Journal of Vaccines Research & Vaccination Category: Medical Type: Research Article

Selection of a Rabies Virus Strain as a New Candidate for Vaccine Development in Thailand

Nitatpattana N1*, Primsirikunawut A2, Buree S1, Chuapudee D2, Rodpai E1, Chaiyo K3, Chansiprasert K1, Nakgoi K3, Poolam K1, Lukebuo A2, Sangkitporn S2, Mandja BM4 and Gonzalez JP5
1 Center For Vaccine Development, Institute Of Molecular Biosciences, Mahidol University At Salaya Putthamonthon, Nakhon Pathom Province, Thailand
2 National Institute Of Health, Thailand Department Of Medical Science MOPH, Bangkok, Thailand
3 Government Pharmaceutical Organization Moph, Bangkok, Thailand
4 Microbiology Department, Faculty Of Medicine, Kinshasa University, Kinshasa, Democratic Republic Of Congo, Chrono-Environment Laboratory, UMR 6249 CNRS, Bourgogne Franche-Comté, University, Besançon, France
5 Georgetown University, School Of Medicine, Centaurus Biotech LLC. / Commonwealth Trading Partner, CTP INC. VA, United States

*Corresponding Author(s):
Nitatpattana N
Center For Vaccine Development, Institute Of Molecular Biosciences, Mahidol University At Salaya Putthamonthon, Nakhon Pathom Province, Thailand
Tel:+1 (301) 332 22 37,
Email:narong.nit@mahidol.ac.th

Received Date: Jul 20, 2020
Accepted Date: Aug 05, 2020
Published Date: Aug 16, 2020

Abstract

Rabies remains a severe viral zoonotic disease worldwide is always fatal once clinical symptoms appear in human and domestic animals. Furthermore, the Rabies virus is commonly transmitted to humans by infected domestic animals. There is no cure available and vaccination is the only way to prevent virus infection. The virus actively circulates in Thailand with several fatal cases and outbreaks in animal; mostly dogs have been recorded for the past decade. Additionally, Thailand import 2.5 Million doses of rabies vaccine yearly, the Thai National Vaccine Institute (Ministry of Public Health, MOPH) decided to initiate research and development on rabies vaccine to set up an effective vaccine to protect against actively circulating viral strains in it and the neighboring countries. The candidate vaccine aims to provide a local cost-effective production with a potential for international licensure.

The main objective of this study was to select a candidate vaccine rabies virus strain that comply with mandatory characteristics, accordingly to WHO’s regulations, including: the ability to efficiently growth in Vero cell line; having an Immunofluorescence companion test using monoclonal anti G protein for antigen detection,  and to be one of the most common strain genotype circulating in Thailand. With such objectives online, selected rabies strain genotype sequences were analyzed and characterized by their identity to the rabies virus strain actively circulating in Thailand. The identified Rabies virus TH2 strain is largely endemic throughout Thailand; it was recognized as a potential candidate for the Rabies vaccine development by showing all required and suitable characteristics. Although, the Rabies virus TH2 strain appears genetically distant from the commercially available vaccine strain in Thailand, it should stimulate immunity and prevent most of the rabies risk due to the actively circulating dog rabies strain in Thailand.

