Journal of Virological Methods, 35 (1991) 227-236 0 1991 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/91/%03.50
227
VIRMET 01254
Rapid detection of hog cholera virus in tissues by the polymerase chain reaction Shih-Tung Liu’, Shui-Nin Li’, Ding-Cheng Wang’, Shu-Fen Chang2, Su-Chuan Chiang’, Wei-Chuang Ho2, Yu-Sun Chang’ and Shiow-Suey Lai2 ‘Department of Microbiology and Immunology, Chang-Gung Medical College, Kwei-Shari.. Taoyuan, Taiwan, and ‘Department of Veterinary Medicine, National Taiwan University, Taipei, Taiwan, R.O.C.
(Accepted 25 July 1991)
Summary A rapid method for the detection of hog cholera virus (HCV) in infected tissues, using polymerase chain reaction (PCR) was developed. Total RNA isolated from HCV-infected tissues was reverse transcribed with AMV reverse transcriptase and the resulting complementary DNA was amplified by Taq DNA polymerase in the presence of two HCV-specific primers. The amplified DNA fragment was detected by agarose gel electrophoresis. The sensitivity of this method was at lo4 TCIDSO of HCV. The sensitivity increased approximately lOOO-foldwhen the DNA was reamplified with a set of nested primers. DNA sequencing analysis of the PCR products revealed that the HCV sequence amplified from a local field isolate was highly homologous to the HCV Alfort strain. This method may be useful for pathological and epidemiological studies of HCV in pigs. Hog cholera
virus; Polymerase
chain reaction
Introduction Hog cholera is a devastating disease of swine which causes serious economical loss in the pig industry annually. The etiological agent of this disease, the hog cholera virus (HCV), is a small enveloped RNA virus Correspondence to: S.-T. Liu, Molecular Genetics Laboratory, Dept. of Microbiology and Immunology, Chang-Gung Medical College, Kwei-Shan, Taoyuan, Taiwan 33332, R.O.C..
228
belonging to the genus Pestivirus of the Togaviridae family (Loan, 1964; Horzinek, 1973; Westaway et al., 1985). Serological evidences have shown that HCV is closely related to bovine diarrhea virus (BVDV) of cattle (Dones and Dubovi, 1987) and border disease virus (BDV) of sheep (Osburn et al., 1973). Recently, the complete genome of HCV has been determined (Meyers et al., 1989). The entire genome is 12 248 nucleotides long and is partially homologous to the genome of BVDV. The polymerase chain reaction (PCR), using Taq DNA polymerase of Thermus aquaticus has been shown to be a rapid and sensitive method for the detection of viral genome in biological samples. The procedure involves repetitive rounds of primer annealing and elongation by the Taq polymerase (Saiki et al., 1985). Amplitied DNA can be analyzed by agarose gel electrophoresis and visualized under UV light after ethidium bromide staining. For example, hepatitis B virus DNA at an initial amount of lo-l4 g could be detected by PCR (Kanebo et al., 1989a). A greater sensitivity (lo-l7 could be attained by Southern blot hybridization of the PCR product with !?IP-labeled HBV probe or by reamplitication with a set of nested primers (Kanebo et al., 1989b). In addition, the PCR method was also useful for the detection of RNA virus such as human immunodeficiency virus (HIV) in human blood (Ou et al., 1988). The procedure involved reverse transcription of viral RNA followed by PCR of the resulting cDNA (Byrne et al., 1988). Recently, this method has also been used for the detection of BVDV in tissue culture cells (Hertig et al., 1991). Currently, the diagnosis of HCV infection relies on the detection of viral antigens in the infected tissues by the immunofluorescent (IF) technique (Mengeling et al., 1963) or the cell culture technique (Lin et al., 1969). However, the immunological technique may lack specificity and the cell culture technique, on the other hand, lacks sensitivity, which may hamper the detection of the virus. We now report the use of the PCR method for the rapid and sensitive detection of HCV and demonstrate that this method is useful for the detection of HCV in pigs.
