Journal of Virological Methods, 44 (1993) 235-240 0 1993 Elsevier Science Publishers B.V. All rights reserved / 0166-0934/93/$06.00

VIRMET 01572

Journal of Virological Methods

The Langat model for ,tick-borne encephalitis virus. Specific detection by RT-PCR

James Campbella, Lauren Iacono-Connorsb, Stephen Walza and Warren Schultza

“Naval Research Laboratory, Code 6900, Washington, D.C. (USA) and bU.S. Army Medical Research Institute of Infectious Diseases, Ft. Detrick, MD (USA)

(Accepted 21 June 1993)

Summary

We have developed a reverse-transcriptase polymerase chain reaction assay for rapid detection of Langat (LGT) virus, a flavivirus that is closely related to the highly pathogenic tick-borne encephalitis (TBE) viruses. Unlike TBE viruses, LGT virus exhibits a significantly lower virulence for man. The assay serves as a safe alternative for the development and optimization of specific assays for the highly pathogenic subtypes of TBE viruses that are endemic throughout much of Europe, the former Soviet Union, and China.

Langat; Reverse transcriptase polymerase chain reaction; Tick-borne encephalitis

Tick-borne encephalitis (TBE), a serious disease with a high mortality rate, is endemic throughout much of Europe, the former Soviet Union (FSU), and China. The etiologic agents of TBE are an antigenically closely related group of flaviviruses. On the basis of antigenic and epidemiologic criteria, TBE viruses have been divided into a Western (TBE W) subtype, endemic in central and eastern Europe and western regions of the FSU, and a Far Eastern (TBE FE) subtype, endemic in eastern FSU and China (Westway et al., 1985; Heinz, 1986). Since flaviviruses other than TBE can also cause encephalitis, rapid and agent specific laboratory tests are needed to guide health care providers and public health officials in devising appropriate clinical public health intervention strategies. While there exists a large body of information on the epidemiology

Correspondence to: S. Walz, Center for Biomolecular Science, Naval Research Laboratory, Code 6900, Washington, D.C. 20375, USA; Fax (202) 767-1295.

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and clinical characteristics of TBE, research on the agents themselves has been restricted due to safety considerations in dealing with the live virus. In the United States these viruses are considered P4 biohazards, and require the most stringent level of biocontainment. Fortunately, not all TBE antigenic complex flavivirus infections routinely produce encephalitis in the human host. Langat (LGT) virus, originally isolated from a tick in Malaysia (Smith, 1956) is particularly interesting in this regard. Although antigenically closely related to the highy virulent TBE viruses (Calisher et al., 1989), LGT virus exhibits a significantly lower virulence in man (Mayer, 1975). Using LGT virus as a model for TBE complex viruses, we have developed a reverse transcriptase- polymerase chain reaction (RT-PCR) assay for detecting specific nucleic acid sequences shared with TBE W and TBE FE, in purified viral RNA preparations and in tissue culture supernatant fluids.

The computer software package OLIGO (National Biosciences, Plymouth, MN) as described by Rychlik and Rhoads (Rychlik and Rhoads, 1989; Rychlik et al., 1990) was used to design a pair of 18-mer PCR amplification primers. These primers bracket a 387 base pair (bp) segment of the LGT virus genome, from nucleotide position 89 to position 476 of the published sequence (Mandl et al., 1991). These LGT primers, designated LVl (5-GGGTCCAAATGC- CAAATG-3) and LV2 (5-CACACCATGATCCCATGT-3’), are 100% homologous with the corresponding regions of the TBE FE genome, and 94% homologous (one internal mismatch each primer pairing, underlined in the sequences above) with the TBE W genome. Based on the published sequences of TBE FE and TBE W (Pletnev et al., 1990; Mandl et al., 1988), the PCR products resulting from amplification of these respective genomes would also be 387 bp. PCR primers were synthesized on an Applied Biosystems Model 320 automated DNA synthesizer and ethanol precipitated prior to use.

Langat virus strain TP21 (kindly supplied by R. Shope, Yale Arbovirus Research Unit, New Haven, CT) was propagated in LLC-MK2 cells, purified by PEG precipitation, concentrated by sedimentation in a linear 30% glycerol- 50% w/v potassium tartrate gradient, and then re-sedimented by rate zonal centrifugation on a lo%-65% w/v sucrose gradient as described by Repik et al. (1983). Viral RNA was recovered from purified virion preparations by lysis in 1% SDS followed by extraction one time with phenol/cresol/8-hydroxyquino- line and two times with phenol/chloroform. Extracted RNA was ethanol precipitated and resuspended to 0.16 pg/pl in deionized, distilled water. A total of 5 ~1 of Langat RNA (0.8 pg) served as template in a random hexamer primed reverse transcription (RT) reaction using a cDNA synthesis kit (Boehringer Mannheim Biochemicals, Indianapolis, IN). The cDNA was then phenol extracted, ethanol precipitated and resuspended in 20 ,ul water. Serial lo-fold dilutions of this LGT virus cDNA were then amplified in a 30 cycle PCR primed with LVl and LV2 (2 PM final concentration) under the following conditions: Denaturation at 94°C for 1 min, annealing at 37°C for 2 min and extension at 72°C for 3 min. Upon completion of the PCR, 5% (5 ~1) of the product was electrophoresed through a 4.0% GTG-NuSieve (FMC BioPro-

