus on ArboChip5.1.</p

Visual analysis of cell-culture derived RVFV strain ZH548.

<p><i>Phlebovirus</i> gene-specific PCR amplicons were identified using oligonucleotide microarray probes grouped by virus genus and segment, (A) <i>Phlebovirus</i> S segment, (B) <i>Phlebovirus</i> M segment, and (C) <i>Phlebovirus</i> L segment, and sorted into subgroups based on phylogenetic clade and target virus(es). ECD signals were converted into z-scores. Subgroups with average z-scores greater than 10 were considered positive and used for virus identification. The plotted maximum z-scores represent the greatest individual probe z-score within a subgroup and were used to determine cross-hybridization. The PCR amplicons hybridized with probes in the RVFV subgroups for all three segment targets and <i>Phlebovirus</i> generic subgroup for L segment. The virus abbreviations are defined in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002349#pntd.0002349.s003" target="_blank">Table S1</a>.</p

Microarray reproducibility.

<p>Microarray analysis using cell-cultured derived viruses were performed in four replicates. The z-scores of the virus-specific probes were averaged and listed for each run. The mean and standard deviation (SD) was calculated to determine the 95% confidence interval (CI) and coefficient of variance (CV) for each virus.</p

PCR amplification and microarray detection of flaviviruses.

<p>Viral RNA derived from cell culture or infected mosquitoes were PCR amplified using GSPs. Amplicons were analyzed using the ArboChip5.1 microarray. JE, Japanese encephalitis; MVE, Murray Valley encephalitis; SLE, St. Louis encephalitis; L1, lineage 1; L2, lineage 2; POS, positive PCR amplification; NEG, negative PCR amplification; yes, detected by microarray; no, not detected by microarray; n.t., not tested.</p>a<p>Viruses propagated in cell culture and identified by microarray to species unless otherwise noted.</p>b<p>Virus-infected mosquito used for microarray evaluations, microarray detected at least one target for each virus.</p>c<p>PCR amplification produced a weak visible band.</p>d<p>ArboChip5.1 does not include probes specific to the target.</p>e<p>Microarray detection with genus-level probes only.</p

Microarray identification of flavivirus RNA from Thailand mosquitoes (2011–2012).

<p>cDNAs from mosquito pools that were found to contain viral RNA by flavivirus screening methods were PCR amplified using GSPs and analyzed using the ArboChip5.1 microarray for virus identification. POS, positive PCR amplification; NEG, negative PCR amplification; n.d., no detection; CxFV, <i>Culex</i> flavivirus; QBV, Quang Binh virus; DENV3, dengue virus type 3; JEV, Japanese encephalitis virus; TMUV, tembusu virus; WTV, Wang Thong virus.</p>a<p>Mosquito pool No. beginning with Th9 were collected in 2011, Th10 were collected in 2012.</p>b<p>Identifcations based on greater than 90% identity using partial NS5 sequences.</p>c<p>PCR amplification produces a weak visible band.</p>d<p>Partial NS5 sequence 78.3% identical to QBV.</p>e<p>WTV targeted probes not included on ArboChip5.1.</p

Visual analysis of cell-culture derived BUNV strain 131B-06.

<p><i>Orthobunyavirus</i> gene-specific PCR amplicons were identified using oligonucleotide microarray probes grouped by virus genus and segment, (A) <i>Orthobunyavirus</i> S segment, (B) <i>Orthobunyavirus</i> M segment, and (C) <i>Orthobunyavirus</i> L segment, and sorted into subgroups based on phylogenetic clade and target virus(es). ECD signals were converted into z-scores. Subgroups with average z-scores greater than 10 were considered positive and used for virus identification. The plotted maximum z-scores represent the greatest individual probe z-score within a subgroup and were used to determine cross-hybridization. The PCR amplicons hybridized with probes in the BUNV subgroups for the M and L segment targets but virus-specific probes could not be designed for the S segment. The BUNV amplicons did have z-scores greater than 10 for seven of the 42 S segment BUNV clade probes, but the average subgroup z-score was less than 10. The virus abbreviations are defined in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002349#pntd.0002349.s003" target="_blank">Table S1</a>.</p

Visual analysis of <i>Ae. aegypti</i> mosquito pool Th9-0122 collected in Thailand.

<p>Screening methods indicated that the mosquito pool contained flavivirus RNA and cDNA from the pool was PCR amplified with <i>Flavivirus</i> (A) E, (B) NS3, and (C) NS5 GSPs. The PCR amplicons hybridized only with probes in the DENV3 subgroups for the (A) E and (C) NS5 gene targets. For the (B) NS3 gene, the amplicons hybridized with probes in the DENV clade and DENV2/3 (hybridizes with DENV2 and DENV3) subgroups, but did not hybridize with the DENV2-specific subgroup. The mosquito pool was identified as containing DENV-3 RNA.</p

PCR amplification and microarray detection of orthobunyaviruses.

<p>Viral RNA derived from cell culture or infected mosquitoes were PCR amplified using GSPs. Amplicons were analyzed using the ArboChip5.1 microarray. POS, positive PCR amplification; NEG, negative PCR amplification; yes, detected by microarray; no, not detected by microarray; n.t., not tested.</p>a<p>Viruses propagated in cell culture and identified by microarray to species unless otherwise noted.</p>b<p>Virus-infected mosquito used for microarray evaluations, microarray detected at least one target for each virus.</p>c<p>ArboChip5.1 does not include probes specific to the target.</p>d<p>GSP set not specific for this clade.</p>e<p>CEV clade detection only.</p