Arctic-like Rabies Virus, Bangladesh.
Subject: Disease transmission
Rabies
Authors: Jamil, Khondoker Mahbuba
Ahmed, Kamruddin
Hossain, Moazzem
Matsumoto, Takashi
Ali, Mohammad Azmat
Hossain, Sohrab
Hossain, Shakhawat
Islam, Aminul
Nasiruddin, Mohammad
Nishizono, Akira
Pub Date: 12/01/2012
Publication: Name: Emerging Infectious Diseases Publisher: U.S. National Center for Infectious Diseases Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2012 U.S. National Center for Infectious Diseases ISSN: 1080-6040
Issue: Date: Dec, 2012 Source Volume: 18 Source Issue: 12
Accession Number: 313345653
Full Text: Rabies virus causes severe encephalitis in a wide range of mammals, including humans. Conservative estimates suggest that 55,000 persons worldwide die of rabies each year (7). Although the case-fatality rate in humans is 100%, rabies is preventable by vaccination. Bangladesh has the world's third highest death rate for human rabies, an estimated 2,100 deaths per year (2). Dogs are the main reservoir of the virus and are responsible for spillover infections in humans (2). Therefore, dogs should be the principal target for successful rabies elimination.

With political will and solid global epidemiologic information, rabies elimination is possible. Molecular typing of circulating rabies viruses is necessary to identify and develop effective control measures, and to understand the spread of certain rabies virus variants and their incursion into new regions (3). For rabies elimination, this knowledge is needed for establishing cooperative approaches between neighboring countries to which the disease is endemic.

Bangladesh is one of several countries in which no molecular study has been conducted to identify types of rabies virus circulating within its boundaries. A lack of knowledge of phylogenetic relationships of Bangladesh rabies virus with viruses in other countries continues to hinder coordinated rabies control efforts in the region. This study was conducted to characterize rabies virus circulating in Bangladesh and to determine its relationship with viruses in neighboring countries to clarify its epidemiologic relationships, origin, and transmission dynamics.

The Study

Seven brain samples were collected from animals with suspected rabies in 3 districts of Bangladesh (Dhaka, Narayanganj, and Narshingdi) in 2010 (Table 1). A portion of brainstem was removed from each sample and preserved in TRizol (Invitrogen, Carlsbad, CA, USA) at -20[degrees]C. Total RNA was extracted from brain homogenate, cDNA was synthesized by using random hexamer primers, reverse transcription PCR was conducted to amplify gene fragments, and nucleotide sequencing of genes was performed (4).

Full-length nucleoprotein (N) and glycoprotein (G) gene sequences from samples were determined. Nucleotide identities of N and G genes were 98%-100%. Amino acid identities of N and G genes were 100% and 98%-100%, respectively. Complete genomic sequencing (11,928 nt) of strain BDR5 was also conducted.

Evolutionary analysis was performed by using fulllength N gene. We created a maximum clade credibility phylogenetic tree using the Bayesian Markov chain Monte Carlo method available in BEAST version 1.6.1 (5). Analysis was conducted by using a relaxed (uncorrelated lognormal) molecular clock and a generalized time reversible + [GAMMA] + proportion invariant model (6). All chains were run for 90 million generations and sampled every 3,000 steps and an effective sample size >1,383 was obtained for all estimated parameters. Posterior densities were calculated with 10% burn-in and checked for convergence by using Tracer version 1.5 in BEAST.

The mean rate of nucleotide substitution estimated for the N gene was 2.3 x [10.sup.4] substitutions/site/year (95% highest posterior density [HPD] 1.4-3.1 x [10.sup.4] substitutions/ site/year). This rate is consistent with that of a previous study (7). The phylogenetic tree showed that rabies viruses in Bangladesh belong to Arctic/Arctic-like group 2 (AAL2) (3) also known as Arctic-like-1 (8), in close association with the strain from Bhutan.

Approximately 397.0 years ago (95% HPD 273.5-589.5 years), AAL and cosmopolitan rabies virus segregated from their most recent common ancestor (Figure 1). Approximately 225.6 years ago (95% HPD 157.4-324.2 years), AAL3 segregated. Approximately 187.4 years ago (95% HPD 129.0-271.9 years), AAL1 and AAL2 segregated. The AAL2 clade had a common progenitor that circulated -133.1 years ago (95% HPD 91.3-193.4 years), which has evolved into several different lineages. One lineage evolved 91.5 years ago (95% HPD 63.1-132.2 years) and currently circulates in Bangladesh, India, and Bhutan. Separate linages circulate in others countries in this region, including Iran, Nepal, Pakistan, and Afghanistan. AAL2 spread into central Bangladesh 32.3 years ago (95% HPD 18.4-50.6 years) in -1978 (95% HPD range 1958-1991).

