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Citation: Tongwei Ren, Xiangling Min, Qingrong Mo, Yuxu Wang, Hao Wang, Ying Chen, Kang Ouyang, Weijian Huang, Zuzhang Wei. Construction and characterization of a full-length infectious clone of Getah virus in vivo [J].VIROLOGICA SINICA, 2022, 37(3) : 348-357.  http://dx.doi.org/10.1016/j.virs.2022.03.007

Construction and characterization of a full-length infectious clone of Getah virus in vivo

  • Corresponding author: Zuzhang Wei, zuzhangwei@gxu.edu.cn
  • Received Date: 02 September 2021
    Accepted Date: 02 March 2022
    Available online: 11 March 2022
  • Getah virus (GETV) is a mosquito-borne virus of the genus Alphavirus in the family Togaviridae and, in recent years, it has caused several outbreaks in animals. The molecular basis for GETV pathogenicity is not well understood. Therefore, a reverse genetic system of GETV is needed to produce genetically modified viruses for the study of the viral replication and its pathogenic mechanism. Here, we generated a CMV-driven infectious cDNA clone based on a previously isolated GETV strain, GX201808 (pGETV-GX). Transfection of pGETV-GX into BHK- 21 cells resulted in the recovery of a recombinant virus (rGETV-GX) which showed similar growth characteristics to its parental virus. Then three-day-old mice were experimentally infected with either the parental or recombinant virus. The recombinant virus showed milder pathogenicity than the parental virus in the mice. Based on the established CMV-driven cDNA clone, subgenomic promoter and two restriction enzyme sites (BamHI and EcoRI) were introduced into the region between E1 protein and 30UTR. Then the green fluorescent protein (GFP), red fluorescent protein (RFP) and improved light-oxygen-voltage (iLOV) genes were inserted into the restriction enzyme sites. Transfection of the constructs carrying the reporter genes into BHK-21 cells proved the rescue of the recombinant reporter viruses. Taken together, the establishment of a reverse genetic system for GETV provides a valuable tool for the study of the virus life cycle, and to aid the development of genetically engineered GETVs as vectors for foreign gene expression.

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    1. Anishchenko, M., Paessler, S., Greene, I.P., Aguilar, P.V., Carrara, A.S., Weaver, S.C., 2004. Generation and characterization of closely related epizootic and enzootic infectious cDNA clones for studying interferon sensitivity and emergence mechanisms of Venezuelan equine encephalitis virus. J. Virol. 78, 1-8.

    2. Aubry, F., Nougairède, A., Gould, E.A., de Lamballerie, X., 2015. Flavivirus reverse genetic systems, construction techniques and applications:a historical perspective.Antivir. Res. 114, 67-85.

    3. Bannai, H., Ochi, A., Nemoto, M., Tsujimura, K., Yamanaka, T., Kondo, T., 2016. A 2015 outbreak of Getah virus infection occurring among Japanese racehorses sequentially to an outbreak in 2014 at the same site. BMC Vet. Res. 12, 98.

    4. Brault, A.C., Foy, B.D., Myles, K.M., Kelly, C.L.H., Higgs, S., Weaver, S.C., Olson, K.E., Miller, B.R., Powers, A.M., 2004. Infection patterns of o'nyong nyong virus in the malaria-transmitting mosquito, Anopheles gambiae. Insect Mol. Biol. 13, 625-635.

    5. Brown, C.M., Timoney, P.J., 1998. Getah virus infection of Indian horses. Trop. Anim. Health Prod. 30, 241-252.

    6. Bryant, J.E., Crabtree, M.B., Nam, V.S., Yen, N.T., Duc, H.M., Miller, B.R., 2005. Isolation of arboviruses from mosquitoes collected in northern Vietnam. Am. J. Trop. Med.Hyg. 73, 470-473.

    7. Caley, I.J., Betts, M.R., Davis, N.L., Swanstrom, R., Johnston, R.E.J.V., 1999. In:Venezuelan equine encephalitis virus vectors expressing HIV-1 proteins:vector design strategies for improved vaccine efficacy, 17, p. 3124.