Materials and Methods Bacterial strains, plasmids, and culture medium Escherichia coli strain HBlOl was used as a cloning host. Plasmid pUC19 was used as a cloning vector (Yanisch-Perron et al., 1985). LB agar or LB broth was used as a general-purpose medium (Maniatis et al., 1982). LB containing 100 pg/ml of ampicillin was used for the selection of recombinant clones. Tissue culture technique and HCV purification HCV strain Taitung,
isolated in Taiwan in 1988, was cultured
in PK-15 cells
229
and was purified according to the sucrose gradient centrifugation previously described (Ho et al., 1987).
methods
Preparation of HCV RNA from viral particles
HCV RNA was purified from viral particles by a phenol-chloroform extraction procedure. This step was carried out by mixing the virus solution (100 ~1) with an equal volume of phenol-chloroform (1: l), followed by one minute of vortexing. The solution was centrifuged in a microfuge at 12 000 rpm for 3 min. The aqueous layer, containing HCV RNA, was transferred into a new microfuge tube and RNA was precipitated with 2.5 vol. of 95% ethanol at - 70°C for 30 min. HCV RNA was pelleted by centrifugation in a microfuge at 12000 rpm for 20 min at 4°C. The pellet was suspended in 60% ethanol and stored at -20°C. Preparation of RNA from tissues
Approximately 100 mg of tissue was minced into small pieces on ice with a sterile stainless surgical blade. The sample was homogenized in a microfuge tube with a micropestle (Eppendorf) in 0.7 ml of ice-cold GTC buffer (4 M guanidium isothiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% Sarkosyl, 0.1 M /&mercaptoethanol). Sodium acetate (pH 4.0) was then added to the solution to a final concentration of 0.1 M. The blood sample was used directly without homogenization, extracted with an equal volume of phenol-chloroform (1:l) and placed on ice for 15 min. The aqueous phase was separated by centrifugation at 4°C in a microfuge at 12000 rpm for 20 min. RNA in the solution was precipitated with an equal volume of isopropanol on dry ice for 20 min and then pelleted by centrifugation at 12000 rpm for 20 min at 4°C. RNA was then suspended in 60% ethanol and stored at -20°C. Primers
The primers were synthesized with a Cruachem DNA synthesizer (Model PS250), and were designed according to the genome of the Alfort strain (Meyers et al., 1989). The sequences of the primers used for PCR were: primer hcv-I, 5’-CTTATCTGGAGGGCCTTCTG-3’ (complementary to nucleotides 1469 to 1488); primer hcv-2, 5’-AGTGACAACGGCACTAATGG-3’ (nucleotides 1189 to 1208). The sequences of the nested primers were: hcv-3, 5’ATTCAGCGAGCCATGTATCT-3’ (nucleotides 1210 to 1229) and hcv-#, 5’TTCATTAACTGAATCCAAGG-3’ (complementary to nucleotides 1429 to 1448). Oligonucleotide hcv-P, 5’-TTTCAGTTCCGTGTCAGTGGCCAGATGAGT-3,’ (nucleotides 1291 to 1320) was used as a probe for DNA hybridization analysis.
230
Reverse
transcription
Reverse transcription of HCV RNA was carried out at 42°C for 30 min in 2.5 x Taq buffer (200 mM NaCl, 15 mM Tris-HCl, pH 7.4, 15 mM MgC12, 15 mM /I-mercaptoethanol, and 0.25 mM each of dATP, dCTP, dGTP, and dTTP). In addition to the Taq buffer, the reaction mixture (40 ~1) also contained HCV RNA, 2.4 U of avian myeloblastosis virus (AMV) reverse transcriptase (Promega) or M-MLV reverse transcriptase (Bethesda Research Laboratory), 16 U of RNasin (Promega), and 0.01 nmol primer hcv-2. The final volume of the reaction mixture was 40 ~1. Amplljkation
of cDNA
After reverse transcription, the following reagents were added to the reverse transcription mixture: 0.02 nmol of each nucleotide triphosphate (dATP, dCTP, dGTP, dTTP); 0.01 nmol of primer hcv-2; 1.5 U of Taq DNA polymerase. Water was then added to a final volume of 100 ~1. The reaction was carried out for 35 cycles in a Thermal Cycler (Perkin Elmer-Cetus). Each PCR cycle consisted of 30 s of denaturation at 94°C 1 min of renaturation at 45°C and 1 min of DNA chain elongation at 72°C. Analysis of ampliJied DNA
Amplified DNA was analyzed by gel electrophoresis on a 6-cm 2% agarose gel at 100 V for 30 min in 0.5 x TBE buffer (Maniatis et al., 1982). DNA bands in the gel were stained with ethidium bromide and examined under UV light. The HaeIII fragments of phage $X174 RF DNA (Promega) were used as molecular weight standards. DNA cloning and DNA sequencing
Amplified DNA was cloned into the SmaI site of pUC19 (Yanisch-Perron et al., 1985). Sequence of the cloned DNA fragment was determined by the chaintermination sequencing method (Sanger et al., 1977). The recombinant plasmids in E. coli were analyzed by the method of Kado and Liu (1981). Slot blot hybridization
Slot blot hybridization was carried out by transferring the PCR products onto a sheet of Zeta-probe membrane (Bio-rad) according to the method recommended by the supplier. Primer hcv-P was labeled with [Y-~~P]ATP (Amersham, 5000 Ci/mmol) by T4 polynucleotide kinase according to the method described by Maniatis et al. (1982).
