Recombination is integral to the evolution of enteroviruses, including poliovirus (PV). The first evidence of recombination of PV was detected in vaccine-related isolates with chimeric sequences excreted by children exposed to the trivalent oral poliovirus vaccine (OPV) (King 1988; Cammack et al. 1988). Similar isolates were later detected in a number of patients with VAPP (Driesel et al. 1995; Li et al. 1996; Martin et al. 2002) as well as in healthy OPV recipients (Cammack et al. 1988; Tatem et al. 1991; Blomqvist et al. 2003). The heterologous sequences of most vaccine- related isolates were derived from the other Sabin OPV strains, with recombinants frequently found among vaccine- related isolates of all serotypes (Cammack et al. 1988; Lipskaya et al. 1991). A small proportion of vaccine-related isolates have capsid sequences derived from the OPV strains and noncapsid sequences derived from other, nonvaccine viruses. Recombination among the OPV strains is readily detectable because the sequences of the parental vaccine strains are well defined (Toyoda et al. 1984). Recombination also occurs during the circulation of wild- type polioviruses and other enteroviruses. Recombination of OPV and circulating wild-type PV was reported in China (Liu et al. 2000; Liu et al. 2003), and recombination of OPV and circulating non-polio enteroviruses were reported in the Dominican Republic and Haiti (Kew et al. 2002), in Madagascar (Rousset et al. 2003), in Egypt (Yang et al. 2003), and in the Philippines (Shimizu et al. 2004). Sequence analysis showed that the above circulating vaccine-derived polioviruses (cVDPVs) were recombinants between PV and unidentified enterovirus that underwent recombination in the nonstructural protein-coding regions of the genome. MICROBIOLOGY INDONESIA, December 2007, p 129-134 Volume 1, Number 3 ISSN 1978-3477 In Vitro Recombination of Poliovirus with Coxsackie A Virus Serotype 18 at Downstream Nonstructural Protein-Coding Regions ANDI UTAMA 1* AND HIROYUKI SHIMIZU 2 1 Research Center for Biotechnology, Lembaga Ilmu Pengetahuan Indonesia, Jalan Raya Bogor Km 46, Cibinong 16911, Indonesia, 2 Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011 Many genetic recombinations of poliovirus (PV) are to be found in excreted viruses, including viruses from vaccine- associated paralytic poliomyelitis (VAPP) as well as healthy vaccine recipients. Most recombinations were among different serotypes of PVs. However, recombination can also occur between PV and other enteroviruses. It was predicted that the hot spot of the recombination is in the nonstructural protein-coding regions, but the exact site is may be different in each recombination. We have demonstrated that the construct recombinant virus between PV and coxsackie A virus serotype 11 (CAV-11), or with CAV-17 with recombination site in the N-term of 2C-coding region, were viable. However, the recombination of PV with CAV-18 at this site was not viable. To determine if the recombination between PV and CAV-18 can occur at other sites, eight chimeric cDNAs (between PV [isolate PJ156] and CAV-18 [PJ156/CAV-18]), all having different recombination sites (2C-8, 2C-133, 2C-235, 2C-268, 2C-287, 2C-327, 3A-67, 3C-60) were constructed using the long-PCR method. The cDNA was then transcribed in vitro and then transfected into the HEp-2 cell-line. As expected, the recombinant virus PJ156/ CAV-18, with recombination sites 2C-327, 3A-67, and 3C-60 were viable, while all the others were not. The recombinant viruses displayed a slightly smaller plaque size, but demonstrated quite similar growth as compared to the parental control PJ156. Since analysis for similarity has shown that the homology between PV and CAV-18 was high around these regions, these results supported the copy-choice mechanism of enterovirus recombination. Key words: poliovirus, CAV-18, recombination _____________________________________________ ________________________ * Corresponding author, Phone: +62-21-8754587, Fax: +62-21-8754588, E-mail: andiutama2002@yahoo.com Although the significance and the mechanism of natural genetic recombination are still not understood, it can be suggested that it has a biological role in genetic recombination in PV evolution, especially for the prolonged circulation of OPV-related PV. Details of the mechanism of PV recombination are not well understood. However, based on current knowledge, the recombination is believed to occur by the ‘copy-choice’ mechanism with homologous genome templates (Wimmer et al. 1993). Generally, for single-stranded RNA viruses such as PV, the mechanism is probably copy choice (template switching during RNA replication) rather than trough true recombination i.e. the mechanism is analogous to gene conversion (Kirkegaard and Baltimore 1986). However, it may be different for the different virus recombinations. Furthermore, the hot spot of recombination in the genome of PV is not well defined. We previously demonstrated that chimeric cDNA constructed between PV and CAV-11 or CAV-17, with the crossover site in the N-term. part of 2C-coding region, resulted in viability of the virus (Utama and Shimizu 2005; Utama and Shimizu 2006). However, the virus was not viable when an RNA transcript derived from chimeric cDNA between PV and CAV-18, with the same crossover site, was transfected into the cell-line. In this study, chimeric cDNAs between PV and CAV-18, with various crossover sites, were artificially constructed. The RNA transcripts were then transfected into HEp-2 cell-line. The viability of the virus was analyzed and the viable recombinant viruses were characterized. MATERIALS AND METHODS Viral RNA Extraction and Construction of Chimeric cDNA. PJ156 isolated from an acute flaccid paralysis case in