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Fig. 1. PCR amplification of LGT virus cDNA. LGT virus RNA (0.8 ng) served as template in a random hexamer primed reverse transcription reaction, and serial dilutions of the cDNA reaction products were amplified by PCR. Lane 1 contains a 1 kb DNA ladder (GIBCO BRL), and lanes 2, 3 and 4 contain PCR- amplified DNA from IO-‘, lo-’ and 10e3 dilutions of the RT reaction products. DNA fragments were

electrophoresed through 4.0% GTG-NuSieve (FMC BioProducts) and stained with ethidium bromide.

ducts, Rockland, ME) gel, stained with ethidium bromide (EtBr) and the fluorescence viewed and photographed over UV light (300 nm). The results of PCR amplification of serial dilutions of LGT cDNA are shown in Fig. 1. All three dilutions yielded a unique, EtBr stained DNA fragment with an apparent molecular size consistent with the predicted 387 bp amplification product. The amplification product from the 10e3 dilution of LGT virus cDNA was separated from the reaction mix by electrophoresis through a 0.7% agarose gel, electroeluted, ethanol precipitated and partial nucleotide sequence was determined. LVl was used to prime the sequence reaction for the ‘virus- sense’ strand of the cDNA, and LV2 was used to prime synthesis of the complementary strand. Sequencing reactions were performed using a Tuq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Fluorescence-based nucleic acid sequence was obtained using an Applied Biosystems 373A DNA Sequencer. Partial sequence data from the virus sense and virus complemen- tary strands of the 387 bp fragment were aligned, revealing 187 bases of identical overlapping sequence. This PCR product nucleic acid sequence was 100% homologous with the published nucleic acid sequence at the correspond- ing position of the LGT virus genome (data not shown). These data confirm that the RT-PCR assay amplifies the intended target LGT virus RNA.

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In order to determine the sensitivity of this RT-PCR for LGT virus particles, as opposed to purified LGT RNA, 50 ml of LGT virus-infected Vero cell tissue culture supernatant fluid (1.4 x lo5 LGT virus plaque forming units per ml) was subjected to centrifugation at 337 000 x g for 45 min. The viral pellet was resuspended in 100 ,ul of TE (10 mM Tris-HCl pH 8.&l mM EDTA) containing 1% NP-40 and 100 pg/ml Proteinase K to yield an approximate virus concentration of 7 x lo7 pfu/ml. Dissolution of the viral proteins and release of RNA was completed by incubation of the LGT virus suspension at 55°C for 1 hour as described by Innis et al. (1989). The RNA preparations were then heated at 95°C for 10 min to destroy the protease (Innis et al., 1989). A total of 2% (2 ~1) of this preparation, containing approximately 1.4 x lo5 copies of the viral genome, provided the viral RNA template for a 20 ~1 random hexamer primed RT reaction using an RT-PCR kit (Perkin Elmer Cetus). The RT reaction was also performed on serial two-fold dilutions of the concentrated viral RNA preparation. The 20 ,ul cDNA product of the RT reaction was combined with 78 ~1 of PCR master mix (deoxynucleosidetripho- sphates, buffer and Taq polymerase as supplied in the RT-PCR kit) and 1 ~1

1 2 3 4 5 6 7 8 2.3 Kb 2.0 Kb

0.56 Kb

Fig. 2. Sensitivity of RT-PCR detection of LGT virus particles from infected tissue culture supernatant fluid. LGT virus present in tissue culture fluid was concentrated to 7 x 10’ pfu/ml. A total of 2 ul of neat (lane 3) and serial two fold dilutions (lanes 4 through 8) of the concentrated virus were subjected to RT- PCR employing random hexamers in the RT step then LGT specific primers in 35 PCR cycles. A positive control RNA (IO4 copies) (Perkin Elmer Cetus) was also subjected to the same RT-PCR amplification using random hexamers and control upper and lower primers to produce a 308 bp fragment (lane 2). Lane 1 contains Lambda Hind111 molecular size. markers. Fragments were electrophoresed through 0.7% agarose

and stained with ethidium bromide.

