Veterinary Microbiology 176 (2015) 358–364
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Segment-2 sequencing and cross-neutralization studies confirm existence of a neutralization resistant VP2 phenotypic variant of bluetongue virus serotype 1 in India Sanchay K. Biswas a, Karam Chand a, Waseem Rehman a, Yella Narasimha Reddy b, Bimalendu Mondal a,* a
Division of Virology, Indian Veterinary Research Institute, Mukteswar Campus, District Nainital-263 138, Uttarakhand, India Department of Veterinary Microbiology, College of Veterinary Science, Sri Venkateswara Veterinary University, Rajendranagar, Hyderabad 500030, India b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 October 2014 Received in revised form 29 January 2015 Accepted 30 January 2015
Segment-2 (seg-2) of a bluetongue virus seropype-1 (BTV-1) isolate WGV104/08/Ind of Indian origin was sequenced and its neutralization behavior was studied to understand the antigenic similarity and relationship with other BTV-1 isolates. Multiple alignments of the coding region of seg-2 of WGV104/08/Ind revealed 97.6–99.0% and 97.2–98.4% similarity with other Indian BTV-1 isolates at nucleotide and deduced amino acid sequence level respectively. Several conservative and non-conservative substitutions were observed on the deduced VP2 amino acid sequence of WGV104/08/Ind. Non-conservative substitution of Lys119Glu on the B-cell epitope and Arg330Gly on the neutralizing epitope of VP2 of this isolate was observed. Using isolate-specific heterologous hyperimmune serum (HIS) the phenotypic antigenic relationship (r) was determined between WGV104/08/Ind and other Indian BTV-1 isolates which ranged from 0.092 to 0.208. The relationship score ranged from 0.203 to 0.295 when neutralization behavior of other Indian BTV-1 isolates was studied with the HIS of WGV104/08/Ind. Antigenic similarity (R) between WGV104/ 08/Ind and other Indian BTV-1 isolates was estimated from a reciprocal crossneutralization study and ranged from 14.70% to 24.80% indicating existence of major subtype antigenic divergence and neutralization resistant behavior of WGV104/08/Ind. ß 2015 Elsevier B.V. All rights reserved.
Keywords: Antigenic similarity Bluetongue virus serotype 1 Segment-2 Neutralization resistance VP2 phenotypic variant
1. Introduction Bluetongue virus (BTV), the type species of the genus Orbivirus within the family Reoviridae, is the causative agent of bluetongue, a major disease of ruminants. So far,
* Corresponding author. Present address: Eastern Regional Station, Indian Veterinary Research Institute, 37 Belgachia Road, Kolkata 700037, West Bengal, India. Tel.: +91 33 2558 2965; fax: +91 33 2556 5725. E-mail address: [email protected] (B. Mondal). http://dx.doi.org/10.1016/j.vetmic.2015.01.023 0378-1135/ß 2015 Elsevier B.V. All rights reserved.
26 distinct serotypes of BTV have been identified worldwide (Maan et al., 2011), but a recent paper describes a possible 27th serotype of the virus which is circulating among goats on Corsica (Zientara et al., 2014). BTV particles consist of three concentric protein layers surrounding the 10 segments of linear double-stranded (ds) RNA genome. The dsRNA segments encode seven structural proteins (VP1 to VP7), with an additional four non-structural (NS1 to NS4) proteins observed during infection and replication (Belhouchet et al., 2011). Segment 2 (seg-2; encoding VP2) is the most variable of
S.K. Biswas et al. / Veterinary Microbiology 176 (2015) 358–364
the BTV segments and VP2 is solely responsible for serotype determination and it is also the key determinant of neutralizing antibody specificity (Shaw et al., 2013). Genetic diversity among the BTV serogroup is clearly established on the VP2 gene, being the most divergent among different BTV serotypes and nucleotypes (Maan et al., 2007). BTV evolves through a combination of genetic drift and shift and therefore, quasispecies evolution coupled with founder effect and evolutionary selective pressure lead to the development of genetic and phenotypic variants of the virus (Bonneau et al., 2001). Phenotypic variations of BTV are constantly generated in the field and neutralization behavior may vary amongst the field strains of the same serotype. Bluetongue is enzootic in India and a wide prevalence of the virus has been reported in domestic ruminants by seroepidemiological studies. Amongst the BTV serotypes circulating in India, BTV-1 is one of the most predominant serotypes as evident by serological surveillance and isolation of a large number of viruses from different parts of the country (Jain et al., 1986; Prasad et al., 1994; Biswas et al., 2010). The present study was conducted to understand seg-2 sequence variability and neutralization behavior of a novel BTV-1 isolate recovered from the southern part of India. 2. Materials and methods
359
2.3. DNA sequencing, comparison of sequence data and phylogenetic analysis The amplified overlapping fragments of DNA were sequenced with Big dye terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Inc., CA, USA) on an automated sequencer following the manufacturer’s instructions. The generated nucleotide sequences were confirmed by BLASTN (http://www.ncbi.nlm.nih.gov/) analysis and the overlapping fragments were assembled with the help of the EditSeq program implemented in the DNASTAR program package (DNASTAR Inc., Madison, USA) to generate a complete seg-2 coding sequence of the virus. Nucleotide sequences were aligned with sequences of other BTV VP2 encoding genes (Table 1) using the CLUSTAL W algorithm (Thompson et al., 1994) implemented in the MegAlign of DNASTAR program package and aligned sequence data was used for phylogenetic analyses. The evolutionary history was inferred using the Maximum Likelihood (ML) method from 1000 replicates (Felsenstein, 1985), taken to represent the evolutionary history of the taxa analyzed. The best suitable model for analysis of evolutionary distances used to infer the phylogenetic tree was selected by the model selection program implemented in MEGA version 6.0 software (Tamura et al., 2013). 2.4. Production of hyperimmune serum and neutralization assay
2.1. Virus isolate The virus used in this study was isolated from a Nellore breed of sheep affected with bluetongue in the West Godavari district of Andhra Pradesh in 2008. Blood samples were collected from sheep showing symptoms of necrotic stomatitis, laminitis, facial edema, nasal discharge and pyrexia. The virus was initially isolated on embryonated chicken egg and subsequently adapted in BHK-21 cells. The isolate (WGV104/08/Ind) was confirmed as BTV-1 and submitted to the Bluetongue virus Repository under ICAR All India Network Program on Bluetongue (AINP-BT). The virus was revived using BHK-21 cultures, seg-2 was sequenced and hyperimmune serum (HIS) was produced. Ten other BTV-1 isolates (Table 1) and their corresponding HIS were also used in neutralization studies. 2.2. RNA isolation, cDNA synthesis and PCR amplification of VP2 gene Total RNA was extracted from BTV-1 (WGV104/08/ Ind)-infected BHK-21 cells (75 cm2 culture flask) using TRI1-Reagent (Sigma) according to manufacturer’s instructions and viral dsRNA was purified by lithium chloride precipitation (Attoui et al., 2000). Purified dsRNA segments were separated by electrophoresis on 1% agarose gel and seg-2 was excised from the gel and extracted using the QIAquick1 Gel extraction kit (Qiagen) according to the manufacturer’s instructions. Gel extracted seg-2 was used for synthesis of cDNA and PCR amplification of complete coding region in multiple overlapping fragments with different oligonucleotide primer pairs as described by Biswas et al. (2010).
