- Open Access
Recombinant Tula hantavirus shows reduced fitness but is able to survive in the presence of a parental virus: analysis of consecutive passages in a cell culture
© Plyusnina and Plyusnin; licensee BioMed Central Ltd. 2005
Received: 01 February 2005
Accepted: 22 February 2005
Published: 22 February 2005
Tula hantavirus carrying recombinant S RNA segment (recTULV) grew in a cell culture to the same titers as the original cell adapted variant but presented no real match to the parental virus. Our data showed that the lower competitiveness of recTULV could not be increased by pre-passaging in the cell culture. Nevertheless, the recombinant virus was able to survive in the presence of the parental virus during five consecutive passages. The observed survival time seems to be sufficient for transmission of newly formed recombinant hantaviruses in nature.
Recombination in RNA viruses serves two main purposes: (i) it generates and spreads advantageous genetic combinations; and (ii) it counters the deleterious effect of mutations that, due to the low fidelity of viral RNA polymerases and lack of proofreading, occur with high frequency . The purging function is, naturally, attributed to the homologous recombination (HRec), i.e. recombination between homologous parental molecules through crossover at homologous sites. HRec was first described for the positive-sense RNA viruses [2, 3] and subsequent studies lead to the widely accepted copy-choice model . HRec was later shown to occur in rotaviruses thus adding double-stranded RNA viruses to the list of viruses capable of recombination . Negative-sense RNA viruses that occupy the largest domain in the virus kingdom until recently were known to undergo non-homologous recombination only, forming either defective genomes, like polymerase "mosaics" of influenza A virus DI-particles  and "copy-backs" of parainfluenza virus  or hybrids between viral and cellular genes  or between different viral genes . The first evidence for HRec in a negative-sense RNA virus has been obtained on hantaviruses [10, 11].
Hantaviruses (genus Hantavirus, family Bunyaviridae) have a tripartite genome comprising the L segment encoding the RNA-polymerase, the M segment encoding two external glycoproteins, and the S segment encoding the nucleocapsid (N) protein . Hantaviruses are maintained in nature in persistently infected rodents, each hantavirus type being predominantly associated with a distinct rodent host species . When transmitted to humans, some hantaviruses cause hemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome, whereas other hantaviruses are apathogenic [14, 15]. Persistent infection in natural hosts allows for the simultaneous presence of more than one genetically distinct hantavirus variant in the same rodent. This may result in hantavirus genome reassortment [16, 17] or recombination, as proposed in the above-mentioned study of Sibold et al  who showed a mosaic-like structure of the S RNA segment and the N protein of Tula hantavirus (TULV). Most recently, we have shown transfection-mediated rescue of TULV with recombinant S segment, in which nt 1–332 originate from the cell culture isolate Moravia/Ma5302V/94 (or TULV02, for short) , nt 369–1853 originate from the strain Tula/Ma23/87 , and nt 333–368, that are identical in both variants, can be of either origin. Both M and L segments of the recombinant virus (recTULV) originate from TULV02 . RecTULV was functionally competent but less competitive than TULV02. One reason for the observed lower fitness of the recTULV might be that it was generated in the presence of the wt variant, with which it has to compete, and thus not given enough time to to establish a well balanced, mature quasi-species population. We, therefore, decided to compare fitness of TULV02 with that of recTULV that underwent several passages in cell culture.
Results and discussion
Although relatively short, the observed survival time of the recTULV in the presence of the original variant TUL02 seems to be sufficient for transmission of a recombinant virus, in a hypothetical in vivo situation, from one rodent to another. If transmission is performed in a sampling-like fashion – and this seems to be the case for hantaviruses  – the recombinant would have fair chances to survive. The existence of wt recombinant strains of TULV  supports this way of reasoning. Evidence for the recombination in the hantavirus evolution continues to accumulate [20, 21].
The data presented in this paper show that the recTULV presents no real match to the original cell adapted variant and that the lower fitness of the recombinant virus can not be increased by pre-passaging in cell culture. The observed survival time of the recTULV in the presence of the parental virus seems to be sufficient for transmission of newly formed recombinant hantaviruses in nature.
RecTULV (clone 5) was purified from the mixture it formed with the original variant, TULV02, using two consequent passages under terminal dilutions . After the purification, recTULV underwent three more passages, performed under standard conditions, i.e. without dilution. The presence of recS-RNA on the passages was monitored by RT-PCR and the isolate appeared to have a stable genotype (data not shown). RecTULV formed foci similar in size to those of the original variant and grew to the titers 5 × 103 – 104 FFU/ml.