Compared with the AAL2 strain from India (AY956319), BDR5 had several amino acid substitutions (Table 2). Sizes of their 2 genomes, leader RNA, trailer RNA, and intergenic regions were similar. The 7V-glycosylation site was predicted by using the NetNGlyc 1.0 server (www.cbs.dtu.dk/server/netnglyc). With the exception of BDR6, the G gene of all strains had potential glycosylation sites at position 37, 146, and 319.

Conclusions

Genetic analysis and phylogenetic studies can contribute to understanding the epidemiology of rabies virus in disease-endemic countries. Molecular analysis of animal rabies viruses showed that AAL2 appeared in central Bangladesh only 32 years ago. A close association between N genes sequences from rabies viruses in Bangladesh and Bhutan indicates that they originated from a common ancestor. If one considers the ease of human movement between countries, AAL2 most likely entered Bangladesh from India rather than from Bhutan.

[FIGURE 1 OMITTED]

Circumstantial evidence suggests that rabies virus spread from India to Bhutan (9). AAL2 circulates in many states of India. It has spread into southern India and has replaced older strains (10,11). It is likely that AAL2 is also circulating in states of India that are between Bhutan and Bangladesh. Estimated time of AAL2 spread is based on 7 samples that are representative of central Bangladesh (Figure 2). Therefore, further surveillance might identify the extent to which AAL2 has spread and the diversity of rabies viruses in other parts of Bangladesh that might alter the estimated date of spread. It has been reported that arctic rabies virus and other variants can co-circulate in the same region (12).

The G protein is the major factor responsible for the pathogenesis of rabies virus and contains 2 glycosylation sites (13). The G protein of strains from Bangladesh uniquely evolved to contain 3 potential glycosylation sites, which has been reported in only fixed (laboratory adapted) strains and proposed to be responsible for their reduced pathogenicity (13). However, the site for additional glycosylation differs between Bangladeshi and fixed strains. Detection of an additional glycosylation site and amino acid substitutions deserve further investigations.

[FIGURE 2 OMITTED]

AAL viruses could have moved southward from Siberia or other northern regions of the former Soviet Union into Nepal, India, and other countries in Asia by a species jump from fox to dog at some point (3). Another possibility is that AAL viruses first emerged in dogs in southern Asia and subsequently spread to northern climes, where they are now maintained in fox populations (3,8). Extensive surveillance of viruses from Iran, Iraq, Afghanistan, and countries north of them is necessary to determine the origin and spread pattern of AAL rabies virus.

The timeline of divergence of different lineages determined in this study was similar to that previously reported (8). That study and our study used the full-length N gene to determine the time of divergence. Another study reported the timeline of divergence as a more recent event (74). This study used partial sequences of N genes, which might be responsible for different results. Rabies virus from Nepal also belongs to AAL2, and as reported in a previous study (75), seemed to be forming a different lineage. However, the speculation was not supported by a significant a posterior density value (0.6355). Thus, a network of countries is urgently needed to exchange information on molecular typing of circulating strains of rabies virus that might be useful in controlling rabies in this region.

This study was supported in part by a Grant-in-Aid Scientific Research B from the Japan Society for the Promotion of Sciences (grant 20406026) and the Research Fund at the Discretion of the President, Oita University (grant 610000-N5010).

Dr Jamil is a physician and virologist in the Department of Virology, Institute of Epidemiology, Disease Control, and Research, Dhaka, Bangladesh. Her research interests are virology and molecular epidemiology of rabies virus.

Author affiliations: Institute of Epidemiology, Disease Control, and Research, Dhaka, Bangladesh (K.M. Jamil); Oita University, Oita, Japan (K. Ahmed, T. Matsumoto, A. Nishizono); Ministry of Health and Family Welfare, Dhaka (M. Hossain); Dhaka City Corporation, Dhaka (M.A. Ali, S. Hossain, A. Islam, M. Nasiruddin); and Tongi Municipality, Tongi, Bangladesh (S. Hossain)

DOI: 10.3201/eid1812.120061

References

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(2.) Hossain M, Ahmed K, Bulbul T, Hossain S, Rahman A, Biswas MN, et al. Human rabies in rural Bangladesh.. Epidemiol Infect. 2011;140:1-8. http://dx.doi.org/10.1017/S095026881100272X

(3.) Nadin-Davis SA, Turner G, Paul JP, Madhusudana SN, Wandeler AI. Emergence of Arctic-like rabies lineage in India. Emerg Infect Dis. 2007;13:111-6. http://dx.doi.org/10.3201/eid1301.060702