The WGV104/08/Ind isolate was purified from a bulk culture of infected BHK-21 cells by ultracentrifugation through discontinuous sucrose gradients (Mertens et al., 1987) and HIS was produced in rabbits against purified virion following a procedure used earlier for hyperimmunization with a different BTV serotype (Pathak et al., 2008). The HIS was clarified, inactivated (at 56 8C for 30 min), filtered through 0.45 mm membrane filters and stored in multiple aliquots at 80 8C to avoid repeated freezing and thawing. WGV104/08/Ind was neutralized with homologous HIS and also with ten heterologous isolate-specific HIS (prepared earlier in our laboratory) against Indian BTV1 strains (Table 1). Serum neutralization test (SNT) was performed on BHK-21 cells according to the method described by MacLachlan et al. (1992) in 96-well tissue culture plate using 200 TCID50 (fixed) of each of 18 viruses (Table 1) and two-fold serial dilution of individual HIS. Serum neutralization titer was calculated by the method of Reed and Muench (1938) and the mean of anti-log values of three individual runs was considered as mean neutralization titer of the HIS against a particular virus isolate. 2.5. Determination of phenotypic antigenic relationship and antigenic similarity Phenotypic antigenic relationship (r) was measured as the ratio of the mean heterologous anti-log titer to the mean of the thomologous anti-log titer as described by Archetti and Horsfall (1950). Reciprocal cross-neutralization data of the isolates was used to determine the antigenic similarity of WGV104/08/Ind with other Indian BTV-1 isolates and calculated in terms of ‘R’ value
S.K. Biswas et al. / Veterinary Microbiology 176 (2015) 358–364
360
Table 1 Characteristics of BTV-1 isolates/strains described in this study. Isolates ID a
WGV104/08/Ind Hisar/85/Indb Avikanagar/94/Indb Chennai/03/Indb KDP15/07/Indb NLG3/07/Indb NRT37/07/Indb SKN7/07/Indb SKN8/07/Indb MKD18/08/Indb MKD25/08/Indb MKD19/08/Ind MKD20/08/Ind MKD21/08/Ind MKD22/08/Ind MKD23/08/Ind MKD24/08/Ind PTG13/11/Ind AJ585116/87/Mal KC879616/79/Chi AJ585178/81/Aus M21844/88/Aus FJ437557/07/Fra KJ019206/13/Ita AJ585110/vac/SA AJ585122/Ref/SA AJ585117/87/Sud AJ585119/82/Cam
GenBank
Species
Year
Place of origin
SNT
Reference
KF563945 KF563929 KF563930 KF563932 KF563933 KF563934 KF563935 KF563936 KF563937 HM014236 KF563944 KF563938 KF563939 KF563940 KF563941 KF563942 KF563943 KF563946 AJ585116 KC879616 AJ585178 M21844 FJ437557 KJ019206 AJ585110 AJ585110 AJ585117 AJ585118
Sheep Sheep Sheep Sheep Sheep Sheep Sheep Culicoides Culicoides Goat Goat Goat Goat Goat Goat Goat Goat Goat nk Sheep nk nk Ovine Ovine nk nk nk ovine
2008 1985 1994 2003 2007 2007 2007 2007 2007 2008 2008 2008 2008 2008 2008 2008 2008 2011 1987 1997 1981 1988 2007 2013 nk nk 1987 1982
West Godavari, AP, Ind Hisar, Haryana, Ind Avikanagar, RJ, Ind Thoothukkudi, TN, Ind Kadappa, AP, Ind Nalgonda, AP, Ind Guntur, AP, Ind Jasdan, Gujarat, Ind S.K. Nagar, Gujarat Mathura, UP, Ind Mathura, UP, Ind Mathura, UP, Ind Mathura, UP, Ind Mathura, UP, Ind Mathura, UP, Ind Mathura, UP, Ind Mathura, UP, Ind Pithoragarh, UK, Ind Kuala Lumpur, Malaysia China Australia Australia France Sardinia, Italy South Africa South Africa Sudan Cameroon
Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y N N N N N N N N N N
Present study Jain et al. (1986) Prasad et al. (1994) AINP-BT (2010) AINP-BT (2010) AINP-BT (2010) AINP-BT (2010) AINP-BT (2010) AINP-BT (2010) Biswas et al. (2010) Biswas et al. (2010) Biswas et al. (2010) Biswas et al. (2010) Biswas et al. (2010) Biswas et al. (2010) Biswas et al. (2010) Biswas et al. (2010) Bisht et al. (2011) Maan et al. (2004b) Zhu et al. (2013) Maan et al. (2004b) Gould (1988) Cetre-Sossah et al. (2011) Lorusso et al. (2014) Maan et al. (2004b) Maan et al. (2004b) Maan et al. (2004b) Maan et al. (2004b)
a
Isolate used for sequencing of seg-2 and preparation of HIS in the present study. HIS prepared earlier in our laboratory against this isolate; GenBank: NCBI GenBank accession number for seg-2/VP2 nucleotide sequence of BTV-1; Year: year of isolation; SNT: Isolate used in the serum neutralization test (SNT) in the present study; AP: Andhra Pradesh; RJ: Rajasthan; TN: Tamil Nadu; UK: Uttarakhand; UP: Uttar Pradesh; Ind: India; nk: not known; Y: yes; N: no. b
[R = (r1 r2)1/2 100, where r1 = (neutralization titre of virus-A with HIS of virus-B)/(neutralization titre of virus-A with HIS of virus-A) and r2 = (neutralization titre of virus-B with HIS of virus-A)/(neutralization titre of virus-B with HIS of virus-B), assuming relatedness of two viruses, namely virus-A and virus-B based on two way crossneutralization, carried out with hyper-immune serum against both viruses]. Antigenic similarity of the two viruses were assessed by the criteria described by Brooksby (1967) as follows: an R-value of 100% or close to 100% indicates antigenic identity between two viruses tested; an R-value >70% indicates little or no difference; an R-value between 33% and 70% indicates a minor subtype difference; an R-value between 11% and 32% indicates a major sub-type difference and an R-value between 0% and 10% indicates a different serotype. 3. Results 3.1. Sequencing and phylogenetic analysis of seg-2 The nucleotide sequence of seg-2 of BTV-1 isolate WGV104/08/Ind has been submitted to GenBank under the accession no. KF563945. BLASTN (NCBI) analysis of the sequence confirmed that it corresponds with seg-2/VP2 sequences that have previously been published for other BTV-1 strains. The coding region of seg-2 of WGV104/08/ Ind was 2886 bp in length (from nucleotide position 18 to 2903 including stop codon) giving rise to VP2 containing
961 amino acids. Analysis revealed unique substitutions of adenine (A) to guanine (G) at nt position 355 and 988. These substitutions do not seem to be sequence artifacts as evident from electropherograms of repeated runs. Nucleotide similarity was found to be 97.6% to 99.0% when compared with other isolates of BTV-1 from India. Phylogenetic tree segregated Indian BTV-1 isolates into a monophyletic cluster at close proximity to other ‘eastern’ topotype viruses. WGV104/08/Ind clustered with other Indian BTV-1 isolates in ML phylogenetic analysis indicating their close ancestral relationship (Fig. 1). 3.2. Amino acid sequence variation in deduced VP2 polypeptide There were twelve cysteine (Cys/C) residues (aa position 136, 162, 262, 281, 363, 446, 617, 784, 851, 870, 928 and 937) in the deduced VP2 amino acid sequence of WGV104/08/Ind. Multiple alignments of the deduced VP2 amino acid sequence revealed 97.2–98.4% similarity with other Indian BTV-1 isolates. Both conservative and non-conservative substitutions (in comparison to the consensus sequence) were evident on the deduced VP2 amino acid sequence of WGV104/08/Ind (Table 2). Nonconservative substitution of Lys119Glu was observed which lies on the proposed B-cell epitope region of BTV1 (Wei et al., 2013). Another non-conservative substitution of Arg330Gly was also observed in the neutralizing epitope of BTV-1 reported earlier (Gould and Eaton, 1990).
S.K. Biswas et al. / Veterinary Microbiology 176 (2015) 358–364
361
SKN7/07/Ind MKD22/08/Ind SKN8/07/Ind MKD25/08/Ind MKD23/08/Ind MKD24/08/Ind MKD19/08/Ind 81 MKD18/08/Ind
MKD21/08/Ind
India
Chennai/03/Ind MKD20/08/Ind
97
'Eastern' topotype
PTG13/11/Ind Hisar/85/Ind
85
Avikanagar/94/Ind 100
WGV104/08/Ind NRT37/07/Ind
100
KDP15/07/Ind
99 100
NLG3/07/Ind AJ585116/87/Mal KC879616/79/Chi
AJ585178/81/Aus 100
M21844/88/Aus
Australia
FJ437557/07/Fra
100
KJ019206/13/Ita
Europe
AJ585119/82/Cam
100
AJ585110/vac/SA
83
AJ585122/Ref/SA 71
'Western' topotype Africa
AJ585117/87/Sud
0.05
Fig. 1. Maximum likelihood (ML) tree (TN93 + I, MEGA 6.0) depicting phylogenetic relationship of WGV104/08/Ind with other BTV-1 isolates at complete coding region of VP2 gene nucleotide sequence. Bootstrap consensus was inferred from 1000 replicates to represent the evolutionary history of the taxa analyzed. Branch lengths are indicative of genetic distances between the sequences.
Table 2 Nucleotide substitutions in VP2 encoding gene and amino acid substitutions in deduced VP2 polypeptide of WGV104/08/Ind isolate of BTV-1. Nucleotide position
8 22 32 295 328 355 454 553 729 749 988 1063 2498
Nucleotide change
Amino acid change
Amino acid position
Consensus
WGV104/08/Ind
Consensus
WGV104/08/Ind
GAG ATC CAA TAC AAA AAG GTC ATA ATA CTG AGA ATA CTT
GGG GTC CGA CAC GAA GAG ATC GTA ATG CCG GGA GTA CCT
Glu Ile Gln Tyr Lys Lys Val Ile Ile Leu Arg Ile Leu
Gly Val Arg His Glu Glu Ile Val Met Pro Gly Val Pro
3 8 11 99 110 119* 152 185 243 250 330* 355 833
Note: Amino acid substitutions were determined in comparison to the consensus sequence generated by multiple alignment (ClustalW) of deduced VP2 sequence of Indian BTV-1 isolates used in neutralization study. Positions of amino acid change in the B-cell epitope and neutralizing epitope of VP2 of BTV-1 are marked with asterisk.
