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TDK conference 2022

Determination of the whole genome of Hungarian West Nile virus strains using a next-generation sequencing method
Horváth András - year 5
University of Veterinary Medicine Budapest, Department of Microbiology and Infectious Diseases
Supervisors: Dr. Anna Nagy, Dr. Petra Forgách


West Nile virus (WNV) is a widely distributed zoonotic arbovirus, a member of the genus Flavivirus, family Flaviviridae and has at least 8 different genetic lineages. Lineages 1 and 2 are co-circulating in Europe and contributed to multiple human and animal outbreaks. Lineage 2 has become more widespread in Central Europe since its first emergence in Hungary in 2004 and caused a vast European epidemic in 2018. In its natural transmission cycle, WNV circulates among avian hosts and mosquito vectors, where mosquitoes can also infect accidental hosts, like humans or horses. However, more than 80% of human infections are asymptomatic, and in less than 1% of cases, severe neurological involvement can be observed. Viral nucleic acid detection by molecular methods, such as polymerase chain reaction (PCR), is an important tool for the laboratory differential diagnosis of acute WNV infections. The most sufficient sample types for PCR are anticoagulant-treated whole blood and urine. The aim of this study was to introduce a novel next-generation sequencing protocol for WNV to obtain whole genome sequences from virus isolates and WNV PCR-positive human clinical specimens collected in Hungary between 2015 and 2021. The protocol was optimized for Illumina MiSeq platform, and the study was carried out at the National Reference Laboratory for Viral Zoonoses of the National Public Health Centre, Hungary. Altogether, 15 WNV isolates and 15 WNV PCR-positive clinical specimens were involved in the study. Samples were examined by reverse transcription (RT) real-time PCR to determine the cycle threshold values, and then the whole WNV genome was amplified by a one-step RT-PCR assay. Samples were evaluated by agarose gel electrophoresis. Whole genome sequencing succeeded in 15 WNV isolates (n=14 cell culture supernatants and n=1 mouse brain homogenate) and 10 WNV PCR-positive human clinical specimens (n=9 urine and n=1 whole blood samples). Maximum likelihood phylogenetic analysis revealed that the major European WNV lineage 2 clades, namely the Eastern European/Russian and the Central European/Hungarian clades, are presented in Hungary. Strains of the Balkan and other European clusters within the Central European clade are co-circulating in the country, following a characteristic geographical distribution. Samples of the 2018 WNV epidemic do not form a monophyletic group in the tree, assuming that certain environmental factors were favourable for WNV transmission rather than the emergence of a new genetic WNV variant. In light of the 2018 WNV outbreak in Europe, the European Centre for Disease Prevention and Control anticipates the need for multi-country outbreak investigations through sequence-based typing. Besides whole blood, the collection of urine samples can highly support virus isolation, viral RNA detection and molecular typing.

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