231
Results
Reverse transcription of HCV RNA and ampliJication of HCV cDNA Because HCV is an RNA virus, it is necessary to reverse transcribe the viral genome into cDNA prior to PCR. This step was carried out in a reaction mixture containing 2.5 x Taq buffer, primer k-1, and either AMV or MMLV reverse transcriptase. After the reverse transcription, primer hcv-1 and Taq DNA polymerase were added to the buffer for DNA amplification. We found that the reaction with AMV enzyme produced a 300-bp band. On the other hand, no DNA product was detected when M-MLV reverse transcriptase was used for reverse transcription. We found that 2 U of Taq polymerase and 0.01 nmol of the primers gave the best results. Sensitivity We have used virus (10’ TCIDS,-J prepared from sucrose gradient centrifugation for the determination of the sensitivity of the PCR technique in detection of HCV. The virus was lo-fold serially diluted in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). RNA was then extracted from the solution, reverse transcribed, and amplified. The results showed that this technique was able to detect RNA isolated from HCV of lo4 TCIDsa (Fig. 1). AmpliJication with nested primers The PCR product (1 ~1) obtained from the amplification with primers hcv-l and hcv-2 was reamplified with a set of nested primers (hcv-3 and hcv-4) containing sequences immediately adjacent to the 3’ ends of hcv-l and hcv-2 of
ABC
DE
Fig. 1. Detection of HCV RNA extracted from infected cell culture by PCR. Lane A: Hat-111fragments of 4X174 RF DNA; lanes B-E: RNA isolated from 100000, 10000, 1000, and 100 TCIDso of HCV, respectively.
232
HCV DNA
Fig. 2. Reamplification of PCR products with nested primers. Lane A: HaeIII fragments of $X174 RF DNA; lanes B-G: RNA isolated from 100000, 10000, 1000, 100, IO, and 1 TCIDsa of HCV, respectively.
AB
Fig. 3. Hybridization
W lATeb.e..e
CD
of PCR products with a 32P-labeled HCV probe. DNA was amplified from (A) 100000, (B) 10000, (C) 1000 (D) 100 TCIDs,, of HCV.
CCCTCCAGAT . .
. . . . . . . .
A&G l
(T8itung
a+r8in)
.. (Altortltr8in)
Fig. 4. Partial sequence of the PCR product amplified from HCV strain Taitung. The sequence of the Alfort strain was obtained from Meyers et al. (1989). Position I of the sequence corresponds to position 1266 of the Alfort strain. (0) indicates identical base; (-) indicates a missing base.
233
ABCDE
Fig. 5. Detection of HCV in pig tissues obtained from a pig infected with HCV. (A) HaeIII fragments of #X174 RF DNA, (B) spleen, (C) lymph node, (D) tonsil, and (E) blood. Total RNA was isolated from tissues, reverse transcribed, and then amplified by PCR. The PCR products were detected by agarose gel electrophoresis. The largest band visible (300 bp) in lanes B-E is the DNA amplified from HCV RNA.
the HCV genome. The results showed that reamplification with these nested primers increased sensitivity by at least IOOO-foldto a level of l-10 TCIDsO (Fig. 2). Analysis
of the PCR product
DNA hybridization studies with 3ZP-labeled oligonucleotide hcv-P showed that the PCR product was homologous to the probe (Fig. 3). We have further cloned and sequenced the 300-bp PCR product. The results confirmed that the DNA fragment had indeed originated from the HCV genome (Fig. 4). Detection of HCV genome in biological specimens
We also examined pig tissues by PCR for the presence of HCV. These tissues, including submaxillary lymph node, spleen, brain, blood, pneumonary lymph node, kidney, inguinal lymph node, adrenal gland, large intestine, liver, heart, mesentery lymph node, tonsil, stomach, hepatogestic lymph node, lung, small intestine, and bladder, were obtained from a pig inoculated with HCV. The virus was detected in spleen, mesentery lymph node, tonsil (Fig. 5) and lung when primers hcv-I and hcv-2 were used for reverse transcription and PCR. On the other hand, HCV genome was detected in all the tissues listed above when the PCR products were reamplified with the nested primers.