Keywords

Fix virus; Local strain; Rabies virus; Seed selection

INTRODUCTION

Rabies is an acute viral infection of the nervous system that presents both encephalitic and paralytic clinical forms. Rabies is transmitted by the rabies virus (Rabies lyssavirus) through saliva of infected mammal (e.g. dog, cat, and domestic ungulate), rarely the human to human transmission occurs, while exceptionally the virus can be transmitted through organ or tissue transplant [1]. The Rabies lyssavirus, is a negative sense, single-stranded enveloped RNA virus of the Rhabdoviridae family presenting in a typical cylindrical morphology in electron-micrograph. The genetic information of 11,615 to 11,966 pair base is packaged as a ribonucleoprotein complex tightly bound to the viral nucleoprotein while the genome encodes for five proteins including: nucleoprotein (N), glycoprotein (G), phosphoprotein (P), matrix protein (M), and polymerase (L) [2]. Although rabies is a well-known ancient disease, its virus appears endemic but particularly in low and medium-income countries (LMIC) while human rabies continues to be an important public health problem particularly in Asia and Africa [3]. The combined annual cost for both continents is estimated to be US$ 583.5 million yearly and mostly due to post exposure prophylaxis [4]. As it’s generally observed in Thailand and LMIC, the canine appears as the primary vector of rabies virus [5]. A human case of Rabies was first identified in Thailand in the 1920s and then in canines. While felines and stray animal populations grew, rabies cases also increased with a record high of 370 people death in 1980 [6]. After several outbreaks that occurred in the 1980s, canine and human rabies decreased due to an efficient post-exposure treatments, education, mas,  dog and cat vaccination campaign [6]. In 2018, Thailand presented one of its major rabies outbreaks spreading among 14 provinces ( Buri ram, Rayong, Songkhla, Surin, Trang, Nakhon Ratchasima, Prachuap Khiri khan, Phatthalung, Nong khai, Yasothon, Klalasin, Mukdahan, Tak, Surat Thani), with a mortality rate of 0.03 per 100,000 inhabitants and 17 fatal cases recorded [7]. In the meanwhile, 1,469 (15.8%) confirmed cases of rabies in animals (mostly dog and cattle) from 54 provinces where reported (Rabies Situation - 7 Oct 19 /2020 - Department of Livestock Development Ministry of Agriculture and Cooperatives). After this recent and unforeseen event, the Thai government decided to put an end to these surprising epidemic outbreaks, the cost of post exposure treatment and animal vaccination remain at a very high cost due to the need to import the anti-rabies vaccine from abroad. Therefore, the National Vaccine Institute (NVI, MOPH, and Thailand) asked the Center for Vaccine Development (Institute of Molecular Biosciences, Mahidol University) and the National Institute of Health (Thailand Department of Medical Science and MOPH) to investigate the potential for the development of a human vaccine using a vaccine strain complying with international regulation. The purpose of this study was to identify and select a rabies virus strain among the entire rabies virus isolated in Thailand and stored at the bio bank of the National Institute of Health (Thai Ministry of Medical Sciences, MOPH). These strains were chosen to exhibit specific and mandatory characteristics as recommended by the World Health Organization [8] and consistently having the ability to replicate in the Vero cell line. After this preliminary phase of identifying with high accuracy a targeted strain, the future development of inactivated human rabies vaccine will be completed under the direction of the MOPH and the Government Pharmaceutical Organization (GPO).

MATERIALS AND METHODS

Cell lines

Two cell lines commercially available from ATCC were selected and use respectively for virus amplification and virus replication effectiveness, including: a mouse cell line derived from mouse (Mus musculus) Neuroblastoma (MNA, ATCC CCL-131); and the Vero cell line derived from normal kidney cells of the adult African green monkey (Cercopithecus aethiops) (ATCC CCL-81).

Rabies virus

Fifty Rabies virus strains were provided by Thai National Institute of Health including thirteen strains from human and thirty-seven strains from canine’s brain. All the strains tested positive by Indirect Fluorescent Assay (IFA) using the FITC Anti-Rabies Monoclonal Globulin (Product number 800-092)

Indirect immunofluorescent antibody test (IFAT)

IFAT was performed using an anti-nucleoprotein monoclonal antibody, commercially available (FITC Anti-Rabies Monoclonal Globulin, Fujirebio US, Inc) following a classical previously described method [9].

Rabies virus Adaptation and Selection

The 50 rabies virus strains were propagated in Mouse Neuroblastoma (MNA) and passaged to time on this cell line at 37°C under 5% CO2. Propagated strains were then selected for their response by IFAT and, only 5 strains only showed a positivity ≥2+ by IFAT (undiluted cell suspension having ≥50% of fluorescent cell were considered as positive). The five selected strains were then propagated in MNA cell line for five or more passages and then used for the virus to be titrated by Reed and Muench method (fluorescence titration) and quantitative evaluated by “Nested RT-PCR” [10]. Ultimately, the five Rabies virus strains, showing a titer >104 Tissue Culture Infective Dose (TCID), were selected and propagated into Vero cell line kept at 5% CO2, 37 °C, and followed by 5 to 10 passages .

Deep sequencing

The RNA extraction was performed on an archived selected TH 13-42-07240 virus strains (i.e. TH2 clusters) sample isolated from canine brain in 2009 [11]. From a Vero cell infected monolayer, i.e. TH 13-42-07240 rabies virus strain tested positive by IFAT, viral RNA was extracted using QIAamp Viral RNA Mini Kit (QIAGEN, Germantown, MD). Total RNA was depleted of host genomic DNA and ribosomal RNA (rRNA) using NEBNext rRNA depletion kit (New England Biolabs, UK) following the manufacturer’s instructions, eluting the final product on in 30 ml molecular gram from the collection, previously de water. The depleted RNA was then purified to remove the enzyme using the RNeasy plus mini kit (QIAGEN, Germantown, MD), without DNase digestion and eluted in 30 ml of molecular grade water. cDNA was synthesized using Maxima H minus Double stranded cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA), following the manufacturer’s instructions.

