Detection and differentiation of old world orthopoxviruses: restriction fragment length polymorphism of the crmB gene region - PubMed (original) (raw)

Detection and differentiation of old world orthopoxviruses: restriction fragment length polymorphism of the crmB gene region

V N Loparev et al. J Clin Microbiol. 2001 Jan.

Abstract

A restriction fragment length polymorphism (RFLP) assay was developed to identify and differentiate Old World, African-Eurasian orthopoxviruses (OPV): variola, vaccinia, cowpox, monkeypox, camelpox, ectromelia, and taterapox viruses. The test uses amplicons produced from virus genome DNA by PCR with a consensus primer pair designed from sequences determined for the cytokine response modifier B (crmB) gene of 43 different OPV strains of known taxonomic origin. The primer pair amplified a single specific product from each of the 115 OPV samples tested. Size-specific amplicons identified and differentiated ectromelia and vaccinia virus strains, which contain a truncated crmB gene, and enabled their differentiation from other OPV species. Restriction digests of amplified products allowed the identification and differentiation of variola, monkeypox, camelpox, vaccinia, and cowpox virus species and strains.

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Figures

FIG. 1

Identification and differentiation of OPVs by PCR amplification of the crmB gene region from genomic DNA. Amplicons of the crmB gene region were obtained with consensus sequence primer pair VL2N-VL33. The sizes of PCR products were determined by direct sequencing of amplified products (18) to be as follows: CPV Brighton, Atlanta (1,369 bp) (lane 1), CPV strain M1 (1,307 bp) (lane 2), CPV strain M5 (1,297 bp) (lane 3), CPV strain M6 (1,322 bp) (lane 4), CPV strain M7 (1,312 bp) (lane 5), CPV strain M8 (1,309 bp) (lane 6), CPV strain M9 (1,291 bp) (lane 7), CPV strain 58 (1,309 bp) (lane 8), ECT MOS (382 bp) (lane 9), ECT MLH (382 bp) (lane 10), TPV Dahomey-1971 (1,303 bp) (lane 11), VAC Lister (1,208 bp) (lane 12), VAC Columbia (1,208 bp) (lane 13), BUF India 81-1985 (1,212 bp) (lane 14), BUF India-3906 (1,208 bp) (lane 15), RPV Utrecht (1,208 bp) (lane 16), VAR major Bangladesh-1975 (1,308 bp) (lane 17), VAR minor alastrim Brazil-Garcia-1966 (1,311 bp) (lane 18), human MPV Congo-8 (1,317 bp) (lane 19), MPV Copenhagen (1,318 bp) (lane 20), CML Somalia-1978 (1,315 bp) (lane 21), and CML (CP-5) Dubai-M4 (1,315 bp) (lane 22).

FIG. 2

Comparative _Nla_III RLFP analysis of PCR-amplified crmB fragments from intact genomes of OPV isolates. _Nla_III fragment sizes noted in parentheses were determined by direct sequence analysis of the crmB gene region (18) and agreed with sizes of fragments calculated by comparison to the 100-bp DNA ladder size marker (M). Lanes: 1, VAR major Bangladesh-1975; 2, VAR minor alastrim Brazil-Garcia-1966; 3, human MPV Congo-8; 4, human MPV 71-0082; 5, CML Somalia-1978; 6, CML (CP-5) Dubai-M4; 7, CPV Brighton; 8, CPV strain M1; 9, CPV strain M5; 10, CPV strain M6; 11, CPV strain M7; 12, CPV strain M8; 13, CPV strain M9; 14, CPV strain 58; 15, VAC Lister; 16, VAC Columbia; 17, BUF India-81-1985; 18, BUF India-3906; 19, RPV Utrecht; 20, TPV Dahomey-1971.

FIG. 3

RLFP analysis after _Aci_I digestion of PCR-amplified crmB fragments from genomes of VAR isolates. Lanes: 1, VAR major strain Bangladesh-1975; 2, VAR major strain Congo-1970; 3, VAR major Harvey-1944; 4, African VAR minor Somalia-1977; 5, “Whitepoxch9-2; 6, “Whitepox” ch9-4; 7, alastrim VAR minor strain Sierra Leone-1968; 8, alastrim VAR minor Garcia-1966.

FIG. 4

RLFP analysis after _Sau_3A digestion of PCR-amplified crmB fragments from genomes of MPV isolates. Lanes: 1, MPV Zaire 74-0226; 2, MPV Zaire 77-0666; 3, MPV Zaire 79-0005; 4, MPV Congo-8; 5, MPV Liberia 70-187; 6, MPV Zaire 96-16; 7, MPV Sierra Leone; 8, MPV Nigeria 71-0082; 9, MPV Benin-1978 78-3945; M, size marker (100-bp DNA ladder).

FIG. 5

RLFP analysis of PCR-amplified crmB fragments from genomes of CML and TPV isolates. _Nla_III digest fragments of the PCR products of CML Mauretania (lane 1), CML Somalia-1978 (lane 2), CML Niger (lane 3), CML (CP-1) Iran-M2 (lane 4), CML-Saudi-M3 (lane 5), and TPV Dahomey-1971 (lane 6) are shown in the left panel. _Nci_I digest fragments of the PCR products of TPV Dahomey-1971 (lane 7), CML Somalia-1978 (lane 8), CML Niger (lane 9), CML Mauretania (lane 10), and CML Iran-M2 (lane 11) are shown in the right panel. Size markers are provided by a 100-bp DNA ladder (lane M).

FIG. 6

_Nla_III RLFP analysis of PCR-amplified crmB fragments from DNA of CPV isolates from human and animals. _Nla_III digest fragments after PCR amplification of OPV85 (human) (lane 1), OPV88/L (cat) (lane 2), OPV88/H (cat) (lane 3), OPV89/1 (cat)-M5 (lane 4), OPV89/2 (cat) (lane 5), OPV89/3 (cat) (lane 6), OPV89/4 (cat)-M6 (lane 7), OPV89/5 (cat)-M7 (lane 8), OPV90/1 (cat)-M8 (lane 9), OPV90/2 (human) (lane 10), OPV90/4 (dog) (lane 11), OPV90/5 (cat)-M9 (lane 12), OPV91/1 (cat) (lane 13), OPV91/2 (human) (lane 14), OPV91/3 (cow) (lane 15), CATPOX3 (lane 16), CATPOX5 (lane 17), RAT Moscow (rat) (lane 18), EP-1 (elephant) (lane 19), EP-2 (elephant)-M1 (lane 20), EP-3 (elephant) (lane 21), EP-4 (elephant) (lane 22), EP-5 (elephant) (lane 23), CPV BRT-Atlanta (lane 24), and CPV BRT-Munich (lane 25) are shown.

FIG. 7

_Nla_III RLFP analysis of PCR-amplified crmB fragments from DNA of VAC variants. _Nla_III digest fragments of the PCR products of VAC COL (lane 1), VAC V (lane 2), VAC LIST (lane 3), VAC VNV (lane 4), VAC VCX (lane 5), VAC Wyeth (lane 6), VAC CV1-78 (lane 7), BUF 81 (lane 8), BUF 3906 (lane 9), RPV UTR (lane 10), Levaditi (lane 11), VAC M1 (lane 12), VAC COP wt (lane 13), VAC COP hr (lane 14), RPV Utrecht (lane 15), VAC Hagen (lane 16), VAC CVA (lane 17), VAC WR (lane 18), VAC Elstree (lane 19), and BUF BP-1 (lane 20) are shown.

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Detection and differentiation of old world orthopoxviruses: restriction fragment length polymorphism of the crmB gene region - PubMed (original) (raw)