Isolation and Identification of a Novel Rabies Virus Lineage in China with Natural Recombinant Nucleoprotein Gene

<div><p>Rabies virus (RABV) causes severe neurological disease and death. As an important mechanism for generating genetic diversity in viruses, homologous recombination can lead to the emergence of novel virus strains with increased virulence and changed host tropism. However, it is still unclear whether recombination plays a role in the evolution of RABV. In this study, we isolated and sequenced four circulating RABV strains in China. Phylogenetic analyses identified a novel lineage of hybrid origin that comprises two different strains, J and CQ92. Analyses revealed that the virus 3′ untranslated region (UTR) and part of the <em>N</em> gene (approximate 500 nt in length) were likely derived from Chinese lineage I while the other part of the genomic sequence was homologous to Chinese lineage II. Our findings reveal that homologous recombination can occur naturally in the field and shape the genetic structure of RABV populations.</p> </div

Recombination confirmation table of different recombination analysis methods.

<p>Recombination confirmation table of different recombination analysis methods.</p

Analysis of the origin of the CQ92 lineage in different regions of the <i>N</i> gene delimited by the putative breakpoints.

<p>(A) A split tree inferred from the complete <i>N</i> gene sequence showing the evolutionary relationship. A networked pattern of mosaic lineage was found to be related to lineages GX4 and SH06. The Neighbor-Net tree was constructed by employing the SplitsTree4 program. (B) Phylogenetic relationships from positions 180 to 598 of CQ92 genome. (D) Phylogenetic relationship of the <i>N</i> gene fragment from positions 1–179 and 599–891. (D) Phylogenetic relationship of positions 892–1423. The evolutionary history of each fragment was inferred using the maximum likelihood method with the Kimura 2-parameter substitution model and Neighbor-Joining (NJ) method with the Maximum Composite Likelihood model. The percentage (>80%) of replicate ML trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown beside the branches. The NJ tree bootstrap values of the branch associated with recombinant strain are shown in parentheses. The tree is drawn to scale, with branch lengths shown in the same units as those of the evolutionary distances used to infer the phylogenetic tree. ▪, mosaics.</p

Analysis of recombination breakpoints.

<p>(A) The bootscan result of CQ92, GX4 and SH06 complete genome. RC-HL was used as the outgroup. The window size was set at 600 bp to avoid noise from gene mutations. (B) The bootscan result of region from positions 1–1423. The parameters used for analysis are shown on the bottom row of the figure. The window size was set at 300 bp. The three crossover sites are represented by vertical lines. (C) The statistical analysis of informative sites. Vertical lines represent the recombination breakpoints with the maximization of χ². χ² of each breakpoint and <i>P</i>-value of Fisher’s exact test are shown under the vertical lines.</p

The evolutionary history of rabies virus based on complete genome sequences inferred using the neighbor-joining method.

<p>The bootstrap consensus tree was inferred from 1000 replicates and was used to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in <50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The phylogenetic tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method and are in the units of the number of base substitutions per site.</p