Genotype sequence analysis:

Characterizing the viral genome, we used a random amplification, deep-sequencing approach (454 Sequencing) as described elsewhere, where 98% of reads matched the Rhabdovirus genome reference (Accession number EU293111) [12]. The multiple sequence alignment was based on Rhabdovirus genome reference strain 8743THA with the Rabies virus strain TH 13-42-07240 (TH2 clusters) and performed using Clustal Omega v1.2.0 [13] with default parameters. The DNA sequence variations between the Rabies virus strain 13-42-07240 sequences and the reference sequence (EU293111) were analyzed with the DnaSP software (version 5.10.1) to identify synonymous and non-synonymous sites.

Phylogenetic Analysis:

The trimmed original sequences were aligned with all Rabies virus sequences available from GenBank (May 2019) using Multiple Alignment of Fast Fourier Transform (MAFFT) [14]. Preliminary phylogenetic analyses were done using PhyML (maximum likelihood), implemented in Seaview Version 4, a multiplatform graphical user interface for sequence alignment and phylogenetic tree building [15]. Main phylogenetic analysis were performed on a subset of sequences, representing the overall genetic diversity of Rabies virus, under a Bayesian statistical framework implemented with BEAST package v.1.8.4 [16] using the model that fits best to the data accordingly with the corrected Bayesian Information Criterion (BICc) obtained in Jmodeltest2® [17].

RESULTS

Following the WHO’s recommendation, Rabies vaccine virus strain candidate should have a titer ≥6 log10 TCID50 (WHO, 2005). From the fifty rabies virus selected strains, after more than five passages in MNA cell and then passaged five to ten times in Vero cell line, only one strain, the Rabies virus strain TH 13-42-07240, was found positive by IFAT (4+, 100%), 1/50 (2%)  and replicates efficiently at first passage in Vero cell (Figure 1) and after the 6th passages, it showed a titer of 6 log10 TCID50 on day 4 (MOI 0.1) and 6.2 log10 TCID50 on day 3 (MOI 0.01) (Table1).

Day

Rabies virus strain TH 13-42-07240

 

Post-Infection

MOI 0.1

MOI 0.01

0

3*

2.1

1

3.6

2.8

2

4.6

4.3

3

5.6

6.2

4

6

5.6

5

5.8

5.4

6

5.5

5.3

7

5

5

Table 1: Replication kinetic of the Rabies virus strain TH 13-42-07240 from Thailand

Altogether, 251 strain sequences were used for phylogenic analysis of the G1 nucleoprotein gene and compared to the rabies virus strain TH 13-42-07240 (Figure F1).

Figure 1: Indirect Immuno-Fluorescent Test on Vero cells monolayer infected by the Rabies virus strain TH 13-42-07240 adapted to Vero ATCC ® CCL-81 cell line

Caption: the observed granular plasmatic fluorescence is characteristic of the monoclonal antibody used for specifically binding to the nucleoprotein product of the virus.

Also, the rabies virus strain TH 13-42-07240 was different from the commonly use Rabies virus strain 13-42-07240 vaccine (Table 2, Figure 2), the TH 13-42-07240 rabies virus strain isolated from a canine brain appears widely distributed throughout Thailand (Table S1). 

Gen Bank Acc. no.

Reference no.

Species

Origin

Year  

EU293121

8743THA

Human

Thailand

1983

EU293111

8764THA

Human

Thailand

1983

EU293115

9147FRA

Fox

France

1991

EU293113

9001FRA

Dog

French Guyana

1990

EU293116

9704ARG

Bat

Argentina

1997

AY705373

SHBRV-18

Bat

USA

1983

EF437215

NNV-RAB-H

Human

India

2006

EF206709

SAD B19

Vaccine

-

-

NC_001542

PV

Vaccine

France

1882

HQ317918

CTN-1-31

Human

China

1956

GQ918139

CVS-11

-

France

1882

EF206707

ERA

Vaccine

-

-

AB085828

HEP-Flury

Vaccine

USA

1939

AY956319

Serotype 1

Saliva

Germany

-

MN075931

TH13-42-07240

Dog

Thailand

1989

Table 2: Complete genome sequences of rabies virus used for genetic analysis

Figure 2: ML phylogeny of 15 selected Rabies virus strain sequences of the complete genome

