Ganna Kovalenko: Nanopore sequencing of emerging viruses in a “hotspot” – African swine fever and avian influenza in Ukraine

Ganna Kovalenko, of The National Academy of Agrarian Sciences of Ukraine, presented her work using Oxford Nanopore technology to sequence two lethal viruses that recently emerged in Ukraine.

The African swine fever virus (ASFV) causes lethal hemorrhagic disease in wild boar and pigs in both backyard and commercial pig farms, presenting a “significant threat to the global pig industry”; outbreaks have spread through Eastern Europe, Russia and China, and there is currently no effective vaccine or antiretroviral therapy available. The virus is 170-190 kbp (“this is huge for a virus”) and has 24 genotypes; it uses the argasid tick as a vector (though this is not seen in Europe) and can infect macrophages and monocytes.

Ganna and her team selected two ASFV genomes, collected in 2014 and 2017 from outbreaks in widely separated regions of Ukraine, for nanopore sequencing. For their pilot experiment, loci providing a “genomic signature” to determine the epidemiological origin of the 2017 sample were enriched via targeted PCR; the library was then prepared using the 1D^2 sequencing kit (SQK-LSK308) and sequenced on the MinION device. The amplicon data was mapped mapped and consensus regions extracted; analysis via BLAST identified the sample as belonging to the virulent ASFV/Georgia/2007 lineage.

Next, the team prepared the 2014 ASFV DNA sample for whole genome sequencing using the Rapid Sequencing Kit (SQK-RAD004), sequencing on the MinION device. Reads were basecalled via Albacore, trimmed with PoreChop, aligned to the 2007 reference genome using Minimap2 and variant-called with Nanopolish. This generated an average of 30x coverage of the ASFV genome. Ganna displayed the few variant substitutions in the data: “ASFV replication is high fidelity!”. The team used these variant substitutions in their annotation of the genome. Construction of a phylogenetic tree showed that the two nanopore-sequenced samples clustered closely, despite their geographic and temporal separation. Ganna showed the multiple clusters of ASFV across Ukraine, and described how the virus appears to have been introduced into the country in several distinct events at different locations.

Ganna then went on to describe the team’s sequencing of the avian influenza A virus (AIV) in Ukraine. AIV is carried by wild birds; however, it can cause “lethal, highly pathogenic outbreaks in poultry (HPAIV).” Over 7,500 samples were collected in the field; initial PCR-based testing suggested the presence of the H5 or H7 subtype of AIV. Viral RNA collected from environmental samples and necropsy tissue from poultry and wild waterfowl was then then reverse transcribed and amplified via MS-RTPCR to generate cDNA libraries; these were then barcoded and sequenced in 1D on the MinION. Each of the eight segments of the viral genome were assembled via Geneious R11; Ganna displayed the alignment of data from one sample to the Eurasian LPAIV M gene with 99% identity, revealing a low pathogenicity subtype. This approach was used to genotype six avian influenza virus samples from mute swans, ducks and chickens in Ukraine via reference-based assemblies and identify reassortment in the segmented genomes.

Ganna concluded that the use of nanopore sequencing enabled “point-of-outbreak” diagnosis of a rapidly spreading, virulent disease. She added that the method that she and her team have developed could be scaled up and applied more widely: “there are many pathogens to sequence in wildlife, livestock and humans.” Their work represents the first time the full genome of an ASFV of the Georgia/2007 strain and full genomes of avian influenza viruses have been sequenced on the MinION, helping further understanding of the evolution and emergence of these viruses.