S.K. Biswas et al. / Veterinary Microbiology 176 (2015) 358–364
Hyperimmune Serum (HIS)
MKD25/08/Ind
MKD18/08/Ind
SKN8/08/Ind
SKN7/07/Ind
NRT37/07/Ind
NLG3/07/Ind
KDP15/07/Ind
isolates. Weak phenotypic antigenic relationship of WGV104/08/Ind was observed in an ex vivo neutralization study with heterologous isolate-specific HIS of other Indian BTV-1 isolates. Antigenic similarity is commonly used for assessment of antigenic relatedness between two viruses in reciprocal cross-neutralization studies. Antigenic similarity (R) between WGV104/08/Ind and other Indian BTV-1 isolates was estimated to be between 14.70% and 24.80% which indicates major subtype antigenic divergence and neutralization resistant behavior of WGV104/ 08/Ind [(an R value between 11% and 32% indicates a major subtype difference (Brooksby, 1967)].
Phenotypic antigenic relationship of Indian BTV-1 isolates with HIS of WGV104/08/Ind
PTG13/11/Ind
MKD24/08/Ind
MKD22/08/Ind
MKD23/08/Ind
MKD20/08/Ind
MKD21/08/Ind
MKD19/08/Ind
MKD25/08/Ind
SKN8/08/Ind
MKD18/08/Ind
SKN7/07/Ind
NRT37/07/Ind
NLG3/07/Ind
KDP15/07/Ind
Hisar/85/Ind
Avikanagar/94/Ind
1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 WGV104/08/Ind
Phenotypic antigenic relationship (r) score
MKD25/08/Ind
MKD18/08/Ind
SKN7/07/Ind
SKN8/08/Ind
NLG3/07/Ind
NRT37/07/Ind
KDP15/07/Ind
Chennai/03/Ind
Hisar/85/Ind
Avikanagar/94/Ind
WGV104/08/Ind
Phenotypiv antigenic relationship (r) score
1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
(b)
Chennai/03/Ind
Fig. 3. Antigenic similarity (R) amongst Indian BTV-1 isolates calculated from reciprocal cross-neutralization studies. Height of the bars indicate antigenic similarity score (R). Homologous similarity score is 100.00. Rvalue of 100% or close to 100%: antigenic identity, >70%: little or no antigenic difference, 33–70%: minor subtype difference, 11–32%: major sub-type difference, 0–10%: different serotype (Brooksby, 1967).
The outer capsid protein VP2, encoded by genome seg-2 is the most variable protein of BTV and is responsible for receptor binding and eliciting neutralizing antibodies to BTV. Phylogenic comparison of seg-2 from 24 South African reference strains and from over 200 other BTV isolates from around the world show a perfect correlation between sequence variation in seg-2 and BTV serotype (Maan et al., 2004a). The present study evaluated sequence data and the neutralization behavior of seg-2 of an Indian BTV-1 isolate WGV104/08/Ind. Although seg-2 is the most variable gene among the BTV genome segments, multiple nucleotide sequence alignments of WGV104/08/Ind revealed a high degree (97.2–98.4%) of similarity. ML tree constructed by using Tamura-Nei (TN93 + I) segregated ‘eastern’ (Indian/Australian) and ‘western’ (African/European) BTV isolates where WGV104/08/Ind clustered into the Indian monophyletic clade indicating a close evolutionary relationship of the isolate with other Indian BTV-1
Phenotypic antigenic relationship (r) of WGV104/08/Ind with isolate-specific HIS
Avikanagar/94/Ind
Virus
4. Discussion
(a)
Antigenic similarity (R) between WGV104/08/Ind and other Indian BTV-1 isolates
WGV104/08/Ind
Neutralization behavior of WGV104/08/Ind was studied against a total of ten isolate-specific heterologous BTV1 HIS along with the homologous HIS of the virus. The phenotypic antigenic relationship (r) of WGV104/08/Ind with other Indian BTV-1 isolates ranged from 0.092 to 0.208 with isolate-specific heterologous HIS where the homologous relationship was 1.0 (Fig. 2a). The relationship score ranged from 0.203 to 0.295 when neutralization behavior of other Indian BTV-1 isolates was studied against the HIS of WGV104/08/Ind (Fig. 2b). Antigenic similarity (R) between WGV104/08/Ind and other Indian BTV-1 isolates calculated from the reciprocal cross-neutralization study ranged from 14.70% to 24.80% where homologous similarity was considered as 100% (Fig. 3).
100 90 80 70 60 50 40 30 20 10 0
Hisar/85/Ind
Antigenic similarity score (R)
3.3. Neutralization behavior and phenotypic antigenic relationship
Chennai/03/Ind
362
BTV-1 isolates
Fig. 2. Phenotypic antigenic relationship (r) of WGV104/08/Ind with isolate specific HIS of Indian BTV-1 isolates (a) and other Indian BTV-1 isolates with HIS of WGV104/08/Ind (b). Homologous relationship score (virus isolate with corresponding HIS) is 1.0.
S.K. Biswas et al. / Veterinary Microbiology 176 (2015) 358–364
Amino acid substitutions in various escape mutant viruses localize mainly in two domains at positions 199– 213 and position 321–346 in VP2, although individual substitutions outside to these positions may also contribute to neutralization of BTV (DeMaula et al., 2000). Amino acid position 328–335 on VP2 is predicted to harbor the major neutralizing epitope of BTV-1 (Gould and Eaton, 1990). Multiple alignments of deduced VP2 amino acid sequences of WGV104/08/Ind with other Indian BTV-1 isolates showed an extreme conservativeness of amino acids in the domain spanned from 199 to 213. Single non-conservative substitution of Arginine (Arg/R) to Glycine (Gly/G) at amino acid position 330 was observed in WGV104/08/Ind. It is due to a point mutation at nucleotide position 988 on VP2 encoding gene sequence which is responsible for alteration of amino acid codon (AGA to GGA), resulted in substitution of Arg330Gly. A similar observation was also reported on VP2 of two different monoclonal antibody resistant Australian BTV-1 variants (Gould and Eaton, 1990). It is likely that the substitution of Arg330Gly is very crucial for phenotypic behavior of BTV-1 and may be responsible for the neutralization resistant behavior of WGV104/08/Ind. Another non-conservative substitution of Lys119Glu was observed which lies on the BTV-1 specific B-cell epitope reported earlier (Wei et al., 2013) although it has been proposed as non-neutralizing epitope. The possible consequences of neutralization resistance may be misclassification of such a variant leading to mistakes in epidemiological studies. Such neutralization resistant VP2 phenotypic variants have been described amongst isolates of BTV-23 and problems of misclassification were faced while serotyping these variants by conventional virus or serum neutralization tests (VNT or SNT) using polyclonal sera (Tembhurne et al., 2010). To avoid these problems, instead of VNT/SNT-based phenotypic serotyping, segment-2 (VP2 encoding gene) based genotypic serotyping has been adopted worldwide and recently an RT-PCR has been developed for identification and differentiation of 26 BTV serotypes by a serotypespecific amplification using seg-2 (Maan et al., 2012). The non-structural (NS) genes/proteins have also been used for development of group-specific RT-PCR and real time RTPCR (Dangler et al., 1990; Orru et al., 2006). Neutralization resistance can also pose problems during vaccination. All the neutralization resistant variants of a BTV serotype need to be incorporated in a vaccine for getting complete protection against the different isolates of the specific serotype. In conclusion, a novel BTV-1 isolate (WGV104/08/Ind) has been characterized that exhibits a special VP2 genotype and phenotype leading to neutralization resistance of this variant. Further studies involving a panel of neutralizing monoclonal antibodies or antibody to defined peptides are required to pinpoint the effect of specific amino acid(s) substitution (s) in neutralization behavior of this variant. Conflict of interest statement The authors declare that they have no competing interest.
363
Acknowledgements This work has been supported by an ICAR-funded project—All India Network Program on Bluetongue. Nucleotide sequencing was carried out at the Central FMDV Typing Lab, PD-FMD, Mukteswar. We thank the Director and Head of the Virology Division, IVRI for providing facilities and support.
