An approach for the sequencing of typed and untyped bluetongue virus isolates
By 
Y. Krishnamohan Reddy

Bluetongue virus (BTV) is the prototype virus of the Orbivirus genus in the Reoviridae family. Within the orbivirus genus, there are 19 serogroups which share antigens detectable in complement fixation tests, agar gel immunodiffusion tests and fluorescent antibody tests and hence there is certain degree of cross reactivity between distinct serogroups.It is a nonenveloped virus with concentric protein layers enclosing a dsRNA genome consisting of 10 segments. The outercapsid consists of two proteins, VP2 and VP5, where VP2 is the major determinant of serotype specificity. VP7 is a major core protein possessing the serogroup determining antigens.

The inner core exhibits icosahedral symmetry and is composed of two major proteins (VP3 and VP7). Three minor proteins (VP1, VP4 and VP6). Ten segments of dsRNA are also located within the core (L1-L3, M4-M6, S7-S10) coding for seven structural proteins and three nonstructural proteins (Fig.1). There are 24 serotypes and also there exists variations within the serotypes. 

More information on the BTV genome sequences will help in determining the degree of homology among various BTV isolates and also their phylogenetic relationship thereby facilitating the design of control programes by suitably incorporating appropriate serotypes of BTV isolates in the production of BTV vaccines. The sequence information will also be useful in identifying the conserved and variable regions in genome segment, which is useful in designing the molecular probes and diagnostics. Various software programmes are available for accessing the information about BTV gene sequences from the public domain data bases, alignment of sequences using clustalW, design of primers, PCR, p Draw 32 for cloning and sequencing.

In addition to the sequencing of L2 segment coding for VP2, sequencing of individual segment gives more accurate information about the phylogenetic relationship of individual isolates of BTV.

Sequencing of BTV genome

There are four approaches for the sequencing of BTV genome of dsRNA

1. Polyadenylation at 3' end of dsRNA and cDNA synthesis

2. Serotype specific and segment specific reverse transcription and cDNA synthesis using forward and reverse primers

3. Single primer amplification technique (SPAT)

4. Single - primer amplification sequence - independent dsRNA cloning procedure

1. Polyadenylation at 3' end of dsRNA and cDNA synthesis 

This is the original protocol followed for sequencing of BTV genome 

To obtain full-length DNA clones representing the entire genome of BTV, the viral dsRNA segments were initially isolated from infected cell extracts by agarose gel electrophoresis. The RNAs were then polyadenylated at their 3' ends and used for cDNA synthesis. Full length cDNA copies of each RNA segment were obtained and each was inserted into Pst I restriction sites of G - tailed pBR 322 plasmid (Fig.2) and then representative clones were sequenced (Polly Roy, 1991; Purdy et al., 1984, 1985; 1986; Lee & Roy, 1986, 1987; Ghiasi et al., 1985, 1987; Yu et al., 1987, 1988; Roy et al., 1988; Fukusho et al., 1987, 1989; Yamaguchi et al., 1988 a, b). Vector specific primers are used for this purpose.

Although they allowed the efficient cloning of dsRNA segments, this method required considerable amounts of RNA (>150΅g) and required insert sizing in recombinant cloning vectors and screening of candidate clones for target genes to be analysed.

The complete sequence of the BTV genome is 19, 218 base pairs (bp) long (13x106 Daltons). The sizes of the individual segments vary from 3944bp (segment L1; 2.7x106 Daltons) to 822bp (segment S10; 5x105 Daltons). The overall base composition of the genome is 21.9% (G+C); the base compositions of the individual segments are all similar and range from 24.6% to 20.7% (G+C). As shown in Table 2, the 5' non-coding regions range from 8 bp (M4) to 34 bp (M6) in length whilst the 3' non-coding regions are longer, ranging from 31 bp (M5) to 116 bp (S10).