234
Discussion We report herein the use of PCR for the detection of HCV in pig tissues. Because HCV is an RNA virus, it is necessary to reverse transcribe the HCV RNA prior to PCR. We used both AMV and M-MLV reverse transcriptase for the synthesis of cDNA. However, we were only able to amplify the HCV genome with a combination of AMV reverse transcriptase and Taq polymerase. It is likely that the M-MLV reverse transcriptase was not compatible with the buffer system that we used for reverse transcription. Because the AMV enzyme was used in Taq buffer and obtained amplification products, it was possible for us to carry out both reverse transcription and DNA amplification reactions in one microfuge tube. Primers hcv-I and hcv-2 were used for the amplification of the HCV cDNA and a detection limit of lo4 TCIDsa was reached (Fig. 1). This sensitivity is equivalent to the sensitivity for detection of hepatitis B virus DNA by PCR (Kanebo et al., 1989a). Kanebo et al. (1989b) were able to increase the PCR sensitivity by approx. lOOO-fold by reamplifying the PCR product with a set of nested primers. We obtained similar results and attained a sensitivity of 1 to 10 TCIDSO when primers hcv-3 and hcv-4 were used for reamplitication (Fig. 2). We also cloned and sequenced the PCR product to demonstrate that the product was indeed amplified from HCV genome. The sequence data revealed that the 300-bp DNA fragment which had been amplified was highly homologous to the corresponding segment in the Alfort strain, indicating that the DNA fragment had indeed originated from HCV. Among the variations, most of them are either A/G or T/C transitions. Results showed that amplification with primers ho-l and hcv-2 could detect virus of lo4 TCID5e. This sensitivity was adequate for most of the applications. For example, spleen is the primary target organ for HCV .infection. A large number of viruses could be found in spleen after infection. Therefore, the detection of HCV genome in the spleen by PCR with the tirst set of primers would be an indication of HCV infection. Further amplification with nested primers enabled us to detect the virus in all the tissues ‘examined. Therefore, amplification with the nested primers may be applicable when the detection of a very low number of viruses is necessary. In addition, PCR can be a valuable tool for the detection of chronic HCV infections in pigs, as the HCV antibody in these pigs may interfere with the detection of the virus by the IF technique. Because this method permits the detection of few HCV particles when nested primers are used, caution must be exercised to avoid any possibility of generating false-positive results due to the contamination of viral RNA or cDNA in any of the tissues, buffers, or primers. Proper positive and negative controls should be included in all PCR experiments.
235
Acknowledgements
We thank the Taiwan Provincial Research Institute for Animal health for supplying us pig tissues. This research was supported by a grant to S.T.L. from the Council of Agriculture, R.O.C.
References Byrne, B.C., Li, J.J. and Sninsky, J. (1988) Detection of HIV-l RNA sequence by in vitro DNA amplification. Nucleic Acids Res. 16, 4165. Dones, R.O. and Dubovi, E.J. (1987) Molecular specificity of the antibody responses of cattle naturally and experimentally infected with cytopathic and noncytopathic bovine diarrhea virus biotypes. Am. J. Vet. Res. 48, 1549-1554. Hertig, C., Pauli, U., Zanoni, R. and Peterhans, E. (1991) Detection of bovine viral diarrhea (BVD) virus using the polymerase chain reaction. Vet. Microbial. 26, 65-76. Ho, WC., Li, N.J. and Lai, S.S. (1987) Purification and electron microscopic observation of hog cholera virus. J. Chin. Sot. Vet. Sci. 13, 89-98. Horzinek, M. (1973) The structure of Togaviruses. Prog. Med. Viral. 16, 109-156. Kado, C.I. and Liu, S.T. (1981) Rapid procedure for detection and isolation of large and small plasmids. J. Bacterial. 