Genetic Characteristics and Phylogeographic Dynamics of Lagoviruses, 1988-2021

Abstract

Rabbit haemorrhagic disease virus (RHDV), European brown hare syndrome virus (EBHSV), rabbit calicivirus (RCV), and hare calicivirus (HaCV) belong to the genus Lagovirus of the Caliciviridae family that causes severe diseases in rabbits and several hare (Lepus) species. Previously, Lagoviruses were classified into two genogroups, e.g., GI (RHDVs and RCVs) and GII (EBHSV and HaCV) based on partial genomes, e.g., VP60 coding sequences. Herein, we provide a robust phylogenetic classification of all the Lagovirus strains based on full-length genomes, grouping all the available 240 strains identified between 1988 and 2021 into four distinct clades, e.g., GI.1 (classical RHDV), GI.2 (RHDV2), HaCV/EBHSV, and RCV, where the GI.1 clade is further classified into four (GI.1a-d) and GI.2 into six sub-clades (GI.2a-f). Moreover, the phylogeographic analysis revealed that the EBHSV and HaCV strains share their ancestor with the GI.1, while the RCV shares with the GI.2. In addition, all 2020-2021 RHDV2 outbreak strains in the USA are connected to the strains from Canada and Germany, while RHDV strains isolated in Australia are connected with the USA-Germany haplotype RHDV strain. Furthermore, we identified six recombination events in the VP60, VP10, and RNA-dependent RNA polymerase (RdRp) coding regions using the full-length genomes. The amino acid variability analysis showed that the variability index exceeded the threshold of 1.00 in the ORF1-encoded polyprotein and ORF2-encoded VP10 protein, respectively, indicating significant amino acid drift with the emergence of new strains. The current study is an update of the phylogenetic and phylogeographic information of Lagoviruses that may be used to map the evolutionary history and provide hints for the genetic basis of their emergence and re-emergence.

Keywords: amino acid variation; evolution; lagoviruses; phylogenetics; phylogeographic analysis; recombination.

Figure 1

The phylogenetic tree based on the full-length genome sequences of Lagovirus strains, 1988–2021. The unrooted ML phylogenetic tree of 240 full-length Lagovirus genomes classifying all the strains into four major clades (GI.1/RHDV, GI.2/RHDV2, RCV, and HaCV/EBHSV). GI.1 can be further classified into four sub-clades (GI1a–d) and RHDV2/GI.2 into six sub-clades (GI.2a–f). The major clades and sub-clades of the RHDV are indicated. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are indicated at each node. The evolutionary distances were computed using the best-fit substitution model (SYM + I + G 4). The red color clades represent Germany (n = 69), green USA (n = 24), blue Poland (n = 22), and the yellow color represents Portugal (n = 13). The tree was visualized and modified to proportion using FigTree v1.4.

Figure 2

Similarity map of the full-length genome sequences of representative Lagoviruses, 1988–2021. (A) Schematic diagram of the Lagovirus complete genome structure. From 5′ end to 3′ end is polyprotein ORF1 that encodes for p16, p23, RNA-helicase, p29, VPg, Peptidase, RdRp, and VP60 and ORF2 that encodes for VP10. (B) SimPlot similarity analysis results using GS/YZ(China-2011-Deer) in GI1c (GenBank ID: MN478485.1) as the query sequence to compare with the fifteen other representative strains of Lagoviruses.

Figure 3

The phylogenetic tree based on VP60 coding sequences of Lagovirus strains, 1988–2021. An unrooted ML phylogenetic tree of 240 VP60 sequences of Lagoviruses classifying all the strains into four major clades (RCV, HaCV/EBHSV, GI.1/RHDV, and GI.2/RHDV2). The RHDVs within GI.1 and GI.2 can be further classified into four sub-clades each (GI.1a–d and GI.2a–d, respectively). The major clades and sub-clades of the Lagoviruses are indicated. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are indicated at each node. The evolutionary distances were computed using the best-fit substitution model (GTR + F + I + G4). The red color clades represent Germany (n = 69), green USA (n = 24), blue Poland (n = 22), and the yellow color represents Portugal (n = 13). The trees were constructed with IQ-TREE v1.6.12 and were visualized and modified to proportion using FigTree v1.4.

Figure 4

Phylogeographic network analysis of the full-length Lagovirus sequences, 1988–2021. The phylogenetic network of 240 full-length genomes of Lagovirus was inferred using the MSN network implemented by PopArt v1.7. The strains isolated in Germany dominate the network, where the 2020–2021 outbreak in USA evolved from a strain isolated in Germany in 2014 (GenBank ID: LR899186.1) and Canada, while the FRG-USA strain (GenBank ID: NC_001543.1) isolated in 2000 clustered as one haplotype with the FRG-Germany strain (GenBank ID: M67473.1) isolated during 1991, connecting all the strains isolated in Australia, three strains from Poland, two from New Zealand, and one strain from Italy. HaCV/ESBHV strains were connected to GI.1, while the RCV was connected to GI.2 strains of RHDVs. The distance of branches is proportional to the number of mutations. Each color represents a different country.

Figure 5

Genetic recombination analysis of 240 full-length Lagovirus genomes. (A) Diagram showing the full-length genome of Lagovirus and the corresponding regions encoding p16, p23, RNA-helicase, p29, VPg, Peptidase, RdRp, VP60, and VP10. The numbers indicate the nucleotide positions relative to the genome of RHDV strain HDV-FRG (Germany-1991) (GenBank ID: M67473.1). (B) Schematic representation of the six potential recombination events listed in Table 2. The serial number of the recombination events and the description of potential recombinants (GenBank ID: virus name/country-collection year) are shown on the left. The filled pink and gray blocks represent the DNA regions from minor and major parent viruses, respectively. The numbers on the top of filled green blocks indicate the nucleotide positions of breakpoints relative to the genome sequence of corresponding recombinant viruses on the left.

Figure 6

Amino acid variability landscape of full-length Lagovirus proteins, 1988–2021. The plot represents amino acid variations in ORF1-encoded polyprotein aa position (A) 1–750, (B) 751–1500, (C) 1501–2385, and (D) ORF2-encoded VP10. The ORF1 and ORF2 nucleotide sequences were used to acquire their consensus amino acid sequence using the Wu-Kabat variability coefficient implemented by PVS. Y axis represents the Wu-Kabat variability coefficient values, where the estimation limit is 1. Above the limit 1 represents variations. X axis represents the amino acid positions.