    8. Chang, C.Y., Huang, C.C., Huang, T.S., Deng, M.C., Jong, M.H., Wang, F.I., 2006. Isolation and characterization of a Sagiyama virus from domestic pigs. In:Journal of Veterinary Diagnostic Investigation:Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc, vol. 18, pp. 156-161.

    9. Chen, R., Wang, E., Tsetsarkin, K.A., Weaver, S.C., 2013. Chikungunya virus 3' untranslated region:adaptation to mosquitoes and a population bottleneck as major evolutionary forces. PLoS Pathog. 9, e1003591.

    10. Davis, N.L., Fuller, F.J., Dougherty, W.G., Olmsted, R.A., Johnston, R.E.J.P., 1986. In:A single nucleotide change in the E2 glycoprotein gene of Sindbis virus affects penetration rate in cell culture and virulence in neonatal mice, 83, pp. 6771-6775.

    11. Davis, N.L., Powell, N., Greenwald, G.F., Willis, L.V., Johnson, B.J., Smith, J.F., Johnston, R.E.J.V., 1991. In:Attenuating mutations in the E2 glycoprotein gene of Venezuelan equine encephalitis virus:construction of single and multiple mutants in a full-length cDNA clone, 183, p. 20.

    12. Davis, N.L., Willis, L.V., Smith, J.F., Johnston, R.E., 1989. In vitro synthesis of infectious venezuelan equine encephalitis virus RNA from a cDNA clone:analysis of a viable deletion mutant. Virology 171, 189-204.

    13. Ferguson, M.C., Saul, S., Fragkoudis, R., Weisheit, S., Cox, J., Patabendige, A., Sherwood, K., Watson, M., Merits, A., Fazakerley, J.K.J., 2015. In:Ability of the encephalitic arbovirus Semliki forest virus to cross the blood-brain barrier is determined by the charge of the E2 glycoprotein, 89, pp. 7536-7549.

    14. Filomatori, C.V., Merwaiss, F., Bardossy, E.S., Alvarez, D.E., 2021. Impact of alphavirus 3'UTR plasticity on mosquito transmission. Semin. Cell Dev. Biol. 111, 148-155.

    15. Fukunaga, Y., Kumanomido, T., Kamada, M., 2000. Getah virus as an equine pathogen.Vet Clin N Am-Equine 16, 605-617.

    16. Garcia-Moreno, M., Sanz, M.A., Carrasco, L.J.S.R., 2016. A viral mRNA motif at the 30-untranslated region that confers translatability in a cell-specific manner. Implications for Virus Evolution. Sci Rep 6, 19217.

    17. Gould, E.A., Coutard, B., Malet, H., Morin, B., Jamal, S., Weaver, S., Gorbalenya, A., Moureau, G., Baronti, C., Delogu, I., Forrester, N., Khasnatinov, M., Gritsun, T., de Lamballerie, X., Canard, B., 2010. Understanding the alphaviruses:recent research on important emerging pathogens and progress towards their control. Antivir. Res. 87, 111-124.

    18. Hawman, D.W., Carpentier, K.S., Fox, J.M., May, N.A., Sanders, W., Montgomery, S.A., Moorman, N.J., Diamond, M.S., Morrison, T.E., 2017. Mutations in the E2 glycoprotein and the 3' untranslated region enhance chikungunya virus virulence in mice. J. Virol. 91, e00816-17.

    19. Higgs, S., Olson, K.E., Klimowski, L., Powers, A.M., Carlson, J.O., Possee, R.D., Beaty, B.J., 1995. Mosquito sensitivity to a scorpion neurotoxin expressed using an infectious Sindbis virus vector. Insect Mol. Biol. 4, 97-103.

    20. Higgs, S.S., Oray, C., Myles, K.K., Olson, K., Beaty, B.J.B., 1999. In:Infecting larval arthropods with a chimeric, double subgenomic Sindbis virus vector to express genes of interest, 27, pp. 908-911.

    21. Hyde, J.L., Chen, R., Trobaugh, D.W., Diamond, M.S., Weaver, S.C., Klimstra, W.B., Wilusz, J.J.V.R., 2015. The 50 and 30 ends of alphavirus RNAs. Non-coding is not nonfunctional 206, 99-107.