Sequencing of the cloned segments has confirmed the presence of conserved terminal sequences on all ten segments as described above (i.e., 5' GUUAAA...3' and 5'...CACUUAC 3' on the positive-sense strands). In addition, nine of the ten segments have another A following the 5' conserved sequence (i.e., at position 7) and six of the segments have two A residues at positions 7 and 8. At the 3' ends of the positive-sense strands the conserved sequence is preceded by an AC in 7 of the 10 segments, and in the remaining 3 segments (4, 5 and 6) it is preceded by CA or CC. In addition, UU dimers are found proximal to the 3' ends of the RNAs in seven of the ten segments (1,2,5,6,7,8 and 9). The 3' non-coding regions of seven of the segments (3,4,5,7,8,9 and 10) are particularly purine-rich; it is not known if this plays any role in transcription, translation or morphogenesis.

Apart from segment 1 the first AUG codon on the positive RNA strand of each of the segments initiates a long open reading frame. Segment 1 has an additional AUG codon upstream of the codon which initiates the open reading frame (residues 7-9); it is not known if this affects the translational efficiency of the gene. Some of the initiating AUG codons have some of the features of the consensus flanking sequences for initiating codons as proposed by Kozak (1981) but none has all the features. For example seven of the segments (1,2,3,5,6,7,8) have a G at position +4 (counting the A of AUG as +1) and all of the segments, except segment 9, have a G, or an A at position - 3. All three possible translation termination codons are used; four gene products terminate with UGA, four with UAG and the remaining two with UAA.

Table 3 represents a summary of the predicted amino acid composition, location, size and net charge of all the primary gene products of BTV-10. The mean composition of all the products is : A, 76.7; R, 72.0; D, 59.7; N,38.1; C, 11.7; E, 74.3; Q, 38.6; G, 59.3; H, 33.1; I, 66.6; L, 81.3; K, 59.2; M, 37.8; F, 37.7; P,, 42.0; S, 56.2; T, 53.3; W, 12.5; Y, 33.5; V, 67.3. When individual gene products are compared to these values, several striking variations are evident. 

2. Serotype specific and segment specific reverse transcription and cDNA synthesis using Forward and Reverse Primers

Presently this method is being widely used for sequencing of genome of serotyped BTV isolates. Forward and Reverse primers are to be designed by drawing the serotype specific and segment specific sequences from public data bases. Then align the sequences of segment of interest of particular serotype by using ClustalW software. It full length of the segment is to be sequenced, then the primer design should be based on 5' and 3' terminal repeats. If there is no homology of one or two or three bases at specific region of interest for the primer sequence, then it may be substituted as follows :

B = C or G or T; D = A or G or T; H = A or C or T;
K = G or T; M = A or C; N = A or C or G or T;
R = A or G; S = C or G; V = A or C or G;
W = A or T; Y = C or T

General guidelines for designing the primers are to be followed. In that G+C/A+T ratio should be about 50%. Tm value of Forward and Reverse primers should be about equal. Annealing Temperature is about 4oC less than Tm value of the primer. Extension time would be 2 minutes for every 1kbp of length of gene.

The method requires purification of dsRNA and separation of individual segments by Agarose gel electrophoresis.

This second generation cloning and sequencing procedures made it possible to clone from much smaller amounts of starting material and PCR-based procedures depend on the prior availability of flanking sequence information of the gene of interest. The conservation of termini within the different reoviridae species allowed amplification of segment termini specific primers.

pDRAW 32 software is widely used for designing cloning strategies

3. Single primer amplification technique (SPAT)

By this protocol, complete genomic sequences can be generated from cultivable dsRNA viruses according to simple rule 'one genome, one month, one person'. In the case of noncultivable viruses (producing very low amounts of genome RNA), the sequence determination is also possible, but more time consuming (Attoui, et al., 2000).

Depending on the situation, different protocols need to be followed

1. Whenever possible, the separation of the genome to individual segments should be obtained (preferentially on agarose gels, or alternatively on polyacrylamide gels if necessary).

