Southernmost Asia Is the Source of Japanese Encephalitis Virus (Genotype 1) Diversity from which the Viruses Disperse and Evolve throughout Asia

<div><p>Background</p><p>Although a previous study predicted that Japanese encephalitis virus (JEV) originated in the Malaysia/Indonesia region, the virus is known to circulate mainly on the Asian continent. However, there are no reported systematic studies that adequately define how JEV then dispersed throughout Asia.</p><p>Methodology/Principal Findings</p><p>In order to understand the mode of JEV dispersal throughout the entire Asian continent and the factors that determine the dispersal characteristics of JEV, a phylogenetic analysis using Bayesian Markov chain Monte Carlo simulations was conducted on all available JEV E gene sequences in GenBank, plus strains recently isolated in China. Here we demonstrate for the first time that JEV lineages can be divided into four endemic cycles, comprising southern Asia, eastern coastal Asia, western Asia, and central Asia. The isolation places of the viruses in each endemic cycle were geographically independent regardless of years, vectors, and hosts of isolation. Following further analysis, we propose that the southernmost region (Thailand, Vietnam, and Yunnan Province, China) was the source of JEV transmission to the Asian continent following its emergence. Three independent transmission routes from the south to north appear to define subsequent dispersal of JEV. Analysis of JEV population dynamics further supports these concepts.</p><p>Conclusions/Significance</p><p>These results and their interpretation provide new insights into our understanding of JEV evolution and dispersal and highlight its potential for introduction into non-endemic areas.</p></div

The spatiotemporal migration of Genotype 1 JEV since the 1970s.

<p>The different panels represent temporal projections of reconstructed migration events in last 40 years (1970, 1978, 1981, 1990, 2000, 2003, 2006 and 2010, respectively). The different colors, yellow, green, light blue, blue, dark blue, purple, orange, red, dark red demonstrates for 1970, 1978, 1981, 1990, 2000, 2003, 2006 and 2010, respectively. The panels only show the tendency of migration events or partial migration events that have occurred up to a particular date, assuming that the virus migrates at a constant rate over the inferred time span of the branch. Blue circles mark the hot point of migration events. AF, Afghanistan; PK, Pakistan; IN, India; NP, Nepal; BT, Bhutan; BG, Bangladesh; BM, Burma; TH, Thailand; LA, Laos; VN, Vietnam; KH, Cambodia; MY, Malaysia; ID, Indonesia; PP, Papua New Guinea; AU, Australia; KP, North Korea; KR, South Korea; JP, Japan. Chinese provinces: HLJ, Heilongjiang Province; JL, Jilin Province; LN, Liaoning Province; NM, Neimenggu; XJ, Xinjiang; BJ, Beijing, TJ, Tianjin; HeB, Hebei Province; SX, Shanxi Province; SaX, Shaanxi Province; GS, Gansu Province; QH, Qinghai Province; NX, Ningxia; SD, Shandong Province; SH, Shanghai; JS, Jiangsu Province; AH, Anhui Province; HeN, Henan Province; XZ, Xizang; ZJ, Zhejiang Province; JX, Jiangxi Province; HuB, Hubei Province; CQ, Chongqing; SC, Sichuan Province; HuN, Hunan Province; GZ, Guizhou Province; YN, Yunnan Province; FJ, Fujian Province; GD, Guangdong Province; GX, Guangxi; HN, Hainan; TW, Taiwan; MG, Mongolia.</p

Temporal distribution of JEV (genotype 1) in four endemic cycles (based on the time spots given by skyline plots).

<p>Temporal distribution of JEV (genotype 1) in four endemic cycles (based on the time spots given by skyline plots).</p

The regional distribution of JEV (genotype 1) isolates in four endemic cycles.

<p>Note: TMRCA, the most recent common ancestor; KH, Cambodia; TH, Thailand; VN, Vietnam; AU, Australia; S-MC, southern mainland China; E-MC, eastern mainland China; N-MC, northern mainland China; W-MC, western mainland China; TW, Taiwan; KR, South Korea; JP, Japan; IN, India.</p

Maximum clade credibility tree for E gene sequences of genotype 1 JEV.