References AINP-BT, 2010. Annual Report 2010–11. ICAR All India Network Program on Bluetongue. Indian Veterinary Research Institute, Izatnagar-243 122, Bareilly, Uttar Pradesh, pp. 56. Archetti, I., Horsfall Jr., F.L., 1950. Persistent antigenic variation of influenza A viruses after incomplete neutralization in ovo with heterologous immune serum. J. Exp. Med. 92, 441–462. Attoui, H., Billoir, F., Cantaloube, F.J., Biagini, P., Micco, P., Lamballerie, X., 2000. Strategies for sequence determination of viral dsRNA genomes. J. Virol. Methods 89, 147–158. Belhouchet, M., Jaafar, M.F., Andrew, E., Grimes, M.J., Mertens, P.P.C., Attoui, H., 2011. Detection of a fourth orbivirus non-structural protein. PLoS One 6, e25697. Bisht, B., Mondal, B., Biswas, S.K., Chand, K., Pandey, A.B., 2011. Isolation and molecular characterization of bluetongue virus from asymptomatic goats. Ind. J. Comp. Microbiol. Immunol. Infect. Dis. 32, 27–29. Biswas, S.K., Chand, K., De, A., Pandey, L.K., Mohapatra, J.K., Prasad, G., Mondal, B., 2010. Isolation of bluetongue virus serotype 1 (BTV-1) from goats and its phylogenetic relationship with other BTV-1 isolates worldwide based on full-length sequence of genome segment-2. Arch. Virol. 155, 2041–2046. Bonneau, K.R., Mullens, B.A., MacLachlan, N.J., 2001. Occurence of genetic drift and founder effect during the quasispecies evolution of the VP2 and NS3/NS3A genes of bluetongue virus upon passage between sheep, cattle and Cuilicoides sonorensis. J. Virol. 75, 8298–8305. Brooksby, J.B., 1967. Variants and immunity: definitions for serological investigation. International symposium on Foot and mouth disease, variants and immunity, Lyon, France. Symposium series. Immunobiol. Standard 8, 1–10. Cetre-Sossah, C., Madani, H., Sailleau, C., Nomikou, K., Sadaoui, H., Zientara, S., Maan, S., Maan, N.S., Mertens, P.P.C., Albina, E., 2011. Molecular epidemiology of bluetongue virus serotype 1 isolated in 2006 from Algeria. Res. Vet. Sci. 91, 486–497. Dangler, C.A., De Mattos, C.A., De Mattos, C.C., Osburn, B.I., 1990. Identifying bluetongue virus ribonucleic acid sequences by polymerase chain reaction. J. Virol. Me 28, 281–292. DeMaula, C.D., Bonneu, K.R., Maclachalan, N.I., 2000. Changes in the outercapsid protein of bluetongue virus serotype ten that abrogate neutralization by monolonal antibodies. Virus Res. 67, 59–66. Felsenstein, J., 1985. Confidence limits on phylogenesis: an approach using the bootstrap. Evolution 39, 783–791. Gould, A.R., 1988. Conserved and non-conserved regions of the outer coat protein VP2, of the Australian bluetongue serotype I virus, revealed by sequence comparison to the VP2 of North American BTV serotype 10. Virus Res. 9, 145–158. Gould, A.R., Eaton, B.T., 1990. The amino acid sequence of the outer coat protein VP2 of neutralizing monoclonal antibody-resistant, virulent and attenuated bluetongue viruses. Virus Res. 17, 161–172. Jain, N.C., Sharma, R., Prasad, G., 1986. Isolation of bluetongue virus from sheep in India. Vet. Rec. 119, 17–18. Lorusso, A., Marcacci, M., Ancora, M., Mangone, I., Leone, A., Marini, V., Camma, C., Savini, G., 2014. Complete genome sequence of bluetongue virus serotype 1 circulating in Italy, obtained through a fast next-generation sequencing protocol. Genome Announc. 2, E00093-14. Maan, N.S., Maan, S., Belaganahalli, M.N., Ostlund, E.N., Johnson, D.J., Nomikou, K., Mertens, P.P.C., 2012. Identification and differentiation of the twenty six bluetongue virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2. PLoS One 7 (2), e32601, http://dx.doi.org/10.1371/journal.pone.0032601. Maan, S., Maan, N.S., Nomikou, K., Veronesi, E., Bachanek-Bankowska, K., Manjunatha, N.B., Attoui, H., Mertens, P.P.C., 2011. Complete genome characterisation of a novel 26th bluetongue virus serotype from Kuwait. PLoS One 6, e26147, http://dx.doi.org/10.1371/journal.pone.0026147.
364
S.K. Biswas et al. / Veterinary Microbiology 176 (2015) 358–364
Maan, S., Maan, N.S., Samuel, A.R., O’Hara, R., Meyer, A.J., Rao, S., Mertens, P.P.C., 2004a. Completion of the sequence analysis and comparison of genome segment 2 (encoding outer capsid protein VP2) from representative isolates of the 24 bluetongue virus serotype. Vet. Ital. 40, 484–488. Maan, S., Maan, N.S., Samuel, A.R., Rao, S., Attoui, H., Mertens, P.P.C., 2007. Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes. J. Gen. Virol. 88, 621– 630. Maan, S., Samuel, A.R., Maan, N.S., attoui, H., Rao, S., Mertens, P.P.C., 2004b. Molecular epidemiology of bluetongue viruses from disease outbreaks in the Mediterranean Basin. Vet. Ital. 40, 489–496. MacLachlan, N.J., Rossitto, P.V., Heidner, H.W., Iezzi, L.G., Yilma, T.D., DeMaula, C.D., Osburn, B.I., 1992. Variation amongst the neutralizing epitopes of bluetongue viruses isolated in the United States in 1979– 1981. Vet. Microbiol. 31, 303–316. Mertens, P.P., Burrough, J.N., Anderson, J., 1987. Purification and properties of viral particles, infectious subviral particles and core of Bluetongue virus serotype 1 and 4. Virology 157, 375–386. Orru, G., Ferrando, M.L., Meloni, M., Liciardi, M., Savini, G., De Santis, P., 2006. Rapid detection and quantitation of Bluetongue virus (BTV) using a Molecular Beacon fluorescent probe assay. J. Virol. Methods 137, 34–42. Pathak, K.B., Biswas, S.K., Tembhurne, P.A., Hosamani, M., Bhanuprakash, V., Prasad, G., Singh, R.K., Rasool, T.J., Mondal, B., 2008. Prokaryotic expression of truncated VP7 of Bluetongue virus (BTV) and reactivity of the purified recombinant protein with all BTV type-specific sera. J. Virol. Methods 152, 6–12. Prasad, G., Garg, A.K., Minakshi Kakker, N.K., Srivastava, R.N., 1994. Isolation of bluetongue virus from sheep in Rajasthan state. Rev. Sci. Tech. Off. Int. Epiz 13, 935–938.
Reed, L.J., Muench, M., 1938. A simple method of estimating fifty percent end points. Am. J. Hyg. 27, 493–497. Shaw, A.E., Ratinier, M., Nunes, S.F., Nomikou, K., Caporale, M., Golder, M., Allan, K., Hamers, C., Hudelet, P., Zientara, S., Breard, E., Mertens, P., Palmarini, M., 2013. Reassortment between two serologically unrelated bluetongue virus strains is flexible and can involve any genome segment. J. Virol. 87, 543–557. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. Tembhurne, P.A., Mondal, B., Pathak, K.B., Biswas, S.K., Sanyal, A., Yadav, M.P., Bandyopadhyay, S.K., Singh, R.K., 2010. Segment-2 sequence analyses and cross-neutralization studies on some Indian bluetongue viruses suggest isolates are VP2-variants of serotype 23. Arch. Virol. 155, 89–95. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Wei, P., Sun, E.C., Liu, N.H., Yang, T., Xu, Q.Y., Zhao, J., Qin, Y.L., Feng, Y.F., Li, J.P., Wang, W.S., Zhang, C.Y., Wu, D.L., 2013. Identification of a novel bluetongue virus 1-specific B-cell epitope using a monoclonal antibody against the VP2 protein. Arch. Virol. 158, 1099–1104. Zhu, J., Yang, H., Li, H., Xiao, L., Wang, J., Li, N., Zhang, N., 2013. Full-genome sequence of bluetongue virus serotype 1 (BTV-1) strain Y863, the first BTV-1 isolate of eastern origin found in China. Genome Announc. 1, E00403–E00413. Zientara, S., Sailleau, C., Viarouge, C., Ho¨per, D., Beer, M., Jenckel, M., Hoffmann, B., Romey, A., Bakkali-Kassimi, L., Fablet, A., Vitour, D., Bre´ard, E., 2014. Novel bluetongue virus in goats, Corsica, France, 2014. Emerg. Infect. Dis. 20, 2123–2125.