Vero E6 cells (5 × 106 cells) were infected with the 1:1 mixture of recTULV and TULV02, approximately 104 FFU altogether. After 7–12 days the supernatant (~20 ml) was collected and RNA was extracted from the cells with TriPure™ isolation reagent, Boehringer Mannheim. Aliquots (2 ml) of the supernatant were used to infect fresh cells; the rest was kept at -70°C. The following nine passages were performed in the same way.
Reverse transcription (RT), polymerase chain reaction (PCR) and sequencing
RT was performed with MuLV reverse transcriptase (New England Biolabs); for PCR, AmpliTaq DNA polymerase (Perkin Elmer, Roche Molecular Systems) was used. To monitor the presence of TULV S RNA on passages, RT-PCR was performed with primers VF738 (5'GCCTGAAAAGATTGAGGAGTTCC3'; nt 738–760) and VR855 (5'TTCACGTCCTAAAAGGTAAGCATCA3'; nt 831–855). To monitor the presence of recTULV S RNA, RT-PCR was performed with primers RECF738 (5'GCCAGAGAAGATTGAGGCATTTC3'; nt 738–760) and RECR855 (5'TTCTCTCCCAATTAGGTAAGCATCA3'; nt 831–855). All four primers were perfect matches to the homologous sequences; to the heterologous sequences, the forward primers have five mismatches while the reverse primers have six. Alternatively, complete S segment sequences of both variants of TULV were amplified using a single universal primer  and then either of the two pairs of primers was used in nested PCR. Authenticity of the PCR amplicons was confirmed by direct sequencing using the ABI PRISM Dye Terminator Sequencing kit (Perkin Elmer Applied Biosystems Division).
The authors thank Prof. Åke Lundkvist for fruitful discussion and Prof. Antti Vaheri for general support. This work was supported by the research grants RFA915 and 202012 from the Academy of Finland.
- Worobey M, Holmes EC: Evolutionary aspects of recombination in RNA viruses. J Gen Virol 1999, 80: 2535-1543.View ArticlePubMedGoogle Scholar
- Hirst GK: Genetic recombination with Newcastle disease virus, polioviruses and influenza virus. Cold Spring Harbor Symp Quant Biol 1962, 27: 303-309.View ArticlePubMedGoogle Scholar
- Ledinko N: Genetic recombination with poliovirus type 1: studies of crosses between a normal horse serum-resistant mutant and several guanidine-resistant mutants of the same strain. Virology 1963, 20: 107-119. 10.1016/0042-6822(63)90145-4View ArticlePubMedGoogle Scholar
- Cooper PD, Steiner-Pryor AS, Scotti PD, Delong D: On the nature of poliovirus genetic recombinants. J Gen Virol 1974, 23: 41-49.View ArticleGoogle Scholar
- Suzuki Y, Gojobori T, Nakagomi O: Intragenic recombination in rotaviruses. FEBS Letters 1998, 427: 183-187. 10.1016/S0014-5793(98)00415-3View ArticlePubMedGoogle Scholar
- Nayak DP, Chambers TM, Akkina RK: Defective interfering RNAs of influenza viruses: origin, structure, expression and interference. Curr Top Microbiol Immunol 1985, 114: 104-151.Google Scholar
- Lamb RA, Kolakofsky D: Paramixoviridae: the viruses and their replication. In Fields Virology. Third edition. Edited by: Fields BN, Knipe DM, Howley PM, Chanock RM, Melnick JL, Monath TP, Roizman B, Straus SE. Philadelphia: Lippincott-Raven publishers; 1996:1177-1204.Google Scholar
- Khatchikian D, Orlich M, Rott R: Increased viral pathogenicity after insertion of a 28S ribosomal RNA sequence into the hemagglutinin gene of influenza virus. Nature 1989, 340: 156-157. 10.1038/340156a0View ArticlePubMedGoogle Scholar
- Orlich M, Gottwald H, Rott R: Nonhomologous recombination between the hemagglutinin gene and the nucleoprotein gene of an influenza virus. Virology 1994, 204: 462-465. 10.1006/viro.1994.1555View ArticlePubMedGoogle Scholar
- Sibold C, Meisel H, Krüger DH, Labuda M, Lysy J, Kozuch O, Pejcoch M, Vaheri A, Plyusnin A: Recombination in Tula hantavirus evolution: analysis of genetic lineages from Slovakia. J Virol 1999, 73: 667-675.PubMed CentralPubMedGoogle Scholar
