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How first rabbit haemorrhagic disease virus case in Sub-Saharan Africa was uncovered

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Again, the African Centre of Excellence for the Genomics of Infectious Disease (ACEGID) at Redeemer’s University (RUN), Ede, Osun State, has blazed the trail on medical diagnosis and research. The Centre has reported the first genome sequences of Rabbit Haemorrhagic Disease Virus (RHDV) in Sub-Saharan Africa.

Redeemer’s University was the first private and public facility in Nigeria with the capacity to diagnose the Ebola Virus Disease (EVD). It was in Christian Happi’s laboratory at Redeemer’s University, where the first case of Ebola from Nigeria was diagnosed in 2014.

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Scientists at ACEGID also developed and patented two rapid diagnosis test kits for the Ebola Virus Disease and Lassa Virus, which are haemorrhagic diseases, in 2017.

Meanwhile, in August 2020, following reports of devastating outbreaks of suspected Rabbit Haemorrhagic Disease (RHD) in Rabbit farms around, Ibadan, South-western region of Nigeria, tissue samples were obtained from five rabbits post mortem for molecular confirmation. Symptoms observed included: anorexia, lacrimal discharge, lethargy, bleeding from the oral and nasal orifices and sudden death in mostly exotic breeds of all ages and sex.

On September 25, 2020, at ACEGID, samples were homogenized in TRIzol and nucleic acid extracted using the QIAamp Viral RNA extraction kit (Qiagen, Hilden, Germany). RT-PCR targeting the VP60 gene of the RHDV genome was carried out using One-step SuperScript™ III One-Step RT-PCR with Platinum™ Taq DNA Polymerase (Invitrogen, USA) kit. RT-PCR products were viewed in 1% agarose gel. Two out of five samples were RT-PCR positive for RHDV. Nextera XT sequencing libraries were prepared from extracted RNA following previously published protocol.

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Libraries were sequenced using the Illumina Miseq machine in the sequencing platform of ACEGID. Following sequencing, raw reads from the next-generation sequencing machine were uploaded to our cloud-based platform (DNAnexus, www.dnanexus.com 2). Quality control check was conducted on the raw reads using fastqc

(https://www.bioinformatics.babraham.ac.uk/projects/fastqc 1). Metagenomics analysis was carried out using Kraken. Three RHDV genomes (One full and two partial) were assembled on October 1, 2020, using publicly available software viral-ngs v2.1.8 (https://github.com/broadinstitute/viral-ngs 1) implemented on DNAnexus. Sequence analysis revealed that the three sequences from this study belong to genotype Lagovirus europaeus/GI.2 also known as RDHV2 or RHDVb. This genotype was first identified in 2010 as a novel pathogenic form of lagovirus in France (Le Gall-Reculé et al., 2011) after which it spread rapidly through Europe and other parts of the world (Oceania, North America and Africa). It has also been reported to replace former circulating GI.1 strain in Australia and Portugal.

Ninety representative RHDV sequences were obtained from the NCBI database and aligned with our three sequences using MAFFT v7.388 with further adjustment made manually as necessary in Geneious. A neighbour-joining tree was generated also in Geneious from a distance matrix corrected for nucleotide substitutions by the Tamura-Nei model. The tree was viewed and manually edited using FigTree (http://tree.bio.ed.ac.uk/software/figtree/). Phylogenetic analysis showed that the sequences from this study belong to the RHDV2 genotype as they clustered together in the same clade with previous RHDV2 sequences from Europe.

To investigate the discrepancy between the PCR and sequencing results, the researchers aligned the three sequences from this study with all sequences in the same RHDV2 clade on the tree to check for amino acid mutations specific to our new sequences from Nigeria. “We also mapped the RT-PCR primers to our full genome obtained from this study to check if any mutation observed occurred in the target regions of the primers. Analysis of the Single Nucleotide Polymorphisms (SNPs) in the RHDV2 genome of a sample (RT5) obtained from our study, revealed 11 unique mutations resulting in amino acid changes. These are: Thr179Ile, Leu216Ser, Leu342Ser, Arg827Lys, Gln928Arg, Thr1337Ile, Thr1742Ile, Leu1929Pro, Ser1978Phe, Val2104Ala, Val2127Ala. Four of these mutations (Leu1929Pro, Ser1978Phe, Val2104Ala, Val2127Ala) occur in the region of target for the RT-PCR primers. Sample RT4 had a unique mutation Pro440Leu compared to other RHDV2 sequences they clustered with on the tree,” the researchers noted.

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