145, 1365-1373. Kanebo, S., Feinstone, S. and Miller, R. (1989a) Rapid and sensitive method for the detection of serum hepatitis B virus DNA using the polymerase chain reaction technique. J. Clin. Microbial. 27, 193&1933. Kanebo, S., Miller, R., Feinstone, S., Unoura, M., Kobayashi, K. Hattori, N. and Purcell, R.H. (1989b) Detection of serum hepatitis B virus DNA in patients with chronic hepatitis using the polymerase chain reaction assay. Proc. Nat]. Acad. Sci. USA 86, 312-316. Lin, TX., Kang, B.J., Shimizu, Y., Kumagai, T. and Sasahara, J. (1969) Evaluation of the fluorescent antibody-cell culture test for detection and titration of hog cholera virus. Natl. Inst. Animal Health Quart. 9, 10-19. Loan, R.W. (1964) Studies of the nucleic acid type and essential lipid content of hog cholera virus. Am. J. Vet. Res. 25, 13661370. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Mengeling, W.L., Gutenkunst, D.E., Fernelius, A.L. and Pirtle, E.C. (1963) Demonstration of an antigenic relationship between hog cholera and bovine viral diarrhea viruses by immunofluorescence. Can. J. Comp. Med. Vet. Sci. 27, 162-164. Meyers, G., Riimenapf, T. and Thiel, H.J. (1989) Molecular cloning and nucleotide sequence of the genome of the hog cholera virus. Virol. 171, 555-567. Osbum, B.L., Clarke, G.L., Stewart, W.C. and Sawyer, M. (1973) Border disease-like syndrome in lambs. Antibodies to hog cholera and bovine virus diarrhea viruses. J. Am. Vet. Med. Assoc. 163, 1165-I 167. Ou, C.Y., Kwok, S., Mitchell, S.W., Mack, D.H., Sninsky, J.J., Krebs, J.W., Feorino, P., Warfield, D. and Schochetman, G. (1988) DNA amplification for direct detection of HIV-I in DNA of peripheral blood mononuclear cells. Science 239, 295-297. Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A. and Arnheim, N. (1985) Enzymatic amplification of /?-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 135&1354. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467.
236 Westaway, E.G., Brinton, M.A., Gaidamovich, S.Y., Horzinek, M.C., Igarashi, A., Kllriainen, L., Lvov, D.K., Portertield, J.S., Russell, P.K. and Trent. D.W. (1985) Togaviridae. Intervirol. 24, 125-139. Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved Ml3 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103-I 19.
227
VIRMET 01254
Rapid detection of hog cholera virus in tissues by the polymerase chain reaction Shih-Tung Liu’, Shui-Nin Li’, Ding-Cheng Wang’, Shu-Fen Chang2, Su-Chuan Chiang’, Wei-Chuang Ho2, Yu-Sun Chang’ and Shiow-Suey Lai2 ‘Department of Microbiology and Immunology, Chang-Gung Medical College, Kwei-Shari.. Taoyuan, Taiwan, and ‘Department of Veterinary Medicine, National Taiwan University, Taipei, Taiwan, R.O.C.
(Accepted 25 July 1991)
Summary A rapid method for the detection of hog cholera virus (HCV) in infected tissues, using polymerase chain reaction (PCR) was developed. Total RNA isolated from HCV-infected tissues was reverse transcribed with AMV reverse transcriptase and the resulting complementary DNA was amplified by Taq DNA polymerase in the presence of two HCV-specific primers. The amplified DNA fragment was detected by agarose gel electrophoresis. The sensitivity of this method was at lo4 TCIDSO of HCV. The sensitivity increased approximately lOOO-foldwhen the DNA was reamplified with a set of nested primers. DNA sequencing analysis of the PCR products revealed that the HCV sequence amplified from a local field isolate was highly homologous to the HCV Alfort strain. This method may be useful for pathological and epidemiological studies of HCV in pigs. Hog cholera
virus; Polymerase
chain reaction
Introduction Hog cholera is a devastating disease of swine which causes serious economical loss in the pig industry annually. The etiological agent of this disease, the hog cholera virus (HCV), is a small enveloped RNA virus Correspondence to: S.-T. Liu, Molecular Genetics Laboratory, Dept. of Microbiology and Immunology, Chang-Gung Medical College, Kwei-Shan, Taoyuan, Taiwan 33332, R.O.C..