    22. Kamada, M., Ando, Y., Fukunaga, Y., Kumanomido, T., Imagawa, H., Wada, R., Akiyama, Y., 1980. Equine Getah virus infection:isolation of the virus from racehorses during an enzootic in Japan. Am. J. Trop. Med. Hyg. 29, 984-988.

    23. Kinney, R.M., Chang, G.J., Tsuchiya, K.R., Sneider, J.M., Trent, D.W.J.J., 1993. In:Attenuation of Venezuelan equine encephalitis virus strain TC-83 is encoded by the 5'-noncoding region and the E2 envelope glycoprotein, 67, p. 1269.

    24. Ksiazek, T.G., Trosper, J.H., Cross, J.H., Basaca-Sevilla, V., 1981. Isolation of Getah virus from nueva ecija province, republic of the Philippines. Trans. R. Soc. Trop. Med. Hyg. 75, 312-313.

    25. Kuhn, R.J., Hong, Z., Strauss, J.H.J.J., 1990. Mutagenesis of the 3' nontranslated region of Sindbis virus. RNA 64, 1465-1476.

    26. Kuhn, R.J., Niesters, H.G., Hong, Z., Strauss, J.H., 1991. Infectious RNA transcripts from Ross River virus cDNA clones and the construction and characterization of defined chimeras with Sindbis virus. Virology 182, 430-441.

    27. Kumanomido, T., Wada, R., Kanemaru, T., Kamada, M., Hirasawa, K., Akiyama, Y., 1988. Clinical and virological observations on swine experimentally infected with Getah virus. Vet. Microbiol. 16, 295-301.

    28. Li, X.D., Qiu, F.X., Yang, H., Rao, Y.N., Calisher, C.H., 1992. Isolation of Getah virus from mosquitos collected on Hainan Island, China, and results of a serosurvey. Southeast Asian J. Trop. Med. Publ. Health 23, 730-734.

    29. Li, Y., Fu, S., Guo, X., Li, X., Li, M., Wang, L., Gao, X., Lei, W., Cao, L., Lu, Z., He, Y., Wang, H., Zhou, H., Liang, G., 2019. Serological survey of Getah virus in domestic animals in yunnan province, China. Vector Borne Zoonotic Dis. 19, 59-61.

    30. Li, Y.Y., Fu, S.H., Guo, X.F., Lei, W.W., Li, X.L., Song, J.D., Cao, L., Gao, X.Y., Lyu, Z., He, Y., Wang, H.Y., Ren, X.J., Zhou, H.N., Wang, G.Q., Liang, G.D., 2017a. Identification of a newly isolated Getah virus in the China-Laos border, China. Biomed. Environ. Sci.:BES 30, 210-214.

    31. Li, Y.Y., Liu, H., Fu, S.H., Li, X.L., Guo, X.F., Li, M.H., Feng, Y., Chen, W.X., Wang, L.H., Lei, W.W., Gao, X.Y., Lv, Z., He, Y., Wang, H.Y., Zhou, H.N., Wang, G.Q., Liang, G.D., 2017b. From discovery to spread:the evolution and phylogeny of Getah virus. Infect.Genet. Evol.:J. Mol. Epidemiol. Evolut. Genet. Infect. disea. 55, 48-55.

    32. Liljestrom, P., Lusa, S., Huylebroeck, D., Garoff, H., 1991. In vitro mutagenesis of a fulllength cDNA clone of Semliki Forest virus:the small 6,000-molecular-weight membrane protein modulates virus release. J. Virol. 65, 4107-4113.

    33. Liu, H., Zhang, X., Li, L.X., Shi, N., Sun, X.T., Liu, Q., Jin, N.Y., Si, X.K., 2019. First isolation and characterization of Getah virus from cattle in northeastern China. BMC Vet. Res. 15, 320.

    34. Lu, G., Ou, J., Ji, J., Ren, Z., Hu, X., Wang, C., Li, S., 2019. Emergence of Getah virus infection in horse with fever in China, 2018. Front. Microbiol. 10, 1416.

