Evidence of two distinct phylogenetic lineages of dog rabies virus circulating in Cambodia

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Introduction
The developing world is severely affected by rabies, causing deaths in humans as well as in animals and resulting in significant economic losses. Indeed, it is estimated that globally canine rabies causes approximately 59,000 human deaths, over 3.7 million disability-adjusted life years (DALYs) and 8.6 billion USD in economic losses annually (Hampson et al., 2015).
Rabies virus (RABV) belongs to the family Rhabdoviridae of the order Mononegavirales which comprises at least 10 genera (http://www.ictvonline.org). The Lyssavirus genus includes classical RABVs, found in a wide range of different animal species throughout the world. All 13 other species of Lyssavirus except two have been isolated from bats, originating from different locations in Europe, Asia, Africa and Australia. RABV has a negative-sense RNA genome of about 12 kb encoding five proteins (Tordo et al., 1986(Tordo et al., , 1993. The nucleoprotein gene (N-gene) is widely used for molecular characterization and phylogenetic analysis of RABVs (Kissi et al., 1995). The large number of N-gene sequences published makes this an obvious region of the genome to study (Johnson et al., 2002, Chupin et al., 2013. The only reliable data on human and animal rabies in Cambodia are available from the Institut Pasteur in Cambodia (IPC) where rabies diagnosis activity has been performed routinely since 1998, but laboratory-confirmed cases of rabies in dogs were recorded since the 1970s (Reynes et al., 1999). In the second half of the 1970s, dogs which are the most important vector for human rabies in Asia (Dodet et al., 2001) almost disappeared from Cambodia because of starvation. However, dog numbers rebounded in subsequent decades and rabies consequently became a serious public health concern. During the mid-1980s, the number of patients who have sought medical assistance after a dog bite has been at least 4000 per year, but the number of reported rabies deaths has been very low, partly because of improved postexposure treatment delivery. Deaths occurring at home are usually not reported to health services. From 1982 to 1991 a total of 5437 animal bites were recorded and 51 human rabies deaths were reported  (Ito et al., 1999) and Laos (Ahmed et al., 2015) were conducted previously.
The objective of the present study was to perform a retrospective molecular and phylogenetic characterization of RABVs from dogs originating from different parts of Cambodia, to compare them to strains originating from other South-East Asian countries and to analyze the circulation of dog RABVs in this region.  Table 1). Dog's heads were usually referred to the IPC lab from people who were consulting for PEP following the animal bite. The animal samples were tested by a standard direct fluorescent antibody test (FAT) (Dean et al., 1996) using an anti-rabies nucleocapsid conjugate (Anti-Rabies Nucleocapsid Conjugate; lyophilizied, adsorbed #357-2112, Bio-Rad, Marnesla-Coquette, France) according to the manufacturer's instructions.  (Table 1) were conducted using the same reaction conditions and cycling protocols as previously described . The first PCR, using the primers RVN- The analysis involved 192 nucleotide sequences, obtained in the present study (Supplementary Table 1) and retrieved from public databases (Supplementary Table 2).

Samples and Viruses.
For the partial N-gene, sequences were analyzed as above with some modifications. The evolutionary history was inferred using the Minimum Evolution (ME) method (Rzhetsky & Nei, 1992).
The 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 number of differences method (Nei & Kumar, 2000) and are in the units of the number of base differences per sequence. The ME tree was searched using the Close-Neighbor-Interchange (CNI) algorithm (Nei & Kumar, 2000) at a search level of 1. The Neighbor-Joining algorithm (Saitou & Nei, 1987) was used to generate the initial tree.

Sampling. The geographical origins of the Cambodian dog samples analyzed in this study
are shown in Figure 1. As there was no information on the exact location where the bite occurred, the cases were attached to the local district health center, which in Cambodia is usually located no more than 5 kilometers from the furthest point of the district.

Phylogenetic analyses.
The complete N-gene sequences of 149 RABV isolates collected in 20 provinces of Cambodia were generated and submitted to GenBank (Supplementary Table 1).
The Model Selection analysis with MEGA6 package revealed that for the dataset of entire and partial nucleoprotein sequences included in the present study, the Tamura

Discussion
Domestic/stray dog rabies is still widespread throughout the world, occurring in over 80 countries/territories (predominantly in the developing world) and is implicated in more than 99% of all cases of human rabies (WHO, 2012). The present study aimed to provide greater clarity on dog rabies molecular epidemiology in South-East Asian countries using the sequences generated here in addition to the limited number of sequence data publicly available. More specific attention was paid to Cambodia, which is severely affected by rabies but where the epidemiology of the disease remains unclear.
Currently, there is no active rabies surveillance system in Cambodia, and the existing data is only available through the rabies diagnosis efforts and the PEP treatment activities of the IPC. As such, reliable analysis of the precise dynamics of dog rabies in Cambodia by year cannot be performed, but the available data confirms that the disease exists and is widespread throughout the country.

Figure 2. Evolutionary relationships of South-East Asian rabies viruses based on complete N-gene sequences.
The tree was built using Maximum Likelihood method based on Tamura 3-parameter method (Tamura, 1992) to compute the evolutionary distances. The tree with the highest log likelihood (-7633.4158) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 192 nucleotide sequences. There were a total of 1346 positions in the final dataset. Bootstrap values lower than 70 are hidden. The estimated TMRCA are indicated next to each node. Branches are color-coded by country of origin: dark blue for Cambodia, pink for Vietnam, red for Thailand, green for Laos, sky-blue for Myanmar and purple for China. The horizontal axis is shown as the number of years from the most recent sample. B. Population dynamics of rabies viruses (complete coding region of nucleoprotein gene sequences). The Bayesian skyline plot shows changes in relative genetic diversity (X axis) and time (Y axis) is shown as the number of years from the most recent sample. The black solid line represents the mean estimate of Neτ, while the 95% HPD intervals are shown in grey solid line. RABV sequence JN786877was excluded from this analysis (absence of information regarding the date of isolation).  Figure 1. Evolutionary relationships of rabies viruses from South-East Asia based on partial N-gene sequences. The tree was built using Minimum Evolution method (Rzhetsky & Nei, 1992). The optimal tree with the sum of branch length = 304.71612549 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (Felsenstein, 1985). Boot strap values lower 70 are hidden. There were a total of 381 positions in the final dataset.