2. If segments cannot be separated (co-migrations cannot be resolved, or limited amounts of co-migrants), cloning should be realized on unseparated genes. In the case of small segments, colony screening permits the identification and sequencing of co-migrants. In case of longer segments, the normal cloning procedure permits the sequencing of one of the co-migrants which can be selectively digested from the cDNA mixture using a single cut site restriction enzyme. This allows the amplification, cloning and sequencing of other components.

3. Whenever 20 ng or more of purified and separated dsRNA are available for each segment, the technique of choice is the SMART™.

4. In other cases the SPAT should be preferred, using 'modified primers' whenever possible.

5. In the case where the genome cannot be visualized on agarose gels, a combination of SPAT and 3SM procedures can be used to determine genomic sequences.

The basic step in the SPAT is the ligation of a 3' blocked DNA oligo neucleotide to the both 3' ends of the ds RNA. This key step is achieved using the DNA-RNA ligation properties of the T4 RNA ligage. A double stranded cDNA is synthesized from the tailed dsRNA using a complementary primer. The cDNA is PCR amplified and the amplicons are inserted in to suitable cloning vectors. This last step is essential since the PCR product is obtained using single primer, a situation that renders direct sequence determination not feasible.

The cloning of the long segments (longer than 2kbp) is achieved using the SPAT with an anchored PCR and B1 and B2 primers using terminal repeat sequences of segment specific dsRNA.

By using the new strategies of sequencing, segment 2 of all the 24 serotypes of BTV were sequenced and compared at Institute for Animal Health, Pirbright, United Kingdom.

4. Single-primer amplification sequence independent dsRNA cloning procedure

Cloning full-length large (>3kb) dsRNA genome segments from small amounts of dsRNA is problematic. A single - primer amplification sequence - independent dsRNA cloning procedure for large genes was developed by Potgieter et al., (2002) without prior knowledge of any sequence information, sequencing of full length gene was possible.

Primer PC1[5' PO4 - GGATCCCGGGAATTCGG(A17)-NH2 3') was ligated to 1-900 ng of dsRNA followed by removal of excess RNA, cDNA annealing and filling in of partial duplexes. Amplification of cDNA was performed using primer PC2 (5' PO4 - CCGAATTCCCGGGATCC - OH3').

The protocol for amplification of the whole genome in one PCR was similar except that the oligo-ligation reactions of the whole genome were cleaned. cDNA was prepared from all the genome segments in one cDNA reaction and the whole genomes were amplified in one tube. Amplified cDNA products were separated on agar gel and individual segments are purified and the purified products are to be cloned in T/A cloning vectors. Positive clones are to identified on the basis of the size of the inserts. The full length sequences with terminal ends are sequenced with Universal primers.

Sequencing

Most DNA sequencing methods presently in use are variations of the chain termination method developed by Sanger and co-workers in the late 1970s. In this method, the DNA to be sequenced acts as a defined primer binding site. In automated sequencing a laser is used to detect DNA fragments labeled with fluorescent dyes. When different dye labels are used to tag each of the four dideoxynucleotides, the reactions can be performed in a single tube and analyzed on a single lane increasing the capacity and throughput of each sequencing gel. Thermostable DNA polymerase that have a high affinity for dideoxynucleotides such as AmplitaqTM FS from ABI and ThermosequenaseTM from Amersham have a modified active site that accommodates dideoxynucleotides more easily than the unmodified enzyme.

References

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2. Fukusho, A., Ritter, G.D. and Roy, P. (1987). Variation in the bluetongue virus neutralization protein VP2. J. Gen. Virol., 68 : 2967-2973.

3. Fukusho, A., Yu, Y., Yamaguchi, Y. and Roy, P. (1989). Completion of the sequence of bluetongue virus serotype 10 by the characterization of structural protein VP6 and a non-structural protein, NS2. J. Gen. Virol., 70:1677-1689.

4. Ghiasi, .H., Purdy, M.A. and Roy, P. (1985). The complete sequence of bluetongue virus serotype 10 segmetn 3 and its predicted VP3 polypeptide compared wth those of BTV serotype 17. Virus Res., 3:181-190.

