Mammalian birnaviruses identified in pigs infected by classical swine fever virus

Abstract

Currently, the Birnaviridae family contains four genera with all members identified from birds, fishes, and insects only. The present study reports a novel birnavirus unexpectedly identified from classical swine fever virus-infected pigs by viral metagenomic analysis, which is, therefore, named as porcine birnavirus (PBRV). Follow-up reverse transcription-polymerase chain reaction (RT-PCR) screening of archived tissues of diseased pigs identified 16 PBRV strains from nine provinces/autonomous regions in China spanning 21 years (1998-2019), and the viral loads of PBRV in clinical samples were 10^5.08-10^7.95 genome copies per 0.1 g tissue, showing the replication of PBRVs in the pigs. Genome-based sequence comparison showed that PBRVs are genetically distant from existing members within the Birnaviridae family with 45.8-61.6 per cent and 46.2-63.2 per cent nucleotide sequence similarities in segments A and B, respectively, and the relatively closed viruses are avibirnavirus strains. In addition, indels of 57, 5, and 18 amino acid residues occurred in 16, 2, and 7 locations of the PBRV polyprotein and VP5 and VP1 proteins, respectively, as compared to the reference avibirnaviruses. Phylogenetic analysis showed that PBRVs formed an independent genotype separated from four other genera, which could be classified into two or three subgenotypes (PBRV-A1-2 and PBRV-B1-3) based on the nucleotide sequences of full preVP2 and VP1 genes, respectively. All results showed that PBRV represents a novel porcine virus species, which constitutes the first mammalian birnavirus taxon, thereby naming as Mambirnavirus genus is proposed.

Keywords: CSFV; classification; genetic diversity; porcine birnavirus; prevalence.

Figure 1.

Geographic distribution of PBRVs. Solid red circles represent the locations of 16 PBRV strains. The number in bracket shows PBRV detection rates in CSFV-positive samples in each province.

Figure 2.

Viral loads of PBRV and CSFV in PBRV-positive samples. The HeNKF3 and SDLK3 were kidney samples. CQYY1, GDFS9, GDPY1, GDZS1, GXBH1, and HeNKF10 were spleen samples. Error bars represent the averages and standard deviations from at least three independent measurements.

Figure 3.

Schematic representation of genomic organization of PBRV genome segments A (A) and B (B) in comparison with those of the four genera. Nine PBRV strains have the same genome organization and the same ORF sizes. PBRV: GDFS9-2018 (MZ080605/MZ080606); IBDV: infectious bursal disease virus (UK661, NC004178/AJ318897) in Avibirnavirus genus; IPNV: infectious pancreatic necrosis virus (WestBuxton, AF078668/AF078669) in Aquabirnavirus genus; BSNV: blotched snakehead virus (BSNV, NC005982/NC005983) in Blosnavirus genus; DXV: Drosophila X virus (DXV, U60650/AF196645) in Entomobirnavirus genus.

Figure 4.

Phylogenetic trees of PBRV strains. Construction of the trees was based on full preVP2 (A) and VP1 (B) genes of nine PBRV strains using MEGA v7.0 with 1,000 repetitions of Bootstrap, and the best fit models for VP1 and preVP2 trees are GTR + G.

Figure 5.

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Figure 6.

Phylogenetic trees of the representatives within the Birnaviridae family. The phylogenetic analysis based on full VP1 (A) and preVP2 (B) genes of PBRVs and other reference strains of four birnavirus genera retrieved from GenBank was performed with MEGA v7.0, and the best fit models for VP1 and VP2 trees are GTR + G.