Legend: Blue circle = the selected vaccine candidate strain TH 13 -42-07240

DISCUSSION

Our study, with respect to the animal, use for Rabies virus amplification for diagnosis appears clearly unnecessary when for the first time NMA cell line were used to efficiently amplify Rabies virus followed by its adaptation to Vero cell culture. With respect to the TH 13-42-07240 rabies virus strain characteristics, a Rabies virus strain comply with WHO recommendation (i.e Vero cell line optimal titer, and availability of the companion IFA test) appears to be a strong candidate for the development of inactivated rabies vaccine that should efficiently stimulated immunity to prevent rabies epidemic in Thailand [21,22]. Furthermore, the rabies virus strain TH 13-42-07240 appears to actively circulate in Thailand and China and has the potential to be used for Rabies vaccine development of interest for the region [18]. Although, it has some genetic distance with the other commercial vaccine in the market, as a local strain isolated in Thailand it has been proven to efficiently by neutralizing circulated rabies virus strain [19,20].

CONCLUSION

Rabies virus strain TH 13-42-07240 originally isolated from Thailand, comply with international recommendation for Rabies candidate vaccine development, it is one of the most abundant strains in the South Eastern Asian peninsula, and therefore appears as a preferred candidate for Rabies vaccine development in Thailand.

ACKNOWLEDGEMENT

Funding for this research study was provided by National Vaccine Institute, Ministry of Public Health. Thank you for staff of Center for Vaccine Development, Institute of Molecular Biosciences, Mahidol University at Salaya and National Institute of Health, Thailand Department of Medical Sciences, Ministry of Public Health. We want to express our gratitude to Ms. Souki Gonzalez for writing assistance, technical and language editing.

REFERENCES

  1. Lu XX, Zhu WY, Wu GZ (2018) Rabies virus transmission via solid organs or tissue allotransplantation. Infect Dis Poverty 7:82.
  2. Dietzgen RG, Kondo H, Goodin MM, Kurath G, Vasilakis N (2017) The family Rhabdoviridae: mono- and bipartite negative-sense RNA viruses with diverse genome organization and common evolutionary origins. Virus Res 227:158-70.
  3. Knobel DL, Cleaveland S, Coleman PG, Fevre EM, Meltzer MI (2005) Re-evaluating the burden of rabies in Africa and Asia. Bull World Health Organ 83:360–368.
  4. Jemberu WT, Molla W, Almaw G, Alemu S (2013) Incidence of rabies in humans and domestic animals and people's awareness in North Gondar Zone, Ethiopia. PLoS Negl Trop Dis 7:5
  5. Mitmoonpitak C, Wilde H, Tepsumetanon W (1997) Current status of animal rabies in Thailand. J Vet Med Sci 59: 457-460.
  6. Janchai (2016) Rabies and Situation in Thailand; 2018. Region 4-5 medical journal 2018; 37. Laboratory technique in rabies, 4th edition, World Health Organization, Geneva.
  7. Weekly Epidemiological Surveillance Report, Thailand. Bureau of Epidemiology, Department of Disease Control, Ministry of Public Health, 2019: 50.
  8. WHO Expert Committee on Rabies. World Health Organization Technical Report Series 709, World Health Organization, Geneva 1984.
  9. Meslin F.X, Kaplan M.H, Koprowski H (1996) Routine Laboratory Procedure in Laboratory Techniques in Rabies 4th Geneva. World Health Organization 55-122.
  10. Kamolvarin N, Tirawatnpong T, Rattanasiwamoke R, Tirawatnpong S, Panpanich T et al (1993) Diagnosis of rabies by polymerase chain reaction with nested primers. J Infect Dis 167:207-210.
  11. Narong Nitatpattana ,Yves Moné , Meriadeg AR Gouilh, Kumchol Chaiyo, Yutthana Joyjinda et al (2011) Genetic Diversity of Dengue-4 Virus Strains Isolated from Patients During a Single Outbreak of Dengue Fever, Thailand. J Fever 2:1009.
  12. Delmas O, Holmes EC, Talbi C, Larrous F, Dacheux L et al (2008) Genomic diversity and evolution of the Lyssaviruses.
  13. Tesh RB (1979) A method for the isolation and identification of dengue viruses, using mosquito cell cultures. Am J Tropical Med Hygiene 1053-1059.
  14. Boheemen S, De Graaf M, Lauber C, Bestebroer TM, Raj VS, et al (2012) Genomic Characterization of a Newly Discovered Coronavirus Associated with Acute Respiratory Distress Syndrome in Humans. mBio 3.
  15. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539.
  16. Katoh K, Standley DM (2013) MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol Biol Evol 30: 772-780.
  17. Gouy M, Guindon S, Gascuel O (2010) SeaView Version 4: A Multiplatform Graphical User Interface for Sequence Alignment and Phylogenetic Tree Building. Mol Biol Evol 27: 221-224.
  18. Denduangboripant J, Wacharapluesadee S, Lumlertdacha B, Ruankaew N, Hoonsuwan W et al (2005) Transmission dynamics of rabies virus in Thailand: Implications for disease control. BMC Infection Diseases 5:52.
  19. Aga AM, Mekonnen Y, Hurisa B, Tesfaye T, Lemma H, et al.(2014) In vivo and in vitro cross neutralization studies of local rabies virus isolates with ERA based cell culture anti-rabies vaccine produced in Ethiopia. J Vaccines 5:256.
  20. World Health Organization, WHO Technical Report Series 931, WHO 2005; Geneva. 1–87.
  21. Minamoto N, Tanaka H, Hoshida M, Goto H,  H Ito et al (1994) Linear and conformation-dependent antigenic sites on the nucleoprotein of rabies virus. Microbiol. Immunol 38:449-455
  22. Chan BK, Wilson T, Fischer KF, Kriesel JD. (2014) Deep sequencing to identify the cause of viral encephalitis.

