Recombination between non-structural and structural genes as a mechanism of selection in lagoviruses: The evolutionary dead-end of an RHDV2 isolated from European hare

Abstract

The genus Lagovirus, belonging to the family Caliciviridae, emerged around the 1980s. It includes highly pathogenic species, rabbit hemorrhagic disease virus (RHDV/GI.1) and European brown hare syndrome virus (EBHSV/GII.1), which cause fatal hepatitis, and nonpathogenic viruses with enteric tropism, rabbit calicivirus (RCV/GI.3,4) and hare calicivirus (HaCV/GII.2). Lagoviruses have evolved along two independent genetic lineages: GI (RHDV and RCV) in rabbits and GII (EBHSV and HaCV) in hares. To be emphasized is that genomes of lagoviruses, like other caliciviruses, are highly conserved at RdRp-VP60 junctions, favoring intergenotypic recombination events at this point. The recombination between an RCV (genotype GI.3), donor of non-structural (NS) genes, and an unknown virus, donor of structural (S) genes, likely led to the emergence of a new lagovirus in the European rabbit, called RHDV type 2 (GI.2), identified in Europe in 2010. New RHDV2 intergenotypic recombinants isolated in rabbits in Europe and Australia originated from similar events between RHDV2 (GI.2) and RHDV (GI.1) or RCV (GI.3,4). RHDV2 (GI.2) rapidly spread worldwide, replacing RHDV and showing several lagomorph species as secondary hosts. The recombination events in RHDV2 viruses have led to a number of viruses with very different combinations of NS and S genes. Recombinant RHDV2 with NS genes from hare lineage (GII) was recently identified in the European hare. This study investigated the first RHDV2 (GI.2) identified in Italy in European hare (RHDV2_Bg12), demonstrating that it was a new virus that originated from the recombination between RHDV2, as an S-gene donor and a hare lagovirus, not yet identified but presumably nonpathogenic, as an NS gene donor. When rabbits were inoculated with RHDV2_Bg12, neither deaths nor seroconversions were recorded, demonstrating that RHDV2_Bg12 cannot infect the rabbit. Furthermore, despite intensive and continuous field surveillance, RHDV2_Bg12 has never again been identified in either hares or rabbits in Italy or elsewhere. This result showed that the host specificity of lagoviruses can depend not only on S genes, as expected until today, but potentially also on some species-specific NS gene sequences. Therefore, because RHDV2 (GI.2) infects several lagomorphs, which in turn probably harbor several specific nonpathogenic lagoviruses, the possibility of new speciation, especially in those other than rabbits, is real. RHDV2 Bg_12 demonstrated this, although the attempt apparently failed.

Keywords: Animal experiments; Lagovirus; RHDV2; Recombination; Virus selection.

Fig. 1

Schematic representation of the experimental trials. A) First preliminary trial; B) Second trial. On the horizontal bars are indicated the days since the beginning of the trial (blood sampling at −3 days before 1st infection) to the end of the experiments (respectively 49 and 33 p.i.). Time points for blood samplings and the different infections are indicated, also including the number and identification of each infected rabbit, route of infection, dose and type of inoculum. The dot lines and the upside-down rabbit figures at days 43 and 45 p.i. indicate respectively in Section A) the death of rabbits #87 and #90 after challenge with the Spanish isolate RHDV2_Bcn14, and in Section B) the death of rabbits #91 and #92 after challenge with RHDV2_TA14.

Fig. 2

Maximum Likelihood (ML) phylogenetic trees performed for: A) the structural genes VP60 (n = 142 sequences; nucleotides 5240–6869; nucleotide substitutions model GTR+G + I) and B) the non-structural genes (n = 142 sequences; nucleotides 1–5239; nucleotide substitutions model GTR+G + I). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Support for each cluster was obtained from 1.000 bootstrap replicates. Bootstrap values >60% are shown. Genotype clusters, which do not include virus variants sequenced in this study, were collapsed and annotated accordingly.

Fig. 3

Similarity plot of the RHDV2_Bg12 recombinant strain. The horizontal axis represents the nucleotide position of the genome, whereas the vertical axis represents the similarity to the two putative parental strains. The black plot indicates the closest major parent, EBHSV strain KC832839, donor of the NS genes, whereas the gray plot indicates RHDV2_N11 KM87868 strain, the closest minor parent, donor of the S gene. A window size of 200 bp with a step size of 20 bp was used.

Fig. 4

Summary of the serological results of the first experimental trial. Titres on the ordinate are expressed as Log2, and the dot-line represents the threshold of positivity (Log2 = 3.3). On the abscissa, the days post the beginning of the trial are reported (T=). Infections were performed as indicated in the upper section of the figure in correspondence to different time points. The upside-down rabbit figures indicate the death of rabbits #87 and #90, respectively, at days 43 and 45 p.i. due to RHD.

Fig. 5

RT-PCR amplification of a portion of p37 and VP60 genes [WOAH, 2023]. RNA amplification from hare liver homogenates used as inoculum for infection (lane 1: Hare_RHDV2_Bg12 and lane 2: Hare_RHDV2_Bcn14) and rabbit liver homogenates experimentally infected (lane 3: rabbit #87 dead at 3 days p.i. and lane 4: rabbit #90 dead at 5 days p.i.). NTC, not-template control; MW, S100 molecular weight markers.

Fig. 6

Serological results of the second experimental trial. On the ordinate, the titres, expressed as Log2, for RHDV2 antibodies detected by c-ELISA and IgM ELISA. The dot-line represents the threshold of positivity (Log2 = 3.3). On the abscissae, the days post the beginning of the trial are reported (T=). Infections were performed as indicated in the upper section of the figure in correspondence to the different time points. RHD indicates the death with clinical signs and lesions consistent with RHD of rabbits #91 and #92, respectively, at days 26 and 27 p.i..

Fig. 7

ELISA sandwich performed with two RHDV2-specific mAbs on liver homogenates. The liver of a rabbit that died of acute RHD , the liver of the Hare_Bcn14 , and the liver of the Hare_Bg12 . Abscissae indicate the inverse of the dilutions of the homogenates (40 means 1/40), whereas ordinates indicate the OD values (absorbance 492 nm = A492).

Fig. 8

(A) Western blotting was performed on liver homogenates using mAb 5G3, recognizing a continuous epitope on the C-terminal half of the VP60 of lagoviruses. Lane 1, RHDV2_Ud11; lane 2, RHDV2_Bg12; lane 3, RHDV2_Bcn14. The samples were diluted at 1:300 (no. 1) and 1:30 (nos. 2 and 3). (B) nsEM of lagovirus particles detected in liver homogenates identified in (1) rabbits in Italy during the first RHDV2 outbreak (RHDV2_Ud11), (2) the hare from Italy (RHDV2_Bg12), and (3) the hare from Spain (RHDV2_Bcn14), respectively. NaPt 2%, TEM FEI Tecnai G2 Spirit operating at 85 kV. Bars, 200 nm.