The possibility of use of a single hybridization temperature instead of different temperature for different probes for detection of human group A rotavirus (HRV) was explored. The sensitivity and specificity of the 5 oligonucleotide probes were assessed using cell culture adapted rotavirus (RV) strains by carrying out hybridization at a fixed temperature of 50℃. The results obtained indicate that a single hybridization temperature may be used for all the probes with virtually no decrease in specificity and sensitivity

Ali Ayub

Bingnan Fu

Hossain Ashfaque

Unicomb Leanne E.

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Trop. Med., 35 (3), 119-124, September, 1993 119Determination of Single Hybridization Temperature for Serotypingof HumanGroup A Rotavirus with Oligonucleotide ProbesAyub ALI1-2, Fu BINGNAN3, Leanne E. UNICOMB4, and Ashfaque HOSSAIN1department of Microbiology, University of Dhaka, Dhaka 1000, BangladeshDepartment of Virology, Institute of Tropical Medicine, Nagasaki University,Nagasaki 852, JapanProvincial Centre for Hygiene and Antiepidemic, Zhenghou, P.R. China4 Virology Laboratory, Laboratory Sciences Division, International Centre for Diar-rhoeal Disease Research, Bangladesh, GPO Box 128, Dhaka 1000, BangladeshAbstract: The possibility of use of a single hybridization temperature instead of differenttemperature for different probes for detection of human group A rotavirus (HRV) was ex-plored. The sensitivity and specificity of the 5 oligonucleotide probes were assessed usingcell culture adapted rotavirus (RV) strains by carrying out hybridization at a fixedtemperature of 50•Ž. The results obtained indicate that a single hybridization temperaturemaybe used for all the probes with virtually no decrease in specificity and sensitivity.Key words: Rotavirus, Hybridization temperature, Oligonucleotide probes, Serotype, Sen-sitivity and SpecificityINTRODUCTIONHumangroup A rotavirus (HRV), members of the family Reoviridae, genus Rotavirus,is the most important pathogen of acute diarrhoea in infants and young children on a globalscale (Kapikian et al, 1985). The virus possess a double stranded RNA (dsRNA) segmentswhich are enclosed within a double capsid protein shell. The outer layer contains two in-dependent serotype-specific neutralization proteins, VP7 and VP4 (Hoshino et al., 1985, Offitand Blavat, 1986). Protein VP7 is encoded by segments 7, 8 or 9, depending on the strains,and defines the VP7 or G-serotype and producing major neutralizing antibody whereas theVP4 protein is encoded by segment 4 and defines the VP4 type or P-type and producingminor neutralizing antibody (Estes and Cohen, 1989). In human and animal rotaviruses, 14VP7 serotypes have been identified so far of which at least 7 (serotype 1-4, 8, 9 and 12) areresponsible for human disease (Kapikian and Chanock, 1990, Urasawa et al., 1990). Based onthe information of nucleotide sequences of VP7 genes, Flores et al (1989) developed a dothybridization assay for distinguishing of RV serotypes 1 to 4 with CDNA probes. Recently aReceived for Publication October 9, 1993Correspondence: Ayub Ali, Department of Virology, Institute of Tropical Medicine, Nagasaki Universi-ty, 1-12-4, Sakamoto-machi, Nagasaki 852, Japan.120method has been reported for serotyping of HRV with synthetic 25 bases of oligonucleotideprobes (Sethabutr et aL, 1990, Bingnan et at., 1991, Ali et al, 1993). These probes aredesignated as