SUPPLEMENTARY MATERIAL

 

Isolation no.

Species

Origin

Year

AY849022

5NBm

Dog

Nonthaburi

2001

AY849023

26NPpmt

Dog

Nakhon Pathom

2001

AY849024

53SPppd

Dog

Samut Prakan

1999

AY849025

67PTtyb

Dog

Pathum Thani

1999

AY849026

80PTllk

Dog

Pathum Thani

1999

AY849027

81NBtn

Dog

Nonthaburi

2001

AY849028

99PTkl

Dog

Pathum Thani

1999

AY849029

79PTm

Dog

Pathum Thani

1999

AY849030

125SSktb

Dog

Samut Sakhon

1999

AY849031

133SSm

Dog

Samut Sakhon

1999

AY849032

176SSktb

Dog

Samut Sakhon

1999

AY849033

187SSm

Dog

Samut Sakhon

1999

AY849034

207SSm

Dog

Samut Sakhon

1999

AY849035

217SSm

Dog

Samut Sakhon

1999

AY849036

218NBbk

Dog

Nonthaburi

2001

AY849037

222NBbbt

Dog

Nonthaburi

1999

AY849038

228NBtn

Dog

Nonthaburi

1999

AY849039

304NPbl

Dog

Nakhon Pathom

2001

AY849040

315NPm

Dog

Nakhon Pathom

2001

AY849041

324NPm

Dog

Nakhon Pathom

2001

AY849042

91PTns

Dog

Pathum Thani

1999

AY849043

95NBtn

Dog

Nonthaburi

1999

AY849044

108PTllk

Dog

Pathum Thani

1999

AY849045

112PTtyb

Dog

Pathum Thani

1999

AY849046

195NBby

Dog

Nonthaburi

1999

AY849047

215NPncs

Dog

Nakhon Pathom

1999

AY849048

232NPsp

Dog

Nakhon Pathom

1999

AY849049

235NBpk

Dog

Nonthaburi

1999

AY849050

263NPm

Dog

Nakhon Pathom

1999

AY849051

C267BKbkn

Cat

Bangkok

2000

AY849052

C269PTm

Cat

Pathum Thani

2000

AY849053

C271BKrtv

Cat

Bangkok

2001

AY849054

C274BKdd

Cat

Bangkok

1999

AY849055

C276BKpv

Cat

Bangkok

1999

AY849056

C277BKkt

Cat

Bangkok

1999

AY849057

303KJtmk

Dog

Kanchanaburi

2001

AY849058

308KJm

Dog

Kanchanaburi

2001

AY849059

318KJtmk

Dog

Kanchanaburi

2001

AY849060

326KJtm

Dog

Kanchanaburi

2001

AY849061

333KJm

Dog

Kanchanaburi

2001

AY849062

335KJpnt

Dog

Kanchanaburi

2001

AY849063

89PTtb

Dog

Pathum Thani

1999

AY849064

38/43

Dog

Chaiyaphum

2000

AY849065

39/43

Dog

Chaiyaphum

2000

AY849066

46/43

Dog

Chaiyaphum

2000

AY849067

23SPppd

Dog

Samut Prakan

2001

AY849068

33/43

Dog

Chaiyaphum

2000

AY849069

34/43

Dog

Chaiyaphum

2000

AY849070

19/43

Dog

Chaiyaphum

2000

AY849071

51BKds

Dog

Bangkok

1999

AY849072

62SPbp

Dog

Samut Prakan

1999

AY849073

48BKpyt

Dog

Bangkok

2001

AY849074

HM65BK

Human

Bangkok

1998

AY849075

HM75BK

Human

Bangkok

1998

AY849076

HM88BKjj

Human

Bangkok

1999

AY849077

HM208BKpv

Human

Bangkok

2001

AY849078

404PLkcs

Cattle

Phatthalung

2002

AY849079

355UThk

Dog

Uthai Thani

2001

AY849080

415PLtm

Dog

Phatthalung

2002

AY849081

294CPm

Dog

Chaiyaphum

2002

AY849082

295CPksb3

Dog

Chaiyaphum

2002

AY849083

351NSm

Dog

Nakhon Sawan

2001

AY849084

353CNmnr

Dog

Chai Nat

2001

 