228
belonging to the genus Pestivirus of the Togaviridae family (Loan, 1964; Horzinek, 1973; Westaway et al., 1985). Serological evidences have shown that HCV is closely related to bovine diarrhea virus (BVDV) of cattle (Dones and Dubovi, 1987) and border disease virus (BDV) of sheep (Osburn et al., 1973). Recently, the complete genome of HCV has been determined (Meyers et al., 1989). The entire genome is 12 248 nucleotides long and is partially homologous to the genome of BVDV. The polymerase chain reaction (PCR), using Taq DNA polymerase of Thermus aquaticus has been shown to be a rapid and sensitive method for the detection of viral genome in biological samples. The procedure involves repetitive rounds of primer annealing and elongation by the Taq polymerase (Saiki et al., 1985). Amplitied DNA can be analyzed by agarose gel electrophoresis and visualized under UV light after ethidium bromide staining. For example, hepatitis B virus DNA at an initial amount of lo-l4 g could be detected by PCR (Kanebo et al., 1989a). A greater sensitivity (lo-l7 could be attained by Southern blot hybridization of the PCR product with !?IP-labeled HBV probe or by reamplitication with a set of nested primers (Kanebo et al., 1989b). In addition, the PCR method was also useful for the detection of RNA virus such as human immunodeficiency virus (HIV) in human blood (Ou et al., 1988). The procedure involved reverse transcription of viral RNA followed by PCR of the resulting cDNA (Byrne et al., 1988). Recently, this method has also been used for the detection of BVDV in tissue culture cells (Hertig et al., 1991). Currently, the diagnosis of HCV infection relies on the detection of viral antigens in the infected tissues by the immunofluorescent (IF) technique (Mengeling et al., 1963) or the cell culture technique (Lin et al., 1969). However, the immunological technique may lack specificity and the cell culture technique, on the other hand, lacks sensitivity, which may hamper the detection of the virus. We now report the use of the PCR method for the rapid and sensitive detection of HCV and demonstrate that this method is useful for the detection of HCV in pigs.
Materials and Methods Bacterial strains, plasmids, and culture medium Escherichia coli strain HBlOl was used as a cloning host. Plasmid pUC19 was used as a cloning vector (Yanisch-Perron et al., 1985). LB agar or LB broth was used as a general-purpose medium (Maniatis et al., 1982). LB containing 100 pg/ml of ampicillin was used for the selection of recombinant clones. Tissue culture technique and HCV purification HCV strain Taitung,
isolated in Taiwan in 1988, was cultured
in PK-15 cells
229
and was purified according to the sucrose gradient centrifugation previously described (Ho et al., 1987).
methods
Preparation of HCV RNA from viral particles
HCV RNA was purified from viral particles by a phenol-chloroform extraction procedure. This step was carried out by mixing the virus solution (100 ~1) with an equal volume of phenol-chloroform (1: l), followed by one minute of vortexing. The solution was centrifuged in a microfuge at 12 000 rpm for 3 min. The aqueous layer, containing HCV RNA, was transferred into a new microfuge tube and RNA was precipitated with 2.5 vol. of 95% ethanol at - 70°C for 30 min. HCV RNA was pelleted by centrifugation in a microfuge at 12000 rpm for 20 min at 4°C. The pellet was suspended in 60% ethanol and stored at -20°C. Preparation of RNA from tissues
Approximately 100 mg of tissue was minced into small pieces on ice with a sterile stainless surgical blade. The sample was homogenized in a microfuge tube with a micropestle (Eppendorf) in 0.7 ml of ice-cold GTC buffer (4 M guanidium isothiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% Sarkosyl, 0.1 M /&mercaptoethanol). Sodium acetate (pH 4.0) was then added to the solution to a final concentration of 0.1 M. The blood sample was used directly without homogenization, extracted with an equal volume of phenol-chloroform (1:l) and placed on ice for 15 min. The aqueous phase was separated by centrifugation at 4°C in a microfuge at 12000 rpm for 20 min. RNA in the solution was precipitated with an equal volume of isopropanol on dry ice for 20 min and then pelleted by centrifugation at 12000 rpm for 20 min at 4°C. RNA was then suspended in 60% ethanol and stored at -20°C. Primers
The primers were synthesized with a Cruachem DNA synthesizer (Model PS250), and were designed according to the genome of the Alfort strain (Meyers et al., 1989). The sequences of the primers used for PCR were: primer hcv-I, 5’-CTTATCTGGAGGGCCTTCTG-3’ (complementary to nucleotides 1469 to 1488); primer hcv-2, 5’-AGTGACAACGGCACTAATGG-3’ (nucleotides 1189 to 1208). The sequences of the nested primers were: hcv-3, 5’ATTCAGCGAGCCATGTATCT-3’ (nucleotides 1210 to 1229) and hcv-#, 5’TTCATTAACTGAATCCAAGG-3’ (complementary to nucleotides 1429 to 1448). Oligonucleotide hcv-P, 5’-TTTCAGTTCCGTGTCAGTGGCCAGATGAGT-3,’ (nucleotides 1291 to 1320) was used as a probe for DNA hybridization analysis.