    35. McKnight, K.L., Simpson, D.A., Lin, S.C., Knott, T.A., Polo, J.M., Pence, D.F., Johannsen, D.B., Heidner, H.W., Davis, N.L., Johnston, R.E., 1996. Deduced consensus sequence of Sindbis virus strain AR339:mutations contained in laboratory strains which affect cell culture and in vivo phenotypes. J. Virol. 70, 1981-1989.

    36. Nemoto, M., Bannai, H., Tsujimura, K., Kobayashi, M., Kikuchi, T., Yamanaka, T., Kondo, T., 2015. Getah virus infection among racehorses, Japan, 2014. Emerg. Infect.Dis. 21, 883-885.

    37. Perrotta, A.T., Been, M.D., 1991. A pseudoknot-like structure required for efficient selfcleavage of hepatitis delta virus RNA. Nature 350, 434-436.

    38. Pfeffer, M., Kinney, R.M., Kaaden, O.R., 1998. The alphavirus 3'-nontranslated region:size heterogeneity and arrangement of repeated sequence elements. Virology 240, 100-108.

    39. Pierro, D.J., Myles, K.M., Foy, B.D., Beaty, B.J., Olson, K.E., 2003. Development of an orally infectious Sindbis virus transducing system that efficiently disseminates and expresses green fluorescent protein in Aedes aegypti. Insect Mol. Biol. 12, 107-116.

    40. Porterfield, J.S., 1975. The basis of arbovirus classification. Med. Biol. 53, 400-405.

    41. Pugachev, K.V., Mason, P.W., Shope, R.E., Frey, T.K.J.V., 1995. Double-subgenomic Sindbis virus recombinants expressing immunogenic proteins of Japanese encephalitis virus induce significant protection in mice against. Lethal JEV Infect. 212, 587-594.

    42. Pugachev, K.V., Tzeng, W.P., Frey, T.K.J.J., 2000. In:Development of a rubella virus vaccine expression vector:use of a picornavirus internal ribosome entry site increases stability of expression, 74, 10811.

    43. Ren, T., Mo, Q., Wang, Y., Wang, H., Nong, Z., Wang, J., Niu, C., Liu, C., Chen, Y., Ouyang, K., Huang, W., Wei, Z., 2020. Emergence and phylogenetic analysis of a Getah virus isolated in southern China. Front. Veterin. Sci. 7, 552517.

    44. Rice, C.M., Levis, R., Strauss, J.H., Huang, H.V., 1987. Production of infectious RNA transcripts from Sindbis virus cDNA clones:mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants.J. Virol. 61, 3809-3819.

    45. Schoepp, R.J., Smith, J.F., Parker, M.D., 2002. Recombinant chimeric western and eastern equine encephalitis viruses as potential vaccine candidates. Virology 302, 299-309.

    46. Sentsui, H., Kono, Y., 1985. Reappearance of Getah virus infection among horses in Japan. Nihon juigaku zasshi. Jpn. J. Vet. Sci. 47, 333-335.

    47. Shi, N., Li, L.X., Lu, R.G., Yan, X.J., Liu, H., 2019. Highly pathogenic swine Getah virus in blue foxes, eastern China, 2017. Emerg. Infect. Dis. 25, 1252-1254.

    48. Shibata, I., Hatano, Y., Nishimura, M., Suzuki, G., Inaba, Y., 1991. Isolation of Getah virus from dead fetuses extracted from a naturally infected sow in Japan. Vet. Microbiol. 27, 385-391.

    49. Simpson, D.A., Davis, N.L., Lin, S.C., Russell, D., Johnston, R.E., 1996. Complete nucleotide sequence and full-length cDNA clone of S.A.AR86 a South African alphavirus related to Sindbis. Virology 222, 464-469.

    50. Sugiyama, I., Shimizu, E., Nogami, S., Suzuki, K., Miura, Y., Sentsui, H., 2009. Serological survey of arthropod-borne viruses among wild boars in Japan. J. Vet. Med. Sci. 71, 1059-1061.

    51. Sun, C., Gardner, C.L., Watson, A.M., Ryman, K.D., Klimstra, W.B.J.J., 2014. In:Stable, high-level expression of reporter proteins from improved alphavirus expression vectors to track replication and dissemination during encephalitic and arthritogenic disease, 88, pp. 2035-2046.