363NScs

Dog

Nakhon Sawan

2001

AY849086

412SKsd

Dog

Songkla

2002

AY849087

414SKm

Dog

Songkla

2002

AY849088

361NSly

Dog

Nakhon Sawan

2001

AY849089

376SHib

Dog

Sing Buri

2001

AY849090

380UTbr

Dog

Uthai Thani

2001

AY849091

393LBm

Dog

Lop Buri

2001

AY849092

384SHib

Dog

Sing Buri

2001

AY849093

411NTht

Dog

Nakhon Si Thammarat

2002

AY849094

374CNspy

Dog

Chai Nat

2001

AY849095

362CNm

Dog

Chai Nat

2001

AY849096

357UTm

Dog

Uthai Thani

2001

AY849097

356UTth

Dog

Uthai Thani

2001

AY849098

381NSttk

Dog

Nakhon Sawan

2001

AY849099

408SKhy

Dog

Songkla

2002

AY849100

332SBm

Dog

Suphan Buri

2001

AY849101

354CNhk

Dog

Chai Nat

2001

AY849102

425PN

Dog

Phisanulok

2002

AY849103

389LBm

Dog

Lop Buri

2001

AY849104

423PN

Dog

Phisanulok

2002

AY849105

424PN

Dog

Phisanulok

2002

AY849106

413SKhy

Cattle

Songkla

2001

AY849107

358CNsbr

Dog

Chai Nat

2001

AY849108

317SBspn

Dog

Suphan Buri

2001

AY849109

334PJsry

Dog

Prachuap Khiri Khan

2001

AY849110

340SBspn

Dog

Suphan Buri

2001

AY849111

400SKm

Dog

Songkla

2001

AY849112

87BKsl

Dog

Bangkok

2001

AY849113

156PTns

Dog

Pathum Thani

1999

AY849114

182CCbnp

Dog

Chachoengsao

1999

AY849115

349PBm

Dog

Phetchaburi

2001

AY849116

307RBptr

Dog

Ratchaburi

2001

AY849117

329PBm

Dog

Phetchaburi

2001

AY849118

406STm

Dog

Satun

2002

AY849119

352NStk

Dog

Nakhon Sawan

2001

AY849120

301RBm

Dog

Ratchaburi

2001

AY849121

157PJsry

Dog

Prachuap Khiri Khan

1999

AY849122

237NYm

Dog

Nakhon Nayok

1999

AY849123

270ATvsc

Cat

Ang Thong

2001

AY849124

281NRm

Dog

Nakhon Ratchasima

2001

AY849125

282NRpc

Dog

Nakhon Ratchasima

2001

AY849126

305RBbp

Dog

Ratchaburi

2001

 AY849127

306RBptr

Dog

Ratchaburi

2001

 AY849128

191AYsn

Dog

Ayutthaya

1999

 AY849129

151SBdc

Dog

Suphan Buri

1999

 AY849130

22CBkhm

Dog

Chanthaburi

2001

 AY849131

162PCm

Dog

Phichit

1999

 AY849132

319PBm

Dog

Phetchaburi

2001

 AY849133

86SPm

Dog

Samut Prakan

2001

 AY849134

136CLblm

Dog

Chon Buri

1999

 AY849135

396YLm

Dog

Yala

2001

 AY849136

250AYbt

Dog

Ayutthaya

1999

 AY849137

302RBpt

Dog

Ratchaburi

2001

 AY849138

316SMm

Dog

Samut Songkhram

2001

 AY849139

426PN

Dog

Phisanulok

2002

 AY849140

296CPksb

Dog

Chaiyaphum

2002

 AY849141

313PBnyp

Dog

Phetchaburi

2001

 AY849142

288NRbl

Dog

Nakhon Ratchasima

2002

 AY849143

505KBlt

Dog

Krabi

2000

 AY849144

507TRrd

Dog

Trang

2000

 AY849145

511NTts

Dog

Nakhon Si Thammarat

2000

AY849146

515KBlt

Dog

Krabi

2000

AY849147

524KBkn

Dog

Krabi

2000

AY849148

559TRpl

Cattle

Trang

2000

AY849149

578KBkt

Dog

Krabi

2001

AY849150

584TRkt

Dog

Satun

2001

AY849151

603KBlt

Dog

Krabi

2001

AY849152

595Ylbns

Dog

Yala

2001

AY849153

493RNm