(4.) Matsumoto T, Ahmed K, Wimalaratne O, Yamada K, Nanayakkara S, Perera D, et al. Whole-genome analysis of a human rabies virus from Sri Lanka. Arch Virol. 2011;156:659-69. http://dx.doi. org/10.1007/s00705-010-0905-8

(5.) Drummond AJ, Rambaut A. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol Biol. 2007;7:214. http:// dx.doi.org/10.1186/1471-2148-7-214

(6.) Posada D, Crandall KA. MODELTEST: testing the model of DNA substitution. Bioinformatics. 1998;14:817-8. http://dx.doi. org/10.1093/bioinformatics/14.9.817

(7.) Bourhy H, Reynes JM, Dunham EJ, Dacheux L, Larrous F, Huong VT, et al. The origin and phylogeography of dog rabies virus. J Gen Virol. 2008;89:2673-81. http://dx.doi.org/10.1099/ vir.0.2008/003913-0

(8.) Kuzmin IV, Hughes GJ, Botvinkin AD, Gribencha SG, Rupprecht CE. Arctic and Arctic-like rabies viruses: distribution, phylogeny and evolutionary history. Epidemiol Infect. 2008;136:509-19. http:// dx.doi.org/10.1017/S095026880700903X

(9.) Tenzin, Dhand NK, Dorjee J, Ward MP. Re-emergence of rabies in dogs and other domestic animals in eastern Bhutan, 2005-2007. Epidemiol Infect. 2011;139:220-5. http://dx.doi.org/10.1017/ S0950268810001135

(10.) Nagarajan T, Nagendrakumar SB, Mohanasubramanian B, Rajalakshmi S, Hanumantha NR, Ramya R, et al. Phylogenetic analysis of nucleoprotein gene of dog rabies virus isolates from southern India. Infect Genet Evol. 2009;9:976-82. http://dx.doi. org/10.1016/j.meegid.2009.04.004

(11.) Reddy GB, Singh R, Singh RP, Singh KP, Gupta PK, Desai A, et al. Molecular characterization of Indian rabies virus isolates by partial sequencing of nucleoprotein (N) and phosphoprotein (P) genes. Virus Genes. 2011;43:13-7. http://dx.doi.org/10.1007/s11262-011 0601-0

(12.) Mansfield KL, Racloz V, McElhinney LM, Marston DA, Johnson N, Ronsholt L, et al. Molecular epidemiological study of Arctic rabies virus isolates from Greenland and comparison with isolates from throughout the Arctic and Baltic regions. Virus Res. 2006;116:1-10. http://dx.doi.org/10.1016/j.virusres.2005.08.007

(13.) Yamada K, Park CH, Noguchi K, Kojima D, Kubo T, Komiya N, et al. Serial passage of a street rabies virus in mouse neuroblastoma cells resulted in attenuation: Potential role of the additional N-glycosylation of a viral glycoprotein in the reduced pathogenicity of street rabies virus. Virus Res. 2012;165:34-45. http://dx.doi. org/10.1016/j.virusres.2012.01.002

(14.) Nadin-Davis SA, Sheen M, Wandeler AI. Recent emergence of the Arctic rabies virus lineage. Virus Res. 2012;163:352-62. http:// dx.doi.org/10.1016/j.virusres.2011.10.026

(15.) Pant GR, Horton DL, Dahal M, Rai JN, Ide S, Leech S, et al. Characterization of rabies virus from a human case in Nepal. Arch Virol. 2011;156:681-4. http://dx.doi.org/10.1007/s00705-0100868-9

Address for correspondence: Kamruddin Ahmed, Research Promotion Institute, Oita University, Yufu 879-5593, Oita, Japan; email: ahmed@ oita-u.ac.jp
Table 1. Characteristics of 7 animal samples tested for
rabies virus, Bangladesh

Sample         Animal              Age, y
no.

BDR1            Dog               Unknown

BDR2            Cow                  8

BDR3            Cow                  10

BDR4            Goat                 3

BDR5            Goat                 2

BDR6            Cow                  6
BDR7            Cow                  5

Sample        District            History
no.

BDR1           Dhaka              Unknown

BDR2         Narsingdi          Calf died of
                              suspected rabies
                                1 wk earlier
BDR3           Dhaka              Unknown

BDR4        Narayanganj       Dog bite 2.5 mo
                                  earlier
BDR5        Narayanganj      Dog bite to head 2
                                 mo earlier
BDR6           Dhaka              Unknown
BDR7        Narayanganj        Dog bite 2 mo
                                  earlier

Sample    Signs and symptoms
no.