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Segment-2 sequencing and cross-neutralization studies confirm existence of a neutralization resistant VP2 phenotypic variant of bluetongue virus serotype 1 in India Sanchay K. Biswas a, Karam Chand a, Waseem Rehman a, Yella Narasimha Reddy b, Bimalendu Mondal a,* a
Division of Virology, Indian Veterinary Research Institute, Mukteswar Campus, District Nainital-263 138, Uttarakhand, India Department of Veterinary Microbiology, College of Veterinary Science, Sri Venkateswara Veterinary University, Rajendranagar, Hyderabad 500030, India b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 October 2014 Received in revised form 29 January 2015 Accepted 30 January 2015
Segment-2 (seg-2) of a bluetongue virus seropype-1 (BTV-1) isolate WGV104/08/Ind of Indian origin was sequenced and its neutralization behavior was studied to understand the antigenic similarity and relationship with other BTV-1 isolates. Multiple alignments of the coding region of seg-2 of WGV104/08/Ind revealed 97.6–99.0% and 97.2–98.4% similarity with other Indian BTV-1 isolates at nucleotide and deduced amino acid sequence level respectively. Several conservative and non-conservative substitutions were observed on the deduced VP2 amino acid sequence of WGV104/08/Ind. Non-conservative substitution of Lys119Glu on the B-cell epitope and Arg330Gly on the neutralizing epitope of VP2 of this isolate was observed. Using isolate-specific heterologous hyperimmune serum (HIS) the phenotypic antigenic relationship (r) was determined between WGV104/08/Ind and other Indian BTV-1 isolates which ranged from 0.092 to 0.208. The relationship score ranged from 0.203 to 0.295 when neutralization behavior of other Indian BTV-1 isolates was studied with the HIS of WGV104/08/Ind. Antigenic similarity (R) between WGV104/ 08/Ind and other Indian BTV-1 isolates was estimated from a reciprocal crossneutralization study and ranged from 14.70% to 24.80% indicating existence of major subtype antigenic divergence and neutralization resistant behavior of WGV104/08/Ind. ß 2015 Elsevier B.V. All rights reserved.
Keywords: Antigenic similarity Bluetongue virus serotype 1 Segment-2 Neutralization resistance VP2 phenotypic variant
1. Introduction Bluetongue virus (BTV), the type species of the genus Orbivirus within the family Reoviridae, is the causative agent of bluetongue, a major disease of ruminants. So far,
* Corresponding author. Present address: Eastern Regional Station, Indian Veterinary Research Institute, 37 Belgachia Road, Kolkata 700037, West Bengal, India. Tel.: +91 33 2558 2965; fax: +91 33 2556 5725. E-mail address: [email protected] (B. Mondal). http://dx.doi.org/10.1016/j.vetmic.2015.01.023 0378-1135/ß 2015 Elsevier B.V. All rights reserved.
26 distinct serotypes of BTV have been identified worldwide (Maan et al., 2011), but a recent paper describes a possible 27th serotype of the virus which is circulating among goats on Corsica (Zientara et al., 2014). BTV particles consist of three concentric protein layers surrounding the 10 segments of linear double-stranded (ds) RNA genome. The dsRNA segments encode seven structural proteins (VP1 to VP7), with an additional four non-structural (NS1 to NS4) proteins observed during infection and replication (Belhouchet et al., 2011). Segment 2 (seg-2; encoding VP2) is the most variable of
S.K. Biswas et al. / Veterinary Microbiology 176 (2015) 358–364
the BTV segments and VP2 is solely responsible for serotype determination and it is also the key determinant of neutralizing antibody specificity (Shaw et al., 2013). Genetic diversity among the BTV serogroup is clearly established on the VP2 gene, being the most divergent among different BTV serotypes and nucleotypes (Maan et al., 2007). BTV evolves through a combination of genetic drift and shift and therefore, quasispecies evolution coupled with founder effect and evolutionary selective pressure lead to the development of genetic and phenotypic variants of the virus (Bonneau et al., 2001). Phenotypic variations of BTV are constantly generated in the field and neutralization behavior may vary amongst the field strains of the same serotype. Bluetongue is enzootic in India and a wide prevalence of the virus has been reported in domestic ruminants by seroepidemiological studies. Amongst the BTV serotypes circulating in India, BTV-1 is one of the most predominant serotypes as evident by serological surveillance and isolation of a large number of viruses from different parts of the country (Jain et al., 1986; Prasad et al., 1994; Biswas et al., 2010). The present study was conducted to understand seg-2 sequence variability and neutralization behavior of a novel BTV-1 isolate recovered from the southern part of India. 2. Materials and methods
359
2.3. DNA sequencing, comparison of sequence data and phylogenetic analysis The amplified overlapping fragments of DNA were sequenced with Big dye terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Inc., CA, USA) on an automated sequencer following the manufacturer’s instructions. The generated nucleotide sequences were confirmed by BLASTN (http://www.ncbi.nlm.nih.gov/) analysis and the overlapping fragments were assembled with the help of the EditSeq program implemented in the DNASTAR program package (DNASTAR Inc., Madison, USA) to generate a complete seg-2 coding sequence of the virus. Nucleotide sequences were aligned with sequences of other BTV VP2 encoding genes (Table 1) using the CLUSTAL W algorithm (Thompson et al., 1994) implemented in the MegAlign of DNASTAR program package and aligned sequence data was used for phylogenetic analyses. The evolutionary history was inferred using the Maximum Likelihood (ML) method from 1000 replicates (Felsenstein, 1985), taken to represent the evolutionary history of the taxa analyzed. The best suitable model for analysis of evolutionary distances used to infer the phylogenetic tree was selected by the model selection program implemented in MEGA version 6.0 software (Tamura et al., 2013). 2.4. Production of hyperimmune serum and neutralization assay
2.1. Virus isolate The virus used in this study was isolated from a Nellore breed of sheep affected with bluetongue in the West Godavari district of Andhra Pradesh in 2008. Blood samples were collected from sheep showing symptoms of necrotic stomatitis, laminitis, facial edema, nasal discharge and pyrexia. The virus was initially isolated on embryonated chicken egg and subsequently adapted in BHK-21 cells. The isolate (WGV104/08/Ind) was confirmed as BTV-1 and submitted to the Bluetongue virus Repository under ICAR All India Network Program on Bluetongue (AINP-BT). The virus was revived using BHK-21 cultures, seg-2 was sequenced and hyperimmune serum (HIS) was produced. Ten other BTV-1 isolates (Table 1) and their corresponding HIS were also used in neutralization studies. 2.2. RNA isolation, cDNA synthesis and PCR amplification of VP2 gene Total RNA was extracted from BTV-1 (WGV104/08/ Ind)-infected BHK-21 cells (75 cm2 culture flask) using TRI1-Reagent (Sigma) according to manufacturer’s instructions and viral dsRNA was purified by lithium chloride precipitation (Attoui et al., 2000). Purified dsRNA segments were separated by electrophoresis on 1% agarose gel and seg-2 was excised from the gel and extracted using the QIAquick1 Gel extraction kit (Qiagen) according to the manufacturer’s instructions. Gel extracted seg-2 was used for synthesis of cDNA and PCR amplification of complete coding region in multiple overlapping fragments with different oligonucleotide primer pairs as described by Biswas et al. (2010).