Dog

Ranong

2000

AY849154

656PLppy

Dog

Phatthalung

2002

AY849155

676KSm

Squirrel

Kalasin

2002

AY849156

513PLm

Dog

Phatthalung

2000

AY849157

589YLm

Dog

Yala

2001

AY849158

608SRm

Dog

Satun

2001

AY849159

HMS152S

Human

Si Sa Ket

2001

AY849160

599TRhy

Dog

Trang

2001

AY849161

473BRpk

Dog

Surin

2001

AY849162

485BRpk

Dog

Buri Ram

2002

AY849163

510NTm

Dog

Nakhon Si Thammarat

2000

AY849164

486UMhsp

Dog

Amnat Chareon

2002

AY849165

487Umm

Dog

Amnat Charoen

2002

AY849166

501RNm

Dog

Nakhon Si Thammarat

2002

AY849167

283NRpm

Dog

Nakhon Ratchasima

2001

AY849168

459KSm

Dog

Kalasin

2002

AY849169

463SRsn

Dog

Surin

2001

AY849170

464SRskp

Dog

Surin

2001

AY849171

465SRm

Dog

Surin

2001

AY849172

466SRrbr

Dog

Surin

2001

AY849173

528NTrpb

Dog

Nakhon Si Thammarat

2000

AY849174

548NTlsk

Dog

Nakhon Si Thammarat

2000

AY849175

289NRht

Dog

Nakhon Ratchasima

2001

AY849176

723KKm

Dog

Khon Kaen

2001

AY849177

481BRhr

Dog

Buri Ram

2002

AY849178

494RNm

Dog

Ranong

2000

AY849179

503SNws

Dog

Surat Thani

2002

AY849180

553STdkl

Dog

Khuan Ka Long/Satun

2000

 AY849181

666STm

Cattle

Satun

2002

 AY849182

458Lam

Dog

RoiEt

2001

AY849183

500SNks

Dog

Surat Thani

2002

AY849184

454Lam

Dog

Roi Et

2001

AY849185

495RNm

Dog

Ranong

2001

AY849186

460Lam

Dog

Roi Et

2002

AY849187

488Umm

Dog

Amnat Charoen

1998

AY849188

489SEkh

Dog

Si Sa Ket

2000

AY849190

502SNcb

Dog

Surat Thani

2002

AY849189

472BRm

Dog

Buri Ram

2001

AY849191

499SNps

Dog

Surat Thani

2002

AY849192

HMS241CL

Human

Chon Buri

2001

AY849193

694LAsp

Dog

Roi Et

2002

AY849194

690NKsps

Dog

Nong Khai

2002

AY849195

689Umm

Dog

Amnat Charoen

2001

AY849196

688Umm

Dog

Amnat Charoen

2002

AY849197

691NKbk

Dog

Nong Khai

2002

AY849198

695Lack

Dog

Roi Et

2002

AY849199

698KSm

Squirrel

Kalasin

2002

AY849200

700KStkt

Dog

Kalasin

2003

AY849201

708YSm

Dog

Yasothon

2000

AY849202

568YLrm

Dog

Yala

2000

AY849203

725MDm

Dog

Mukdahan

2001

AY849204

726UBbt

Dog

Ubol Ratchathani

2001

AY849205

738KKm

Dog

Khon Kaen

2002

AY849206

742MDm

Dog

Mukdahan

2002

AY849207

740MDdt

Dog

Mukdahan

2002

AY849208

711SLm

Dog

Sakon Nakhon

2000

AY849209

709SEktr

Dog

Si Sa Ket

2000

AY849210

678cBRm

Cat

Buri Ram

2000

AY849211

728UBm

Cattle

Ubol Ratchathani

2001

AY849212

685BRppc

Dog

Buri Ram

2002

AY849213

714YSm

Dog

Yasothon

2001

AY849214

715YSm

Water buffalo

Yasothon

2001

AY849215

718YSlkk

Dog

Yasothon

2001

AY849216

705MDm

Dog

Mukdahan

2000

AY849217

731UBvrc

Dog

Ubol

2001

AY849218

704KKcp

Dog

Khon Kaen

2000

AY849219

703KKm

Dog

Khon Kaen

2000

AY849220

732KSm

Cattle

Kalasin

2001

AY849221

741YSm

Dog

Yasothon

2002

AY849222

744UBdud