HuGlAc, HuG2Ac, HuGSAc, HuG4Ac and HuGSAc and three differenttemperatures allowed hybridization of them. However use of different temperatures for dif-ferent probes is often inconvenient.In this report, we present our findings that a single hybridization temperature can beused for 5 different oligonucleotide probes for serotyping of HRV.MATERIALS AND METHODSCell and VirusesThe rhesus monkey kidney cell line, MA104 (Microbiological Associates, Bethesda,Maryland), was used to cultivate the rotavirus strains. MA104 cells were grown in theDulbecco's modified medium (GIBCO Laboratories, New York) supplimented with 10% foetalcalf serum (GIBCO Laboratories, New York), 100 units/ml penicillin and 100 jt/g/ml strep-tomycin. Rotavirus strains Wa (serotype 1), RV5 (serotype 2), RV3 (serotype 3), ST3(serotype 4) and B37 (serotype 8) were used in this experiment. All the virus strains weregrown following the method of Albert et al. (1987).Extraction of RNA from tissue culture supernatantVirus particles from tissue culture supernatants was concentrated by 20% polyethyleneglycol (MW 4000)/2.5 M NaCl. RNA extraction from concentrated virus preparation was car-ried out following the method of Herring et al. (1982). Briefly, a 450 /A volume of concen-trated virus preparation in phosphate buffered saline (PBS), pH 7.2, was mixed with 50 /d of1 M sodium acetate with 1% sodium dodecyl sulphate, pH 5.0, and then mixed with 500 piphenol: chloroform: isoamyl alcohol (25:24:1). After 1 min of vortexing, the mixture was cen-trifuged at 10,000 rpm for 4 min at room temparature. The supernatant was harvested andextracted once more with equal volume of phenol: chloroform: isoamyl alcohol. The extractedRNA was pelleted down with ethanol and purity of the ds RNA preparation was derterminedby spectrophotometer from the ratio of ODs at 260/280nm (Maniatis et aL, 1982).Dot hybridizationThe purified dsRNA was denatured with equal volume of 6.15 M formaldehyde-lOxSSC (l xSSC=150 mMsodium chloride plus 15 mM sodium citrate) for 15 min at 65°Cand serially diluted dsRNA from 5 rotavirus serotypes were spotted onto 5 separate pieces of0.45 pm pore nitrocellulose filter (Bio-Rad Laboratories, Richmond, CA). Filters were airdried and baked at 80°C for 2 hours. The five synthetic oligonucleotide probes (serotype 1-4,and 8) were kindly provided by Dr. Peter Escheverria of the Armed Forces Research In-stitute of Medical Sciences, Bangkok, Thailand. The 5' end labelling with T4 polynucleotidekinase (Bethesda Research Laboratories, USA) and |>-32P]-ATP (Amersham, UK) was carriedout as previously described (Sethabutr et al., 1990). The dotted membranes were hybridizedwith the serotype specific oligonucleotide probes at hybridization temparatures 46, 41, 41,121 ^^^^l^^lM^M^^M^^M^/^/M        W?;tffi^!^^^t!:: ^i^: ;m^ ^        rj »'.-/1'.;-?'à"; « * : * !-,-:-/ ,;?à"/ '/ s.'.r.< « ;..<à"'à"*':.* ;:; -<:i^Bh       v. ///,';:</s ,;:;;/,; :|'/r/;,/f;".;à"//,à"^.: ,<-/<v ;/ , //^jj% >-  iiiilllll!p:   ''ffiiL£* $££?*l*i$Ji^f*^?*?*~Ir*7**r?*;?i!«*?:??*?*< W^^^-f^- -. :'  '; -f ^^i-  ^!MJW&M^'!i(^'':'/^^ ^PE -' lv;..'.: ' 'à";.- à"' ',;';::'.'i:.  jfi:;fff^;:i;:(^.;H\^à"à":;':à",'s,-à"'à"'à".-,:.à" " à" à" !à"à"/:a;.;à"à"à".^;, '4\ ^a' ll ll  III  ;.-..:.,:41c, à" à""    à".s0°e:.",.,;.  K:&i:".:-':.. ,::.-- ;=r-.;r';SERgTYFB:fà"".,;£';.,à".'. ^.i^..'!;..'. ;:.:;à". 'à"  .-1-': à":-«(à"?à" ; ;^i; ':;-i::W :.