230
Reverse
transcription
Reverse transcription of HCV RNA was carried out at 42°C for 30 min in 2.5 x Taq buffer (200 mM NaCl, 15 mM Tris-HCl, pH 7.4, 15 mM MgC12, 15 mM /I-mercaptoethanol, and 0.25 mM each of dATP, dCTP, dGTP, and dTTP). In addition to the Taq buffer, the reaction mixture (40 ~1) also contained HCV RNA, 2.4 U of avian myeloblastosis virus (AMV) reverse transcriptase (Promega) or M-MLV reverse transcriptase (Bethesda Research Laboratory), 16 U of RNasin (Promega), and 0.01 nmol primer hcv-2. The final volume of the reaction mixture was 40 ~1. Amplljkation
of cDNA
After reverse transcription, the following reagents were added to the reverse transcription mixture: 0.02 nmol of each nucleotide triphosphate (dATP, dCTP, dGTP, dTTP); 0.01 nmol of primer hcv-2; 1.5 U of Taq DNA polymerase. Water was then added to a final volume of 100 ~1. The reaction was carried out for 35 cycles in a Thermal Cycler (Perkin Elmer-Cetus). Each PCR cycle consisted of 30 s of denaturation at 94°C 1 min of renaturation at 45°C and 1 min of DNA chain elongation at 72°C. Analysis of ampliJied DNA
Amplified DNA was analyzed by gel electrophoresis on a 6-cm 2% agarose gel at 100 V for 30 min in 0.5 x TBE buffer (Maniatis et al., 1982). DNA bands in the gel were stained with ethidium bromide and examined under UV light. The HaeIII fragments of phage $X174 RF DNA (Promega) were used as molecular weight standards. DNA cloning and DNA sequencing
Amplified DNA was cloned into the SmaI site of pUC19 (Yanisch-Perron et al., 1985). Sequence of the cloned DNA fragment was determined by the chaintermination sequencing method (Sanger et al., 1977). The recombinant plasmids in E. coli were analyzed by the method of Kado and Liu (1981). Slot blot hybridization
Slot blot hybridization was carried out by transferring the PCR products onto a sheet of Zeta-probe membrane (Bio-rad) according to the method recommended by the supplier. Primer hcv-P was labeled with [Y-~~P]ATP (Amersham, 5000 Ci/mmol) by T4 polynucleotide kinase according to the method described by Maniatis et al. (1982).
231
Results
Reverse transcription of HCV RNA and ampliJication of HCV cDNA Because HCV is an RNA virus, it is necessary to reverse transcribe the viral genome into cDNA prior to PCR. This step was carried out in a reaction mixture containing 2.5 x Taq buffer, primer k-1, and either AMV or MMLV reverse transcriptase. After the reverse transcription, primer hcv-1 and Taq DNA polymerase were added to the buffer for DNA amplification. We found that the reaction with AMV enzyme produced a 300-bp band. On the other hand, no DNA product was detected when M-MLV reverse transcriptase was used for reverse transcription. We found that 2 U of Taq polymerase and 0.01 nmol of the primers gave the best results. Sensitivity We have used virus (10’ TCIDS,-J prepared from sucrose gradient centrifugation for the determination of the sensitivity of the PCR technique in detection of HCV. The virus was lo-fold serially diluted in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). RNA was then extracted from the solution, reverse transcribed, and amplified. The results showed that this technique was able to detect RNA isolated from HCV of lo4 TCIDsa (Fig. 1). AmpliJication with nested primers The PCR product (1 ~1) obtained from the amplification with primers hcv-l and hcv-2 was reamplified with a set of nested primers (hcv-3 and hcv-4) containing sequences immediately adjacent to the 3’ ends of hcv-l and hcv-2 of
ABC
DE
Fig. 1. Detection of HCV RNA extracted from infected cell culture by PCR. Lane A: Hat-111fragments of 4X174 RF DNA; lanes B-E: RNA isolated from 100000, 10000, 1000, and 100 TCIDso of HCV, respectively.
232
HCV DNA
Fig. 2. Reamplification of PCR products with nested primers. Lane A: HaeIII fragments of $X174 RF DNA; lanes B-G: RNA isolated from 100000, 10000, 1000, 100, IO, and 1 TCIDsa of HCV, respectively.
AB
Fig. 3. Hybridization
W lATeb.e..e
CD
of PCR products with a 32P-labeled HCV probe. DNA was amplified from (A) 100000, (B) 10000, (C) 1000 (D) 100 TCIDs,, of HCV.
CCCTCCAGAT . .
. . . . . . . .