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each of 20 PM LVl and 20 PM LV2 (final primer concentration 0.2 PM). PCR amplification was performed as follows: 2 min at 95°C for 1 cycle; 1 min at 95°C and 1 min at 54°C for 35 cycles; and 7 min at 60°C for 1 cycle. Positive control RNA, provided in the kit (lo4 copies), was also subjected to RT-PCR amplification as above using random hexamers and control upper and lower primers. RT-PCR amplified products were visualized by electrophoresis of 6.5 ~1 of each reaction through a 0.7% agarose, followed by EtBr staining and UV illumination. A comparison of test and control RT-PCR amplification products is presented in Fig. 2. A 1:8 dilution of the concentrated viral preparation (approximately 2 x lo4 copies of the LGT viral genome) yielded a sharp EtBr stained band with an apparent molecular size consistent with a 387 bp fragment (Fig. 2, lane 6). This band is slightly larger and of equivalent intensity to the control RNA RT-PCR product of 308 bp (Fig. 2, lane 2). Test template dilutions greater than 1:8 yielded no detectable PCR product of the predicted size (lanes 7 and S), while more concentrated templates yielded more intensely staining bands (lanes 3,4, and 5). The DNA bands of lower molecular weight evident in lanes 6, 7, and 8 represent spurious amplification products which apparently form when the target template is limiting.

Our results clearly indicate that detergent lysis and proteinase K digestion of test samples followed by RT-PCR assay as described above can be used to detect LGT virus particles in fluid specimens at concentrations equal to or in excess of lo7 pfu/ml. This level of sensitivity approaches the performance level of a widely used commercial RT-PCR kit (Perkin Elmer Cetus) for detecting positive control RNA template. Because the LGT primers are nearly 100% homologous with their target sites on the TBE W and TBE FE genomes, we predict that our assay system would detect these pathogens. The complete genomic sequence of Powassan virus, another member of the TBE serocomplex, has recently been published (Mandl et al., 1993). Significant homology exists between LVl, LV2 and target sites on the Powassan genome suggesting that this virus would also be detected using the described LGT assay. Computer homology searches of LVl and LV2 primer sequences against published sequence of flaviviruses not belonging to the tick-borne encephalitis serocomplex (dengue, yellow fever, Japanese encephalitis, and St. Louis encephalitis) failed to detect complementary sequence which would yield PCR amplification products under the stringent annealing temperature of 54°C.

Rapid and sensitive assays for detecting and identifying the various tick- borne encephalitis viruses are needed to reduce the morbidity and mortality associated with these diseases. To minimize the risk to laboratory personnel in developing and optimizing such assays, a relevant, non-pathogenic model is desirable. The LGT virus provides such a model, and the 4 h RT-PCR assay we describe allows research and development to be carried out safely under P2 biocontainment conditions. Studies to validate this assay with TBE virus isolates are in progress.

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References

Calisher, C. H., Karabatsos, N., Dalrymple, J., Shope, R.E., Porterfield, J. S. and Westaway, E. G. (1989) Antigenic relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera. J. Gen. Virol. 70, 3743.

Heinz, R. (1986) Epitope mapping of flavivirus glycoproteins. Adv. Virus Res. 31, 103-168. Innis, M., Gelfand, D., Sninsky, J. and White, T. PCR Protocols: A Guide to Methods and

Applications. Academic Press, 1989. Mandl, C. W., Heinz, F. X. and Kunz, C. (1988) Sequence of structural proteins of tick-borne

encephalitis virus (western subtype) and comparative analysis with other flaviviruses. Virology 166, 197-20s.

Mandl, C., Iacono-Connors, L., Wallner, G., Holzmann, H., Kunz, C. and Heinz, F. (1991). Sequence of the genes encoding the structural proteins of the low-virulence tick-borne flaviviruses Langat TP21 and Yelantsev. Virology 185, 891-895.

Mandl, C., Holzmann, H., Kunz, C. and Heinz, F. (1993). Complete genomic sequence of Powassan virus: evaluation of genetic elements in tick-borne versus mosquito-borne flaviviruses. Virology 194, 173-184.

Mayer, V. (1975) A live vaccine against tick-borne encephalitis: Integrated studies 1. Basic properties and behavior of the E514 clone (Langat virus). Acta Virol. 19, 209-218.

Pletnev, A. G., Yamshchikov, V. F. and Blinov, V. M. (1990) Nucleotide sequence of the genome and complete amino acid sequence of the polyproteins of tick-borne encephalitis virus. Virology 174, 250-263.

Repik, P., Dalrymple, J., Brandt, W., McCown, J. and Russell, P. (1983). RNA fingerprinting as a method for distinguishing dengue 1 virus strains. Am. J. Trop. Med. Hyg. 32, 577-589.

Rychlik, W. and Rhoads, R.E. (1989) A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucl. Acids Res. 17, 8543-8551.

Rychlik, W., Spencer, W. J. and Rhoads, R. E.(l990). Optimization of the annealing temperature for DNA amplification in vitro. Nucl. Acids Res. 18, 64096412.

Smith, C. E. G. (1956) A virus resembling Russian spring-summer encephalitis virus from an ixodid tick in Malaya. Nature 178 (4533), 581-582.

Westaway, E., Brinton, M., Gaidamovitch, S., et al. (1985). Flaviviridae. Intervirology 24, 183-192.