à"»à"? :^;v ;.r. >'.:?»::7^sfr°T;-*;,à"> j4^;-;'. a:;! VVV.-'f';à":- à"-« ;.'.'. -.'. :-yiv^-;:Sr::-' .à"à"V'>.;-^.' -v'.:o- " V-..:v';,i:'*;. ":i:"- '' : à"à"- -à"*..-.'.* , ,  '.'.' -'::-';:'r :i«:..--'-:3-::":':'    à"PiHll!    ilfi:-!;;  fllllMP-eiflHn^Jn^ii  '  à" -so°c               SEKOtY.PE, 3122Fig. Dot hybridization assay of five reference rotavirus dsRNA (A)Wa(serotype 1), (B) RV5 (serotype 2), (C) RV3 (serotype 3), (D)ST3 (serotype 4) and (E) B37 (serotype 8). Two-fold dilution ofdsRNA spotted onto nitrocellulose filters and hybridized withserotype 1-, 2-, 3-, 4-, and 8-specific probes at 46, 41, 41, 46,and 49°C for HuGlAc, HuG2Ac, HuG3Ac, HuG4Ac andHuGSAc, respectively and 50°C for all the probes. Serotype ofstrains are indicated at the top of the figure.46, and 49°C for HuGlAc, HuG2Ac, HuGSAc, HuG4Ac and HuG8Ac (Bingnan et al., 1991,All et al., 1993) and at 50°C for all the probes.123RESULTS AND DISCUSSIONThe result of hybridizations carried out at two temperatures (those used by Bingnan etal, 1991, Ali et al., 1993 and 50°C proposed by us) for all five probes are shown in thefigure. At a hybridization temperature of 50°C, it was observed that at this temperaturecross-reaction was satisfactorily removed and similar sensitivity to that of temperatures usedby Bingnan et al. (1991) and Ali et al. (1993) was observed. The probes did not show cross-reactivity with concentrated RNA preparations from other serotypes with the exception thatat 41 °C, HuG3Ac (see figure C). However the cross-reactivity signal was significantly weakerthan the positive signal. But when the hybridization was performed at 50°C, the probes didnot show any cross-reactivity. In addition, partial cross-reactivity exhibited by HuG3Ac at41°C was also eliminated when hybridization was carried out at our proposed commontemperature i,e. 50°C. When hybridization was performed with the extracts from Shigellaspp, Escherichia colt, isolates from five different diarrhoeagenic categories, Giardia spp andEntamoeaba histolytica, and it was observed that at both temperatures, probes did not reactwith either of the extract (data not shown).The sensitivity of each probe was determined at two hybridization temperatures andcompared. At hybridization temperature of 50°C , it was observed that at this temperature thesensitivity of the probes were also unaltered with the exception of HuGlAc and HuGSAc(see figure A and E). At 50°C, HuGlAc and HuGSAc showed slightly weaker signal than at46 and 49°C, although the difference between the two signals does not affect the identity ofthe serotype. Therefore, 50°C can be chosen as an appropriate temperature for hybridizationof all probes as opposed to the use of a range of temperatures for different probes byBingnan et al. (1991) and Ali et al (1993).Although the salt concentration of the hybridization buffer can adversely affect thespecificity of the probe but we did not experiment with different salt concentration of thehybridization buffer because we used the standard salt concentration described elsewhere.This study was thus shown that a single temperature can be used for serotyping ofHRV using synthetic oligonucleotide probes. This might prove to be an useful technical ad-vantage in handling large number of samples.ACKNOWLEDGEMENTSThis study was funded by the United States Agency for International Developmentunder grant No. DPE-5986-A-00-1009-00 and by the International Centre for DiarrhoealDisease