The WGV104/08/Ind isolate was purified from a bulk culture of infected BHK-21 cells by ultracentrifugation through discontinuous sucrose gradients (Mertens et al., 1987) and HIS was produced in rabbits against purified virion following a procedure used earlier for hyperimmunization with a different BTV serotype (Pathak et al., 2008). The HIS was clarified, inactivated (at 56 8C for 30 min), filtered through 0.45 mm membrane filters and stored in multiple aliquots at 80 8C to avoid repeated freezing and thawing. WGV104/08/Ind was neutralized with homologous HIS and also with ten heterologous isolate-specific HIS (prepared earlier in our laboratory) against Indian BTV1 strains (Table 1). Serum neutralization test (SNT) was performed on BHK-21 cells according to the method described by MacLachlan et al. (1992) in 96-well tissue culture plate using 200 TCID50 (fixed) of each of 18 viruses (Table 1) and two-fold serial dilution of individual HIS. Serum neutralization titer was calculated by the method of Reed and Muench (1938) and the mean of anti-log values of three individual runs was considered as mean neutralization titer of the HIS against a particular virus isolate. 2.5. Determination of phenotypic antigenic relationship and antigenic similarity Phenotypic antigenic relationship (r) was measured as the ratio of the mean heterologous anti-log titer to the mean of the thomologous anti-log titer as described by Archetti and Horsfall (1950). Reciprocal cross-neutralization data of the isolates was used to determine the antigenic similarity of WGV104/08/Ind with other Indian BTV-1 isolates and calculated in terms of ‘R’ value
S.K. Biswas et al. / Veterinary Microbiology 176 (2015) 358–364
360
Table 1 Characteristics of BTV-1 isolates/strains described in this study. Isolates ID a
WGV104/08/Ind Hisar/85/Indb Avikanagar/94/Indb Chennai/03/Indb KDP15/07/Indb NLG3/07/Indb NRT37/07/Indb SKN7/07/Indb SKN8/07/Indb MKD18/08/Indb MKD25/08/Indb MKD19/08/Ind MKD20/08/Ind MKD21/08/Ind MKD22/08/Ind MKD23/08/Ind MKD24/08/Ind PTG13/11/Ind AJ585116/87/Mal KC879616/79/Chi AJ585178/81/Aus M21844/88/Aus FJ437557/07/Fra KJ019206/13/Ita AJ585110/vac/SA AJ585122/Ref/SA AJ585117/87/Sud AJ585119/82/Cam
GenBank
Species
Year
Place of origin
SNT
Reference
KF563945 KF563929 KF563930 KF563932 KF563933 KF563934 KF563935 KF563936 KF563937 HM014236 KF563944 KF563938 KF563939 KF563940 KF563941 KF563942 KF563943 KF563946 AJ585116 KC879616 AJ585178 M21844 FJ437557 KJ019206 AJ585110 AJ585110 AJ585117 AJ585118
Sheep Sheep Sheep Sheep Sheep Sheep Sheep Culicoides Culicoides Goat Goat Goat Goat Goat Goat Goat Goat Goat nk Sheep nk nk Ovine Ovine nk nk nk ovine
2008 1985 1994 2003 2007 2007 2007 2007 2007 2008 2008 2008 2008 2008 2008 2008 2008 2011 1987 1997 1981 1988 2007 2013 nk nk 1987 1982
West Godavari, AP, Ind Hisar, Haryana, Ind Avikanagar, RJ, Ind Thoothukkudi, TN, Ind Kadappa, AP, Ind Nalgonda, AP, Ind Guntur, AP, Ind Jasdan, Gujarat, Ind S.K. Nagar, Gujarat Mathura, UP, Ind Mathura, UP, Ind Mathura, UP, Ind Mathura, UP, Ind Mathura, UP, Ind Mathura, UP, Ind Mathura, UP, Ind Mathura, UP, Ind Pithoragarh, UK, Ind Kuala Lumpur, Malaysia China Australia Australia France Sardinia, Italy South Africa South Africa Sudan Cameroon
Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y N N N N N N N N N N
Present study Jain et al. (1986) Prasad et al. (1994) AINP-BT (2010) AINP-BT (2010) AINP-BT (2010) AINP-BT (2010) AINP-BT (2010) AINP-BT (2010) Biswas et al. (2010) Biswas et al. (2010) Biswas et al. (2010) Biswas et al. (2010) Biswas et al. (2010) Biswas et al. (2010) Biswas et al. (2010) Biswas et al. (2010) Bisht et al. (2011) Maan et al. (2004b) Zhu et al. (2013) Maan et al. (2004b) Gould (1988) Cetre-Sossah et al. (2011) Lorusso et al. (2014) Maan et al. (2004b) Maan et al. (2004b) Maan et al. (2004b) Maan et al. (2004b)
a
Isolate used for sequencing of seg-2 and preparation of HIS in the present study. HIS prepared earlier in our laboratory against this isolate; GenBank: NCBI GenBank accession number for seg-2/VP2 nucleotide sequence of BTV-1; Year: year of isolation; SNT: Isolate used in the serum neutralization test (SNT) in the present study; AP: Andhra Pradesh; RJ: Rajasthan; TN: Tamil Nadu; UK: Uttarakhand; UP: Uttar Pradesh; Ind: India; nk: not known; Y: yes; N: no. b
[R = (r1 r2)1/2 100, where r1 = (neutralization titre of virus-A with HIS of virus-B)/(neutralization titre of virus-A with HIS of virus-A) and r2 = (neutralization titre of virus-B with HIS of virus-A)/(neutralization titre of virus-B with HIS of virus-B), assuming relatedness of two viruses, namely virus-A and virus-B based on two way crossneutralization, carried out with hyper-immune serum against both viruses]. Antigenic similarity of the two viruses were assessed by the criteria described by Brooksby (1967) as follows: an R-value of 100% or close to 100% indicates antigenic identity between two viruses tested; an R-value >70% indicates little or no difference; an R-value between 33% and 70% indicates a minor subtype difference; an R-value between 11% and 32% indicates a major sub-type difference and an R-value between 0% and 10% indicates a different serotype. 3. Results 3.1. Sequencing and phylogenetic analysis of seg-2 The nucleotide sequence of seg-2 of BTV-1 isolate WGV104/08/Ind has been submitted to GenBank under the accession no. KF563945. BLASTN (NCBI) analysis of the sequence confirmed that it corresponds with seg-2/VP2 sequences that have previously been published for other BTV-1 strains. The coding region of seg-2 of WGV104/08/ Ind was 2886 bp in length (from nucleotide position 18 to 2903 including stop codon) giving rise to VP2 containing
961 amino acids. Analysis revealed unique substitutions of adenine (A) to guanine (G) at nt position 355 and 988. These substitutions do not seem to be sequence artifacts as evident from electropherograms of repeated runs. Nucleotide similarity was found to be 97.6% to 99.0% when compared with other isolates of BTV-1 from India. Phylogenetic tree segregated Indian BTV-1 isolates into a monophyletic cluster at close proximity to other ‘eastern’ topotype viruses. WGV104/08/Ind clustered with other Indian BTV-1 isolates in ML phylogenetic analysis indicating their close ancestral relationship (Fig. 1). 3.2. Amino acid sequence variation in deduced VP2 polypeptide There were twelve cysteine (Cys/C) residues (aa position 136, 162, 262, 281, 363, 446, 617, 784, 851, 870, 928 and 937) in the deduced VP2 amino acid sequence of WGV104/08/Ind. Multiple alignments of the deduced VP2 amino acid sequence revealed 97.2–98.4% similarity with other Indian BTV-1 isolates. Both conservative and non-conservative substitutions (in comparison to the consensus sequence) were evident on the deduced VP2 amino acid sequence of WGV104/08/Ind (Table 2). Nonconservative substitution of Lys119Glu was observed which lies on the proposed B-cell epitope region of BTV1 (Wei et al., 2013). Another non-conservative substitution of Arg330Gly was also observed in the neutralizing epitope of BTV-1 reported earlier (Gould and Eaton, 1990).
S.K. Biswas et al. / Veterinary Microbiology 176 (2015) 358–364
361
SKN7/07/Ind MKD22/08/Ind SKN8/07/Ind MKD25/08/Ind MKD23/08/Ind MKD24/08/Ind MKD19/08/Ind 81 MKD18/08/Ind
MKD21/08/Ind
India
Chennai/03/Ind MKD20/08/Ind
97
'Eastern' topotype
PTG13/11/Ind Hisar/85/Ind
85
Avikanagar/94/Ind 100
WGV104/08/Ind NRT37/07/Ind
100
KDP15/07/Ind
99 100
NLG3/07/Ind AJ585116/87/Mal KC879616/79/Chi
AJ585178/81/Aus 100
M21844/88/Aus
Australia
FJ437557/07/Fra
100
KJ019206/13/Ita
Europe
AJ585119/82/Cam
100
AJ585110/vac/SA
83
AJ585122/Ref/SA 71
'Western' topotype Africa
AJ585117/87/Sud
0.05
Fig. 1. Maximum likelihood (ML) tree (TN93 + I, MEGA 6.0) depicting phylogenetic relationship of WGV104/08/Ind with other BTV-1 isolates at complete coding region of VP2 gene nucleotide sequence. Bootstrap consensus was inferred from 1000 replicates to represent the evolutionary history of the taxa analyzed. Branch lengths are indicative of genetic distances between the sequences.
Table 2 Nucleotide substitutions in VP2 encoding gene and amino acid substitutions in deduced VP2 polypeptide of WGV104/08/Ind isolate of BTV-1. Nucleotide position
8 22 32 295 328 355 454 553 729 749 988 1063 2498
Nucleotide change
Amino acid change
Amino acid position
Consensus
WGV104/08/Ind
Consensus
WGV104/08/Ind
GAG ATC CAA TAC AAA AAG GTC ATA ATA CTG AGA ATA CTT
GGG GTC CGA CAC GAA GAG ATC GTA ATG CCG GGA GTA CCT
Glu Ile Gln Tyr Lys Lys Val Ile Ile Leu Arg Ile Leu
Gly Val Arg His Glu Glu Ile Val Met Pro Gly Val Pro
3 8 11 99 110 119* 152 185 243 250 330* 355 833
Note: Amino acid substitutions were determined in comparison to the consensus sequence generated by multiple alignment (ClustalW) of deduced VP2 sequence of Indian BTV-1 isolates used in neutralization study. Positions of amino acid change in the B-cell epitope and neutralizing epitope of VP2 of BTV-1 are marked with asterisk.
