Sequencing of the VP1–2A Genome Segment for Hepatitis A Subtyping

Zalesskikh A.A., Bystrova T.N.

Key words: hepatitis A; genotyping; sequencing.

2020, volume 12, issue 1, page 52.

DOI: https://doi.org/10.17691/stm2020.12.1.06

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The aim of the study was to improve the VP1–2A sequencing method and facilitate the hepatitis A virus genotyping technique.

Materials and Methods. Nucleic acids were extracted by using commercial kits for nucleic acid extraction and reverse transcription in a manual and automatic version. Primer sequences were selected using the DNASTAR software. Oligonucleotides were synthesized by the amidophosphite method followed by purification with polyacrylamide gel electrophoresis. The sought fragments of the genome were obtained with PCR. Commercial components were used to prepare the reaction mixture. The results were verified by agarose gel electrophoresis with ethidium bromide DNA staining. After amplification, the DNA fragments were purified by adsorption on silica columns. Sequence identification of the purified fragments was performed by the Sanger sequencing method. The obtained sequences of the VP1–2A fragment were analyzed by the MEGA 6 software package. The optimized technique for sequencing of the VP1–2A segment was tested with 171 clinical and environmental samples containing hepatitis A virus RNA.

Results. The sequencing technique has been optimized at the stage of amplification of the variable VP1–2A structural genome segment. To obtain this result, we created original primer sequences and selected the polymerase reaction conditions (length, localization and annealing temperature, concentration of the primers and template). The sought fragment with a length of 249 nucleotide pairs was located between the 2988^th and 3237^th base pairs as per the reference HM175 strain. Using this optimized method of VP1–2A fragment sequencing, the hepatitis A genotype was detected in 76 from 171 samples containing hepatitis A virus RNA in the city of Nizhny Novgorod from 2000 to 2017. The prevalence of the IA subtype was found in 97% of cases (74 of 76 samples), and only 2 samples contained subtype IB.

Conclusion. The sequencing technique optimized at the stage of VP1–2A fragment amplification allows one to determine the hepatitis A virus genotype, identify imported virus strains, and also establish an epidemiological relationship between different cases of the disease.