Research, Bangladesh which is supported by governments of Australia, Bangladesh,Belgium, Canada, Denmark, France, Japan, the Netherland, Norway, Saudi Arabia, Sweden,Switzerland, the United Kingdom and the United States and the international organizationsincluding the United Nations Children's Fund, the World Health Organization, the Ford Foun-dation and the Sasakawa Foundation.Wethank P. Echeverria for providing the oligonucleotides. Special thanks to Professor124Akira Igarashi for valuable advice and careful reading the manuscript.This study was performed as partial fulfilment of the M. Sc. requirements for theUniversity of Dhaka (Ayub Ali).REFERENCES1 ) Albert, M.J., Unicomb, L.E. & Bishop, R.F. (1987): Cultivation and characterization of humanrotaviruses with "short" RAN pattern. J. Clin. MicrobioL, 25, 183-185.2 ) Ali, A., Bingnan, F., Unicomb, L.E., Rahim, Z., Hossain, A., & Tzipori, S. (1993): Evaluation of aprobe hybridisation serotyping method for group A rotavirus. J. Diarrhoeald Dis. Res. ll(3):153-156.3 ) Bingnan, F., Unicomb, L.E., Rahim, Z., Banu, N.N., Fodder, G.; Clemens, J., Van Loon, F.P.L.,Rao, M.R., Malek, A. & Tzipori, S (1991): Rotavirus associated diarrhoea in rural Bangladesh: Two-years study of incidence and serotype distribution. J. Clin. MicrobioL, 29, 1359-1363.4 ) Estes, M. & Cohen, J. (1989): Rotavirus genes structure and function. MicrobioL Rev., 53,410-449.5 ) Flores, J., Green, K.Y., Garcia, D., Sears, J., Perez-Schael, I., Avendano, L.F., Rodriguez, W.B.,Taniguchi, K., Urasawa, S. & Kapikian, A.Z. (1989): Dot hybridization assay for distinction ofrotavirus serotypes. J. Clin. MicrobioL 19, 273-278.6 ) Herring, A.J., Inglis, N.F., Ojeh, C.K., Snodgrass, D.R. & Menzies, J.D. (1982): Rapid diagnosis ofrotavirus infection by direct detection of viral nucleic acid in silver stained polyacrylamide gels. J.Clin. MicrobioL, 16, 473-477.7 ) Hoshino, Y., Sereno, M. M., Midthun, K., Flores, J., Kapikian, A.Z. & Chanock, R.M. (1985): In-dependent segregation of two antigenic specificites (VPS and VP7) involved in neutralization ofrotavirus infectivity. Proc. Nat Aca. ScL, U.S.A., 82, 8701-8704.8 ) Kapikian, A.Z. & Chanock, R.M. (1990): Rotaviruses. In Virology, 2nd edn, pp. 1353-1404. Editedby B.N. Fields & D.M. Knipe. New York: Raven Press.9 ) Kapikian, A.Z. & Chanock, R.M. (1985): Rotavirus. In Virology, pp 863-906, Edited by B.N.Fields. New York: Raven Press.10) Maniatis, T., Fritsch, E.F. & Sambrook, J (eds). (1982): Molecular Cloning: A laboratory Manual.Cold Spring Harbour Laboratory, New York.ll) Offit, P.A. & Blavat, G. (1986): Identification of the two rotavirus genes determining neutralizationspecificites. J. ViroL, 57, 376-381.12) Sethabutr, 0., Unicomb, L.E., Holmes, I.H., Taylor, D.N., Bishop, R.F. & Echeverria, P. (1990):Serotyping of human group A rotavirus with oligonucleotide probes. J. Infect. Dis., 162, 368-372.13) Urasawa, S., Urasawa, T., Wakasugi, F., Kobayashi, N., Taniguchi, K., Lintag, I.C., Saniel, M.C. &Goto, H. (1990): Presumptive seventh serotype of human rotavirus. Arch. ViroL, 113, 279-282.

Determination of Single Hybridization Temperature for Serotyping of Human Group A Rotavirus with Oligonucleotide Probes

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Determination of Single Hybridization Temperature for Serotyping of Human Group A Rotavirus with Oligonucleotide Probes

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