S.K. Biswas et al. / Veterinary Microbiology 176 (2015) 358–364
Hyperimmune Serum (HIS)
MKD25/08/Ind
MKD18/08/Ind
SKN8/08/Ind
SKN7/07/Ind
NRT37/07/Ind
NLG3/07/Ind
KDP15/07/Ind
isolates. Weak phenotypic antigenic relationship of WGV104/08/Ind was observed in an ex vivo neutralization study with heterologous isolate-specific HIS of other Indian BTV-1 isolates. Antigenic similarity is commonly used for assessment of antigenic relatedness between two viruses in reciprocal cross-neutralization studies. Antigenic similarity (R) between WGV104/08/Ind and other Indian BTV-1 isolates was estimated to be between 14.70% and 24.80% which indicates major subtype antigenic divergence and neutralization resistant behavior of WGV104/ 08/Ind [(an R value between 11% and 32% indicates a major subtype difference (Brooksby, 1967)].
Phenotypic antigenic relationship of Indian BTV-1 isolates with HIS of WGV104/08/Ind
PTG13/11/Ind
MKD24/08/Ind
MKD22/08/Ind
MKD23/08/Ind
MKD20/08/Ind
MKD21/08/Ind
MKD19/08/Ind
MKD25/08/Ind
SKN8/08/Ind
MKD18/08/Ind
SKN7/07/Ind
NRT37/07/Ind
NLG3/07/Ind
KDP15/07/Ind
Hisar/85/Ind
Avikanagar/94/Ind
1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 WGV104/08/Ind
Phenotypic antigenic relationship (r) score
MKD25/08/Ind
MKD18/08/Ind
SKN7/07/Ind
SKN8/08/Ind
NLG3/07/Ind
NRT37/07/Ind
KDP15/07/Ind
Chennai/03/Ind
Hisar/85/Ind
Avikanagar/94/Ind
WGV104/08/Ind
Phenotypiv antigenic relationship (r) score
1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
(b)
Chennai/03/Ind
Fig. 3. Antigenic similarity (R) amongst Indian BTV-1 isolates calculated from reciprocal cross-neutralization studies. Height of the bars indicate antigenic similarity score (R). Homologous similarity score is 100.00. Rvalue of 100% or close to 100%: antigenic identity, >70%: little or no antigenic difference, 33–70%: minor subtype difference, 11–32%: major sub-type difference, 0–10%: different serotype (Brooksby, 1967).
The outer capsid protein VP2, encoded by genome seg-2 is the most variable protein of BTV and is responsible for receptor binding and eliciting neutralizing antibodies to BTV. Phylogenic comparison of seg-2 from 24 South African reference strains and from over 200 other BTV isolates from around the world show a perfect correlation between sequence variation in seg-2 and BTV serotype (Maan et al., 2004a). The present study evaluated sequence data and the neutralization behavior of seg-2 of an Indian BTV-1 isolate WGV104/08/Ind. Although seg-2 is the most variable gene among the BTV genome segments, multiple nucleotide sequence alignments of WGV104/08/Ind revealed a high degree (97.2–98.4%) of similarity. ML tree constructed by using Tamura-Nei (TN93 + I) segregated ‘eastern’ (Indian/Australian) and ‘western’ (African/European) BTV isolates where WGV104/08/Ind clustered into the Indian monophyletic clade indicating a close evolutionary relationship of the isolate with other Indian BTV-1
Phenotypic antigenic relationship (r) of WGV104/08/Ind with isolate-specific HIS
Avikanagar/94/Ind
Virus
4. Discussion
(a)
Antigenic similarity (R) between WGV104/08/Ind and other Indian BTV-1 isolates
WGV104/08/Ind
Neutralization behavior of WGV104/08/Ind was studied against a total of ten isolate-specific heterologous BTV1 HIS along with the homologous HIS of the virus. The phenotypic antigenic relationship (r) of WGV104/08/Ind with other Indian BTV-1 isolates ranged from 0.092 to 0.208 with isolate-specific heterologous HIS where the homologous relationship was 1.0 (Fig. 2a). The relationship score ranged from 0.203 to 0.295 when neutralization behavior of other Indian BTV-1 isolates was studied against the HIS of WGV104/08/Ind (Fig. 2b). Antigenic similarity (R) between WGV104/08/Ind and other Indian BTV-1 isolates calculated from the reciprocal cross-neutralization study ranged from 14.70% to 24.80% where homologous similarity was considered as 100% (Fig. 3).
100 90 80 70 60 50 40 30 20 10 0
Hisar/85/Ind
Antigenic similarity score (R)
3.3. Neutralization behavior and phenotypic antigenic relationship
Chennai/03/Ind
362
BTV-1 isolates
Fig. 2. Phenotypic antigenic relationship (r) of WGV104/08/Ind with isolate specific HIS of Indian BTV-1 isolates (a) and other Indian BTV-1 isolates with HIS of WGV104/08/Ind (b). Homologous relationship score (virus isolate with corresponding HIS) is 1.0.
S.K. Biswas et al. / Veterinary Microbiology 176 (2015) 358–364
Amino acid substitutions in various escape mutant viruses localize mainly in two domains at positions 199– 213 and position 321–346 in VP2, although individual substitutions outside to these positions may also contribute to neutralization of BTV (DeMaula et al., 2000). Amino acid position 328–335 on VP2 is predicted to harbor the major neutralizing epitope of BTV-1 (Gould and Eaton, 1990). Multiple alignments of deduced VP2 amino acid sequences of WGV104/08/Ind with other Indian BTV-1 isolates showed an extreme conservativeness of amino acids in the domain spanned from 199 to 213. Single non-conservative substitution of Arginine (Arg/R) to Glycine (Gly/G) at amino acid position 330 was observed in WGV104/08/Ind. It is due to a point mutation at nucleotide position 988 on VP2 encoding gene sequence which is responsible for alteration of amino acid codon (AGA to GGA), resulted in substitution of Arg330Gly. A similar observation was also reported on VP2 of two different monoclonal antibody resistant Australian BTV-1 variants (Gould and Eaton, 1990). It is likely that the substitution of Arg330Gly is very crucial for phenotypic behavior of BTV-1 and may be responsible for the neutralization resistant behavior of WGV104/08/Ind. Another non-conservative substitution of Lys119Glu was observed which lies on the BTV-1 specific B-cell epitope reported earlier (Wei et al., 2013) although it has been proposed as non-neutralizing epitope. The possible consequences of neutralization resistance may be misclassification of such a variant leading to mistakes in epidemiological studies. Such neutralization resistant VP2 phenotypic variants have been described amongst isolates of BTV-23 and problems of misclassification were faced while serotyping these variants by conventional virus or serum neutralization tests (VNT or SNT) using polyclonal sera (Tembhurne et al., 2010). To avoid these problems, instead of VNT/SNT-based phenotypic serotyping, segment-2 (VP2 encoding gene) based genotypic serotyping has been adopted worldwide and recently an RT-PCR has been developed for identification and differentiation of 26 BTV serotypes by a serotypespecific amplification using seg-2 (Maan et al., 2012). The non-structural (NS) genes/proteins have also been used for development of group-specific RT-PCR and real time RTPCR (Dangler et al., 1990; Orru et al., 2006). Neutralization resistance can also pose problems during vaccination. All the neutralization resistant variants of a BTV serotype need to be incorporated in a vaccine for getting complete protection against the different isolates of the specific serotype. In conclusion, a novel BTV-1 isolate (WGV104/08/Ind) has been characterized that exhibits a special VP2 genotype and phenotype leading to neutralization resistance of this variant. Further studies involving a panel of neutralizing monoclonal antibodies or antibody to defined peptides are required to pinpoint the effect of specific amino acid(s) substitution (s) in neutralization behavior of this variant. Conflict of interest statement The authors declare that they have no competing interest.
363
Acknowledgements This work has been supported by an ICAR-funded project—All India Network Program on Bluetongue. Nucleotide sequencing was carried out at the Central FMDV Typing Lab, PD-FMD, Mukteswar. We thank the Director and Head of the Virology Division, IVRI for providing facilities and support.