BDR1      Angry, biting tendency,
          excessive salivation, gradually
          became drowsy
BDR2      Angry, salivation, drooping of
          tongue, inability to drink or eat

BDR3      Angry, salivation, frequent
          micturition, inability to drink or
BDR4      Angry, inability to eat and drink,
          biting tendency
BDR5      Angry, salivation, inability to eat
          and drink
BDR6      Angry, salivation, trying to attack
BDR7      Angry, salivation, trying to attack

              GenBank
Sample       accession
no.            no. *

BDR1       Not determined

BDR2          AB699208

BDR3          AB699209

BDR4          AB699210

BDR5      AB699220 (whole
              genome)
BDR6          AB699212
BDR7          AB699213

* For glycoprotein gene.

Table 2. Substitutions in genome sequence of rabies
virus BDR5 from Bangladesh compared with genome
sequence of strain from India (AY956319), 2010 *

  Protein, amino acid                            Site/domain/region
  substitution                                   of protein ([dagger])

N
  [Asp.sub.378] [right arrow] [Glu.sub.378]      Antigenic site IV
  [Gln.sub.422] [right arrow] [Arg.sub.422]      --
P [Ser.sub.64] [right arrow] [Pro.sub.64]        Variable domain I
  [Gln.sub.71] [right arrow] [Thr.sub.71]        Variable domain I
  [Asn.sub.90] [right arrow] [Ser.sub.90]        N protein binding site
                                                 in variable domain II
  [Pro.sub.159] [right arrow] [Ser.sub.159]      N protein binding site
                                                 in variable domain II
  [His.sub.162] [right arrow] [Ser.sub.162]      N protein binding site
                                                 in variable domain II
  [Asn.sub.166] [right arrow] [Ser.sub.166]      N protein binding site
                                                 in variable domain II
  [Ala.sub.174] [right arrow] [Val.sub.174]      N protein binding site
M                                                in variable domain II
  [Leu.sub.21] [right arrow] [Pro.sub.21]        --
  [Ser.sub.46] [right arrow] [Gly.sub.46]        --
  [Ile.sub.168] [right arrow] [Val.sub.168]      --
G
  [Ala-.sub.(minus)15]                           Signal peptide
  [right arrow] [Val.sub.-15]
  [Val-.sub.(minus)6]                            Signal peptide
  [right arrow] [Phe.sub.-6]
  [Val.sub.7] [right arrow] [Ile.sub.7]          --
  [Asp.sub.146] [right arrow] [Asn.sub.146]      Putative additional
                                                 W-glycosylation: NKS
  [Val.sub.427] [right arrow] [Ile.sub.427]      --
  [Arg.sub.462] [right arrow] [Gly.sub.462]      Transmembrane domain
  [His.sub.465] [right arrow] [Arg.sub.465]      Transmembrane domain
  [Gly.sub.473] [right arrow] [Ser.sub.473]      Transmembrane domain
L
  [Asp.sub.18] [right arrow] [Glu.sub.18]        --
  [Ala.sub.19] [right arrow] [Thr.sub.19]        --
  [Arg.sub.315] [right arrow] [Lys.sub.315]      Conserved domain I
  [Val.sub.361] [right arrow] [Leu.sub.361]      Conserved domain I
  [His.sub.640] [right arrow] [Gln.sub.640]      Conserved domain III
  [Lys.sub.657] [right arrow] [Arg.sub.657]      Conserved domain III
  [Ala.sub.966] [right arrow] [Thr.sub.966]      Conserved domain IV
  [Pron.sub.33] [right arrow] [Sern.sub.33]      Conserved domain V
  [Arg.sub.1307] [right arrow] [Lys.sub.1307]    Conserved domain IV
  [Asp.sub.1373] [right arrow] [Gly.sub.1373]    --
  [Leu.sub.1626] [right arrow] [Val.sub.1626]    --
  [Leu.sub.1654] [right arrow] [Ser.sub.1654]    --
  [Val.sub.1755] [right arrow] [Ile.sub.1755]    --
  [Cys.sub.1825] [right arrow] [Tyr.sub.1825]    --
  [Asn.sub.1841] [right arrow] [Lys.sub.1841]    --
  [Gln.sub.1845] [right arrow] [His.sub.1845]    --
  [Cys.sub.1872] [right arrow] [Phe.sub.1872]    --
  [Asn.sub.2091] [right arrow] [Ser.sub.2091]    --

* N, nucleoprotein; P, phosphoprotein; M, matrix protein; G,
glycoprotein; L, polymerase.

([dagger]) - indicates that the amino acid substitution
was in a location that has no site/domain/region name.
NKS, asparagine-lysine-serine.
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