Dog

Ubol Ratchathani

2002

AY849223

713KKcp

Dog

Khon Kaen

2001

AY849224

717UBm

Dog

Ubol Ratchathani

2001

AY849225

707LYm

Dog

Loei

2000

AY849226

131SPpsj

Dog

Samut Prakan

1999

AY849227

HMS223RY

Human

Rayong

2002

AY849228

747PGtp

Dog

Phangnga

2003

AY849229

766PRm

Dog

Phetchabun

2003

AY849230

769PRcd

Dog

Phetchabun

2003

AY849232

774NNm

Dog

Nan

2002

AY849231

773CMcp

Dog

Chiang Mai

2002

AY849233

775PYp

Dog

Phayao

2003

AY849234

776NStk

Dog

Nakhon Sawan

2002

AY849235

795NScs

Dog

Nakhon Sawan

2003

AY849236

801SUsk

Dog

Sukhothai

2002

AY849237

802KPlkb

Dog

Kamphaeng Phet

2002

AY849238

811SUkrm

Dog

Sukhothai

2002

AY849239

806PNnm

Dog

Phisanulok

2002

AY849240

814UDm

Dog

Uttaradit

2002

AY849241

807UDts

Dog

Uttaradit

2002

AY849242

800Udm

Dog

Uttaradit

2002

AY849243

815PRls

Dog

Phetchabun

2002

AY849244

805PCbmn

Dog

Phichit

2002

AY849245

780NSm

Dog

Nakhon Sawan

2003

AY849246

762PRm

Dog

Phetchabun

2003

AY849247

813PNbrk

Dog

Phisanulok

2002

AY849248

796PNbrk

Dog

Phisanulok

2003

AY849249

816PRcd

Dog

Phetchabun

2002

AY849250

778LBtv

Dog

Lop Buri

2003

AY849251

794SUssr

Dog

Sukhothai

2003

AY849252

784Sum

Dog

Sukhothai

2003

AY849253

785SUkkl

Dog

Sukhothai

2003

AY849254

788SUsk

Dog

Sukhothai

2003

AY849255

808SUssn

Dog

Sukhothai

2002

AY849256

793SUsn

Dog

Sukhothai

2003

AY849257

777LBm

Dog

Lop Buri

2003

AY849258

779NSly

Dog

Nakhon Sawan

2003

AY849259

804UDm

Dog

Uttaradit

2002

AY849260

338PJm

Dog

Prachuap Khiri Khan

2001

HQ317918

CTN-1-31

Human

China

1956

EU293115

9147FRA

Fox

France

1991

EU293113

9001FRA

 Dog

French Guyana

1990

AY705373

SHBRV-18

 Bat

USA

1983

EU293116

9704ARG

  Bat

Argentina

1997

EF206709

SAD B19

Vaccine

-

-

EF206707

ERA

Vaccine

-

-

NC_001542

PV

Vaccine

France

1882

AB085828

HEP-Flury

Vaccine

USA

1939

GQ918139

CVS-11

-

France

1882

EF437215

NNV-RAB-H

Human

India

2006

AY956319

Serotype 1

Saliva

Germany

-

MN075931

TH13-42-07240

 Dog

Thailand

1989

Table S1: Nucleoprotein Gene sequences of the selected rabies virus strains used for genetic analysis 

Figure S1: ML phylogeny of xxx sequences from the N-coding region of Rabies virus strains.

Caption: Sequences were obtained from Gen Bank and the TH strain sequenced for this study.

Citation: Nitatpattana N, Primsirikunawut A, Buree S, Chuapudee D, Rodpai E, Chaiyo K, Chansiprasert K, Nakgoi K, Poolam K, Lukebuo A, Sangkitporn S,Mandja BM, Gonzalez JP (2020) Selection of a Rabies Virus Strain as a New Candidate for Vaccine Development in Thailand. J Vaccines Res Vaccin , 6: 015.

Copyright: © 2020  Nitatpattana N, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

© 2021, Copyrights Herald Scholarly Open Access. All Rights Reserved!