A&G l
(T8itung
a+r8in)
.. (Altortltr8in)
Fig. 4. Partial sequence of the PCR product amplified from HCV strain Taitung. The sequence of the Alfort strain was obtained from Meyers et al. (1989). Position I of the sequence corresponds to position 1266 of the Alfort strain. (0) indicates identical base; (-) indicates a missing base.
233
ABCDE
Fig. 5. Detection of HCV in pig tissues obtained from a pig infected with HCV. (A) HaeIII fragments of #X174 RF DNA, (B) spleen, (C) lymph node, (D) tonsil, and (E) blood. Total RNA was isolated from tissues, reverse transcribed, and then amplified by PCR. The PCR products were detected by agarose gel electrophoresis. The largest band visible (300 bp) in lanes B-E is the DNA amplified from HCV RNA.
the HCV genome. The results showed that reamplification with these nested primers increased sensitivity by at least IOOO-foldto a level of l-10 TCIDsO (Fig. 2). Analysis
of the PCR product
DNA hybridization studies with 3ZP-labeled oligonucleotide hcv-P showed that the PCR product was homologous to the probe (Fig. 3). We have further cloned and sequenced the 300-bp PCR product. The results confirmed that the DNA fragment had indeed originated from the HCV genome (Fig. 4). Detection of HCV genome in biological specimens
We also examined pig tissues by PCR for the presence of HCV. These tissues, including submaxillary lymph node, spleen, brain, blood, pneumonary lymph node, kidney, inguinal lymph node, adrenal gland, large intestine, liver, heart, mesentery lymph node, tonsil, stomach, hepatogestic lymph node, lung, small intestine, and bladder, were obtained from a pig inoculated with HCV. The virus was detected in spleen, mesentery lymph node, tonsil (Fig. 5) and lung when primers hcv-I and hcv-2 were used for reverse transcription and PCR. On the other hand, HCV genome was detected in all the tissues listed above when the PCR products were reamplified with the nested primers.
234
Discussion We report herein the use of PCR for the detection of HCV in pig tissues. Because HCV is an RNA virus, it is necessary to reverse transcribe the HCV RNA prior to PCR. We used both AMV and M-MLV reverse transcriptase for the synthesis of cDNA. However, we were only able to amplify the HCV genome with a combination of AMV reverse transcriptase and Taq polymerase. It is likely that the M-MLV reverse transcriptase was not compatible with the buffer system that we used for reverse transcription. Because the AMV enzyme was used in Taq buffer and obtained amplification products, it was possible for us to carry out both reverse transcription and DNA amplification reactions in one microfuge tube. Primers hcv-I and hcv-2 were used for the amplification of the HCV cDNA and a detection limit of lo4 TCIDsa was reached (Fig. 1). This sensitivity is equivalent to the sensitivity for detection of hepatitis B virus DNA by PCR (Kanebo et al., 1989a). Kanebo et al. (1989b) were able to increase the PCR sensitivity by approx. lOOO-fold by reamplifying the PCR product with a set of nested primers. We obtained similar results and attained a sensitivity of 1 to 10 TCIDSO when primers hcv-3 and hcv-4 were used for reamplitication (Fig. 2). We also cloned and sequenced the PCR product to demonstrate that the product was indeed amplified from HCV genome. The sequence data revealed that the 300-bp DNA fragment which had been amplified was highly homologous to the corresponding segment in the Alfort strain, indicating that the DNA fragment had indeed originated from HCV. Among the variations, most of them are either A/G or T/C transitions. Results showed that amplification with primers ho-l and hcv-2 could detect virus of lo4 TCID5e. This sensitivity was adequate for most of the applications. For example, spleen is the primary target organ for HCV .infection. A large number of viruses could be found in spleen after infection. Therefore, the detection of HCV genome in the spleen by PCR with the tirst set of primers would be an indication of HCV infection. Further amplification with nested primers enabled us to detect the virus in all the tissues ‘examined. Therefore, amplification with the nested primers may be applicable when the detection of a very low number of viruses is necessary. In addition, PCR can be a valuable tool for the detection of chronic HCV infections in pigs, as the HCV antibody in these pigs may interfere with the detection of the virus by the IF technique. Because this method permits the detection of few HCV particles when nested primers are used, caution must be exercised to avoid any possibility of generating false-positive results due to the contamination of viral RNA or cDNA in any of the tissues, buffers, or primers. Proper positive and negative controls should be included in all PCR experiments.
235
Acknowledgements
We thank the Taiwan Provincial Research Institute for Animal health for supplying us pig tissues. This research was supported by a grant to S.T.L. from the Council of Agriculture, R.O.C.
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