    52. Thomas, J.M., Klimstra, W.B., Ryman, K.D., Heidner, H.W.J.J., 2003. Sindbis virus vectors designed to express a foreign protein as a cleavable component of the. Viral Struct.Polypr. 77, 5598-5606.

    53. Vanlandingham, D.L., Tsetsarkin, K., Hong, C., Klingler, K., Mcelroy, K.L., Lehane, M.J., Higgs, S.J.I.B., Biology, M., 2005. In:Development and characterization of a double subgenomic chikungunya virus infectious clone to express heterologous genes in Aedes aegypti mosquitoes, 35, pp. 1162-1170.

    54. Wang, C.Y., Dominguez, G., Frey, T.K., 1994. Construction of rubella virus genome-length cDNA clones and synthesis of infectious RNA transcripts. J. Virol. 68, 3550-3557.

    55. Wang, H., Niu, C., Nong, Z., Quan, D., Chen, Y., Kang, O., Huang, W., Wei, Z., 2020. Emergence and phylogenetic analysis of a novel Seneca Valley virus strain in the Guangxi Province of China. Res. Vet. Sci. 130, 207-211.

    56. Weaver, S.C., 2005. Host range, amplification and arboviral disease emergence. Arch.virol. Suppl. 33-44.

    57. Xing, C., Jiang, J., Lu, Z., Mi, S., He, B., Tu, C., Liu, X., Gong, W., 2020. Isolation and characterization of Getah virus from pigs in Guangdong province of China. Transbound Emerg Dis. https://doi.org/10.1111/tbed.13567.

    58. Yago, K., Hagiwara, S., Kawamura, H., Narita, M., 1987. A fatal case in newborn piglets with Getah virus infection:isolation of the virus. Nihon juigaku zasshi Japn. J. Veterin. Sci. 49, 989-994.

    59. Yang, T., Li, R., Hu, Y., Yang, L., Zhao, D., Du, L., Li, J., Ge, M., Yu, X., 2018. An outbreak of Getah virus infection among pigs in China, 2017. Transbound. Emerg. Dis. 65, 632-637.

    60. Zhai, Y.G., Wang, H.Y., Sun, X.H., Fu, S.H., Wang, H.Q., Attoui, H., Tang, Q., Liang, G.D., 2008. Complete sequence characterization of isolates of Getah virus (genus Alphavirus, family Togaviridae) from China. J. Gen. Virol. 89, 1446-1456.

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    Construction and characterization of a full-length infectious clone of Getah virus in vivo

      Corresponding author: Zuzhang Wei, zuzhangwei@gxu.edu.cn
    • Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, 530005, China

    Abstract: Getah virus (GETV) is a mosquito-borne virus of the genus Alphavirus in the family Togaviridae and, in recent years, it has caused several outbreaks in animals. The molecular basis for GETV pathogenicity is not well understood. Therefore, a reverse genetic system of GETV is needed to produce genetically modified viruses for the study of the viral replication and its pathogenic mechanism. Here, we generated a CMV-driven infectious cDNA clone based on a previously isolated GETV strain, GX201808 (pGETV-GX). Transfection of pGETV-GX into BHK- 21 cells resulted in the recovery of a recombinant virus (rGETV-GX) which showed similar growth characteristics to its parental virus. Then three-day-old mice were experimentally infected with either the parental or recombinant virus. The recombinant virus showed milder pathogenicity than the parental virus in the mice. Based on the established CMV-driven cDNA clone, subgenomic promoter and two restriction enzyme sites (BamHI and EcoRI) were introduced into the region between E1 protein and 30UTR. Then the green fluorescent protein (GFP), red fluorescent protein (RFP) and improved light-oxygen-voltage (iLOV) genes were inserted into the restriction enzyme sites. Transfection of the constructs carrying the reporter genes into BHK-21 cells proved the rescue of the recombinant reporter viruses. Taken together, the establishment of a reverse genetic system for GETV provides a valuable tool for the study of the virus life cycle, and to aid the development of genetically engineered GETVs as vectors for foreign gene expression.

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