<p>Seven clusters were identified and estimated TMRCAs of these clusters (with their 95% HPD values in parentheses) are shown. Four endemic cycles were grouped based on the geographical locations of JEV strains in each cluster. Red, blue, olive and turquoise were used to mark strains from southernmost Asia, eastern coastal Asia, western Asia, and central Asia, respectively. Posterior probability values of each cluster and endemic cycles were showed in the right of the nodes. In order to present all available sequences in the tree, black triangles were used to condense strains with the same isolation place and similar isolation times.</p

Bayesian skyline plots for genotype 1 JEV.

<p>Highlighted areas correspond to 95% HPD intervals.</p

Dispersal characteristics of genotype 1 JEV based on phylogenetic analysis.

<p>Gray shadow presents the location of JEV strains used in this study; emboldened circle represents southern Asia endemic cycle established in the JEV transmission source area; red circles presented as dashed lines, intact lines circle and square dot lines represent west Asia, central Asia and east coastal Asia JEV endemic cycles, respectively; blue boxes presented as dashed lines, intact lines and square dots represent bird migration routes in Asia and they coincide with JEV endemic cycles; green dotted lines illustrate the potential dispersal directions of JEV in the future. CN, China; JP, Japan; KR, South Korea; IN, India; VN, Vietnam; TH, Thailand; KH, Cambodia; MY, Malaysia; ID, Indonesia; AU, Australia.</p

Maximum likelihood tree of 211 RABV from China partial N gene sequences generated by PHYML.

<p>Bootstrap values are indicated at the main nodes. Most sequences are contained in clade I and clade II. Underlined provinces are from the southwest, provinces with a line above the name are from the east. New isolates are marked with a blue diamond, human isolates are marked with a green cross. Clade I shows statistically significant geographic subdivision (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001640#pntd.0001640.s004" target="_blank">Table S4</a>) according to east and southwest China with older sequences generally isolated from the southwest and sequences from the east only appearing in the younger subgroups. No such division appears in clade II. Subgroup II-A and II-C correspond to ferret badger samples (marked in red). A full list of sequences in each clade with background information is given in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001640#pntd.0001640.s001" target="_blank">Table S1</a>.</p

Evolutionary and transmission history of clade I and clade II.

<p>Bayesian skyline plots showing the evolutionary and transmission histories of a) clade I and b) clade II and their corresponding trees. (a) also shows the number of human rabies cases recorded by year (bottom) and (b) shows the skyline plot for clade I on the same time scale (insert bottom right). Clade I shows greater variation in genetic diversity compared to clade II. Although both clades show a drop in genetic diversity around 2003 ((a) blue arrow on left), this is not correlated to number of human cases as they were still increasing rapidly and didn't peak until 2007 (red arrow on right of (a)). However, the drop appears to coincide with the introduction of translocation events, (a) and (b) top, as at this time multiple events appear in the trees.</p

National Borders Effectively Halt the Spread of Rabies: The Current Rabies Epidemic in China Is Dislocated from Cases in Neighboring Countries

<div><p>China has seen a massive resurgence of rabies cases in the last 15 years with more than 25,000 human fatalities. Initial cases were reported in the southwest but are now reported in almost every province. There have been several phylogenetic investigations into the origin and spread of the virus within China but few reports investigating the impact of the epidemic on neighboring countries. We therefore collected nucleoprotein sequences from China and South East Asia and investigated their phylogenetic and phylogeographic relationship. Our results indicate that within South East Asia, isolates mainly cluster according to their geographic origin. We found evidence of sporadic exchange of strains between neighboring countries, but it appears that the major strain responsible for the current Chinese epidemic has not been exported. This suggests that national geographical boundaries and border controls are effective at halting the spread of rabies from China into adjacent regions. We further investigated the geographic structure of Chinese sequences and found that the current epidemic is dominated by variant strains that were likely present at low levels in previous domestic epidemics. We also identified epidemiological linkages between high incidence provinces consistent with observations based on surveillance data from human rabies cases.</p> </div