References AINP-BT, 2010. Annual Report 2010–11. ICAR All India Network Program on Bluetongue. Indian Veterinary Research Institute, Izatnagar-243 122, Bareilly, Uttar Pradesh, pp. 56. Archetti, I., Horsfall Jr., F.L., 1950. Persistent antigenic variation of influenza A viruses after incomplete neutralization in ovo with heterologous immune serum. J. Exp. Med. 92, 441–462. Attoui, H., Billoir, F., Cantaloube, F.J., Biagini, P., Micco, P., Lamballerie, X., 2000. Strategies for sequence determination of viral dsRNA genomes. J. Virol. Methods 89, 147–158. Belhouchet, M., Jaafar, M.F., Andrew, E., Grimes, M.J., Mertens, P.P.C., Attoui, H., 2011. Detection of a fourth orbivirus non-structural protein. PLoS One 6, e25697. Bisht, B., Mondal, B., Biswas, S.K., Chand, K., Pandey, A.B., 2011. Isolation and molecular characterization of bluetongue virus from asymptomatic goats. Ind. J. Comp. Microbiol. Immunol. Infect. Dis. 32, 27–29. Biswas, S.K., Chand, K., De, A., Pandey, L.K., Mohapatra, J.K., Prasad, G., Mondal, B., 2010. Isolation of bluetongue virus serotype 1 (BTV-1) from goats and its phylogenetic relationship with other BTV-1 isolates worldwide based on full-length sequence of genome segment-2. Arch. Virol. 155, 2041–2046. Bonneau, K.R., Mullens, B.A., MacLachlan, N.J., 2001. Occurence of genetic drift and founder effect during the quasispecies evolution of the VP2 and NS3/NS3A genes of bluetongue virus upon passage between sheep, cattle and Cuilicoides sonorensis. J. Virol. 75, 8298–8305. Brooksby, J.B., 1967. Variants and immunity: definitions for serological investigation. International symposium on Foot and mouth disease, variants and immunity, Lyon, France. Symposium series. Immunobiol. Standard 8, 1–10. Cetre-Sossah, C., Madani, H., Sailleau, C., Nomikou, K., Sadaoui, H., Zientara, S., Maan, S., Maan, N.S., Mertens, P.P.C., Albina, E., 2011. Molecular epidemiology of bluetongue virus serotype 1 isolated in 2006 from Algeria. Res. Vet. Sci. 91, 486–497. Dangler, C.A., De Mattos, C.A., De Mattos, C.C., Osburn, B.I., 1990. Identifying bluetongue virus ribonucleic acid sequences by polymerase chain reaction. J. Virol. Me 28, 281–292. DeMaula, C.D., Bonneu, K.R., Maclachalan, N.I., 2000. Changes in the outercapsid protein of bluetongue virus serotype ten that abrogate neutralization by monolonal antibodies. Virus Res. 67, 59–66. Felsenstein, J., 1985. Confidence limits on phylogenesis: an approach using the bootstrap. Evolution 39, 783–791. Gould, A.R., 1988. Conserved and non-conserved regions of the outer coat protein VP2, of the Australian bluetongue serotype I virus, revealed by sequence comparison to the VP2 of North American BTV serotype 10. Virus Res. 9, 145–158. Gould, A.R., Eaton, B.T., 1990. The amino acid sequence of the outer coat protein VP2 of neutralizing monoclonal antibody-resistant, virulent and attenuated bluetongue viruses. Virus Res. 17, 161–172. Jain, N.C., Sharma, R., Prasad, G., 1986. Isolation of bluetongue virus from sheep in India. Vet. Rec. 119, 17–18. Lorusso, A., Marcacci, M., Ancora, M., Mangone, I., Leone, A., Marini, V., Camma, C., Savini, G., 2014. Complete genome sequence of bluetongue virus serotype 1 circulating in Italy, obtained through a fast next-generation sequencing protocol. Genome Announc. 2, E00093-14. Maan, N.S., Maan, S., Belaganahalli, M.N., Ostlund, E.N., Johnson, D.J., Nomikou, K., Mertens, P.P.C., 2012. Identification and differentiation of the twenty six bluetongue virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2. PLoS One 7 (2), e32601, http://dx.doi.org/10.1371/journal.pone.0032601. Maan, S., Maan, N.S., Nomikou, K., Veronesi, E., Bachanek-Bankowska, K., Manjunatha, N.B., Attoui, H., Mertens, P.P.C., 2011. Complete genome characterisation of a novel 26th bluetongue virus serotype from Kuwait. PLoS One 6, e26147, http://dx.doi.org/10.1371/journal.pone.0026147.
364
S.K. Biswas et al. / Veterinary Microbiology 176 (2015) 358–364
Maan, S., Maan, N.S., Samuel, A.R., O’Hara, R., Meyer, A.J., Rao, S., Mertens, P.P.C., 2004a. Completion of the sequence analysis and comparison of genome segment 2 (encoding outer capsid protein VP2) from representative isolates of the 24 bluetongue virus serotype. Vet. Ital. 40, 484–488. Maan, S., Maan, N.S., Samuel, A.R., Rao, S., Attoui, H., Mertens, P.P.C., 2007. Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes. J. Gen. Virol. 88, 621– 630. Maan, S., Samuel, A.R., Maan, N.S., attoui, H., Rao, S., Mertens, P.P.C., 2004b. Molecular epidemiology of bluetongue viruses from disease outbreaks in the Mediterranean Basin. Vet. Ital. 40, 489–496. MacLachlan, N.J., Rossitto, P.V., Heidner, H.W., Iezzi, L.G., Yilma, T.D., DeMaula, C.D., Osburn, B.I., 1992. Variation amongst the neutralizing epitopes of bluetongue viruses isolated in the United States in 1979– 1981. Vet. Microbiol. 31, 303–316. Mertens, P.P., Burrough, J.N., Anderson, J., 1987. Purification and properties of viral particles, infectious subviral particles and core of Bluetongue virus serotype 1 and 4. Virology 157, 375–386. Orru, G., Ferrando, M.L., Meloni, M., Liciardi, M., Savini, G., De Santis, P., 2006. Rapid detection and quantitation of Bluetongue virus (BTV) using a Molecular Beacon fluorescent probe assay. J. Virol. Methods 137, 34–42. Pathak, K.B., Biswas, S.K., Tembhurne, P.A., Hosamani, M., Bhanuprakash, V., Prasad, G., Singh, R.K., Rasool, T.J., Mondal, B., 2008. Prokaryotic expression of truncated VP7 of Bluetongue virus (BTV) and reactivity of the purified recombinant protein with all BTV type-specific sera. J. Virol. Methods 152, 6–12. Prasad, G., Garg, A.K., Minakshi Kakker, N.K., Srivastava, R.N., 1994. Isolation of bluetongue virus from sheep in Rajasthan state. Rev. Sci. Tech. Off. Int. Epiz 13, 935–938.
Reed, L.J., Muench, M., 1938. A simple method of estimating fifty percent end points. Am. J. Hyg. 27, 493–497. Shaw, A.E., Ratinier, M., Nunes, S.F., Nomikou, K., Caporale, M., Golder, M., Allan, K., Hamers, C., Hudelet, P., Zientara, S., Breard, E., Mertens, P., Palmarini, M., 2013. Reassortment between two serologically unrelated bluetongue virus strains is flexible and can involve any genome segment. J. Virol. 87, 543–557. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. Tembhurne, P.A., Mondal, B., Pathak, K.B., Biswas, S.K., Sanyal, A., Yadav, M.P., Bandyopadhyay, S.K., Singh, R.K., 2010. Segment-2 sequence analyses and cross-neutralization studies on some Indian bluetongue viruses suggest isolates are VP2-variants of serotype 23. Arch. Virol. 155, 89–95. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Wei, P., Sun, E.C., Liu, N.H., Yang, T., Xu, Q.Y., Zhao, J., Qin, Y.L., Feng, Y.F., Li, J.P., Wang, W.S., Zhang, C.Y., Wu, D.L., 2013. Identification of a novel bluetongue virus 1-specific B-cell epitope using a monoclonal antibody against the VP2 protein. Arch. Virol. 158, 1099–1104. Zhu, J., Yang, H., Li, H., Xiao, L., Wang, J., Li, N., Zhang, N., 2013. Full-genome sequence of bluetongue virus serotype 1 (BTV-1) strain Y863, the first BTV-1 isolate of eastern origin found in China. Genome Announc. 1, E00403–E00413. Zientara, S., Sailleau, C., Viarouge, C., Ho¨per, D., Beer, M., Jenckel, M., Hoffmann, B., Romey, A., Bakkali-Kassimi, L., Fablet, A., Vitour, D., Bre´ard, E., 2014. Novel bluetongue virus in goats, Corsica, France, 2014. Emerg. Infect. Dis. 20, 2123–2125.