5. Ghiasi, H., Fukusho, A., Eshita, Y. and Roy, P.(1987). Identification and characterization of conserved and variable regions in the neutralization VP2 gene of bluetongue virus. Virology, 160:100-109.

6. Lee, J. and Roy, P. (1986). Nucleotide sequence of a cDNA clone of RNA segment 10 of bluetongue virus (serotype 10). J. Gen Virol., 67:2833-2837.

7. Lee, J. and Roy, P. (1987). Complete sequence of the NS1 gene (M6 RNA) of US bluetongue virus erotype 10. Nucleic Acids Res., 15:7207.

8. Mertens, P.P.C., Brown, F and Sangar, D.V. (1984) Assignment of the genome segments of bluetongue virus type 1 to the proteins which they encode. Virology 135 : 207-217

9. Potgieter, A.C., Steele, A.D and van Dijk, A.A. (2002) Cloning of complete genome sets of six dsRNA viruses using an improved cloning method for large dsRNA genes J. Gen. Virol., 83 : 2215-2223. 

10. Purdy, M.A., Ghiasi, H., Rao, C.D. and Roy, P. (1985). Complete sequence of bluetongue virus L2 RNA that codes for the antigen recognized by neutralizing antibodies. J. Virol., 55:826-839.

11. Purdy, M.A., Petre, J. and Roy, P. (1984). Cloning of the bluetongue virus L3 gene. J. Virol., 51:754-759.

12. Purdy, M.A., Ritter, G.D., Roy, P. (1986). Nucleotide sequence of cDNA clones encoding the outer capsid protein, VP5, of bluetongue virus serotype 10. J. Gen. Virol., 67:957-962.

13. Roy, P. (1991). Towards the control of emerging bluetongue disease. Oxford Virology Publications, London.

14. Roy, P., Fukusho, A., Ritter, D.G., Lyons, D.(1988). Evidence for genetic relationship between RNA and DNA viruses from the sequence homology of a putative polymerase gene of bluetongue virus with that of vaccinia virus : Conservation of RNA polymerase genes from diverse species. Nucleic Acids Res., 16:11759-11767.

15. Samuel, A., Maan, S., Zientara, S., Sailleau, C., Knowles, N.J., Breard, Hammoumi, E.S., Mellor, P.S. and Mertens, P.P.C. (2002) Molecular epidemiology of bluetongue viruses from disease outbreaks in the Mediterranean basin. Page 9 Abstracts XII International congress of Virology, Paris

16. Yamaguchi, S., Fukusho, A., Roy, P. (1988a). Complete sequence of neutralization protein VP2 of the recent US isolate Bluetongue viru serotype 2: its relationship with VP2 species of other US serotypes. Virus Res., 11:49-58.

17. Yu, Y., Fukusho, A., Ritter, D.G., Roy, P. (1988). Complete nucleotide sequence of the group-reactive antigen VP7 gene of bluetongue virus. Nucleic Acids Res., 16:1620.

18. Yu, Y., Fukusho, A, Roy, P. P (1987). Nucleotide sequence of the VP4 core protein gene (M4 RNA) of US bluetongue virus serotype 10. Nucleic Acids Res., 15:7206.

Acknowledgement
Schematic structure of bluetongue virus is provided by Professor Polly Roy,Department of Infectious and tropical Diseases,London School of Hygiene and Tropical Medicine,Universty of London and Comparison of BTV serotypes is from the web site of Institute of Animal Health,Pirbright,United Kingdom.

Authors Corresponding address: 

Y. Krishnamohan Reddy
Vaccine Research Centre-Viral Vaccines,
Tamil Nadu Veterinary and Animal Sciences University,
Centre for Animal Health Studies, Madhavaram Milk Colony, Chennai - 600 051. India

Email : drykmreddy@hotmail.com 

The views expressed in this article are of the author(s), and any clarifications can be obtained from the author(s).