Vaz J., Floyd C., Mason B., Shankar A.G., Lewis H. Control of a community outbreak of hepatitis A in an area of low endemicity, Wales, 2016. Hum Vaccin Immunother 2017; 13(10): 2352–2356, https://doi.org/10.1080/21645515.2017.1347242.
Michaelis K., Wenzel J.J., Stark K., Faber M. Hepatitis A virus infections and outbreaks in asylum seekers arriving to Germany, September 2015 to March 2016. Emerg Microbes Infect 2017; 6(4): e26, https://doi.org/10.1038/emi.2017.11.
Gallone M.F., Desiante F., Gallone M.S., Barbuti G., Tafuri S., Germinario C. Serosurveillance of hepatitis A in a region which adopted the universal mass vaccination. Medicine (Baltimore) 2017; 96(9): e5884, https://doi.org/10.1097/md.0000000000005884.
Gepatit A: etiologiya, epidemiologiya, diagnostika, profilaktika [Hepatitis A: etiology, epidemiology, diagnosis, prevention]. Pod red. Shkarina V.V. [Shkarin V.V. (editor)]. Nizhny Novgorod: NizhGMA; 2015.
Virusnye gepatity v Rossiyskoy Federatsii: analiticheskiy obzor. Pod red. Pokrovskogo V.I., Totolyana A.A. [Pokrovskiy V.I., Totolyan A.A. (editors)]. Saint Petersburg: FBUN NIIEM im. Pastera; 2016.
Karetkina G.N. Viral hepatitis A: past, present and future. Infektsionnye bolezni: novosti, mneniya, obuchenie 2014; 3(8): 38–48.
Mukomolov S.L., Mikhailov M.I., Semenenko T.A., Sel’kova E.P., Gerasimova I.E. Review: burden of hepatitis a in Russian Federation. Epidemiologiya i vaktsinoprofilaktika 2014; 6(79): 24–34.
Chernov A.V., Brusenskaya T.Y., Korchagin V.V. Analysis of the incidence of acute viral hepatitis in the Voronezh region. Territoriya innovatsiy 2017; 4(8): 117–120.
WHO position paper on hepatitis A vaccines. Wkly Epidemiol Rec 2012; 87(28/29): 261–276. URL: http://www.who.int/wer/2012/wer8728_29/en/.
Karandashova I.V., Pimenov N.N., Neverov A.D., Mikhailovskaya G.V., Dolgin V.A., Braslavskaya S.I., Komarova S.V., Lytkina I.N., Shulakova N.I., Shipulin G.A., Chulanov V.P. Molecular epidemiological characteristics of hepatitis a outbreak in Moscow. Epidemiologiya i infektsionnye bolezni. Aktual’nye voprosy 2012; 3: 9–14.
Bondarenko T.Y., Ternovoi V.A., Netesov S.V. Hepatitis a virus: structure-functional features of genome, molecular diagnostics, and cultivation. Molecular Genetics, Microbiology and Virology 2013; 28(3): 99–109, https://doi.org/10.3103/s0891416813030038.
Mukomolov C.L., Kalinina O.V. The molecular epidemiology of viral hepatitis. Mir virusnykh gepatitov 2003; 11: 2–7.
Chulanov V.P., Karandashova I.V., Pimenov N.N., Molochniy V.P., Tomilka G.S., Sleptsova S.S., Semenova V.K., Malyi V.P., Boiko V.V., Kamolov B.D., Volchkova E.V. Clinical significance of genetic diversity of hepatitis A virus. Epidemiologiya i infektsionnye bolezni 2014; 19(4): 12–17.
Nainan O.V., Xia G., Vaughan G., Margolis H.S. Diagnosis of hepatitis A virus infection: a molecular approach. Clin Microbiol Rev 2006; 9(1): 63–79, https://doi.org/10.1128/cmr.19.1.63-79.2006.
Vaughan G., Goncalves Rossi L.M., Forbi J.C., de Paula V.S., Purdy M.A., Xia G., Khudyakov Y.E. Hepatitis A virus: host interactions, molecular epidemiology and evolution. Infect Genet Evol 2014; 21: 227–243, https://doi.org/10.1016/j.meegid.2013.10.023.
Costa-Mattioli M., Di Napoli A., Ferré V., Billaudel S., Perez-Bercoff R., Cristina J. Genetic variability of hepatitis A virus. J Gen Virol 2003; 84(Pt 12): 3191–3201, https://doi.org/10.1099/vir.0.19532-0.
Sánchez G., Bosch A., Pintó R.M. Genome variability and capsid structural constraints of hepatitis A virus. J Virol 2003; 77(1): 452–459, https://doi.org/10.1128/jvi.77.1.452-459.2003.
Robertson B.H., Jansen R.W., Khanna B. Genetic relatedness of hepatitis A virus strains recovered from different geographical regions. J Gen Virol 1992; 73 (Pt 6): 1365–1677, https://doi.org/10.1099/0022-1317-73-6-1365.
Mukomolov S., Kontio M., Zheleznova N., Jokinen S., Sinayskaya E., Stalevskaya A., Davidkin I. Increased circulation of hepatitis A virus genotype IIIA over the last decade in St. Petersburg, Russia. J Med Virol 2012; 84(10): 1528–1534, https://doi.org/10.1002/jmv.23378.
Bruni R., Taffon S., Equestre M., Cella E., Lo Presti A., Costantino A., Chionne P., Madonna E., Golkocheva-Markova E., Bankova D., Ciccozzi M., Teoharov P., Ciccaglione A.R. Hepatitis A virus genotypes and strains from an endemic area of Europe, Bulgaria 2012–2014. BMC Infect Dis 2017; 17(1): 497, https://doi.org/10.1186/s12879-017-2596-1.

Zalesskikh A.A., Bystrova T.N. Sequencing of the VP1–2A Genome Segment for Hepatitis A Subtyping. Sovremennye tehnologii v medicine 2020; 12(1): 52, https://doi.org/10.17691/stm2020.12.1.06