- Plyusnin A, Kukkonen SKJ, Plyusnina A, Vapalahti O, Vaheri A: Transfection-Mediated Generation of Functionally Competent Tula Hantavirus with Recombinant S RNA Segment. EMBO Journal 2002, 21: 1497-1503. 10.1093/emboj/21.6.1497PubMed CentralView ArticlePubMedGoogle Scholar
- Elliott RM, Bouloy M, Calisher CH, Goldbach R, Moyer JT, Nichol ST, Pettersson R, Plyusnin A, Schmaljohn CS: Family Bunyaviridae . In Virus taxonomy VIIth report of the International Committee on Taxonomy of Viruses. Edited by: van Regenmortel MHV, Fauquet CM, Bishop DHL, Carsten EB, Estes MK, Lemon SM, Maniloff J, Mayo MA, McGeoch DJ, Pringle CR, Wickner RB. San Diego: Academic Press; 1999:599-621.Google Scholar
- Plyusnin A, Morzunov S: Virus evolution and genetic diversity of hantaviruses and their rodent hosts. Curr Top Microbiol Immunol 2001, 256: 47-75.PubMedGoogle Scholar
- Nichol ST, Ksiazek TG, Rollin PE, Peters CJ: Hantavirus pulmonary syndrome and newly described hantaviruses in the United States. In The Bunyaviridae. Edited by: Elliott RM. New York: Plenum Press; 1996:269-280.View ArticleGoogle Scholar
- Lundkvist Å, Plyusnin A: Molecular epidemiology of hantavirus infections. In The Molecular Epidemiology of Human Viruses. Edited by: Leitner T. Boston-Dordrecht: Kluwer Academic Publishers; 2002:351-384.View ArticleGoogle Scholar
- Henderson WW, Monroe MC, St Jeor SC, Thayer WP, Rowe JE, Peters CJ, Nichol. ST: Naturally occurring Sin Nombre virus genetic reassortants. Virology 1995, 214: 602-610. 10.1006/viro.1995.0071View ArticlePubMedGoogle Scholar
- Li D, Schmaljohn AL, Anderson K, Schmaljohn CS: Complete nucleotide sequences of the M and S segments of two hantavirus isolates from California: evidence for reassortment in nature among viruses related to hantavirus pulmonary syndrome. Virology 1995, 206: 973-983. 10.1006/viro.1995.1020View ArticlePubMedGoogle Scholar
- Vapalahti O, Lundkvist Å, Kukkonen SKJ, Cheng Y, Gilljam M, Kanerva M, Manni T, Pejcoch M, Niemimaa J, Kaikusalo A, Henttonen H, Vaheri A, Plyusnin A: Isolation and characterization of Tula virus: a distinct serotype in genus Hantavirus , family Bunyaviridae . J Gen Virol 1996, 77: 3063-3067.View ArticlePubMedGoogle Scholar
- Plyusnin A, Vapalahti O, Lankinen H, Lehväslaiho H, Apekina N, Myasnikov Yu, Kallio-Kokko H, Henttonen H, Lundkvist Å, Brummer-Korvenkontio M, Gavrilovskaya I, Vaheri A: Tula virus: a newly detected hantavirus carried by European common voles. J Virol 1994, 68: 7833-7839.PubMed CentralPubMedGoogle Scholar
- Sironen T, Vaheri A, Plyusnin A: Molecular evolution of Puumala hantavirus. Journal of Virology 2001, 75: 11803-11810. 10.1128/JVI.75.23.11803-11810.2001PubMed CentralView ArticlePubMedGoogle Scholar
- Chare ER, Gould EA, Holmes EC: Phylogenetic analysis reveals a low rate of homologous recombination in negative-sense RNA viruses. J Gen Virol 2003, 84: 2691-703. 10.1099/vir.0.19277-0View ArticlePubMedGoogle Scholar
- Banner LR, Lai MMC: Random nature of coronavirus RNA recombination in the absence of selection pressure. Virology 1991, 185: 441-445. 10.1016/0042-6822(91)90795-DView ArticlePubMedGoogle Scholar
- Jarvis TC, Kirkegaard K: Poliovirus RNA recombination: mechanistic studies in the absence if selection. EMBO Journal 1992, 11: 3135-3145.PubMed CentralPubMedGoogle Scholar
- Tolskaya EA, Romanova LI, Blinov VM, Viktorova EG, Sinyakov AN, Kolesnikova , Agol VI: Studies on the reecombination between RNA genomes of poliovirus: the primary structure and nonrandom distribution of crossover regions in the genomes of intertypic poliovirus recombinants. Virology 1987, 161: 54-61. 10.1016/0042-6822(87)90170-XView ArticlePubMedGoogle Scholar
- Nagy PD, Simon AE: New insights into the mechanisms of RNA recombination. Virology 1997, 235: 1-9. 10.1006/viro.1997.8681View ArticlePubMedGoogle Scholar
- Negroni M, Buc H: Mechanisms of retroviral recombination. Ann Rev Genet 2001, 35: 275-302. 10.1146/annurev.genet.35.102401.090551View ArticlePubMedGoogle Scholar
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