Analysis of genome determinants of virulence and setting-up of a library of full-size genome copies of the attenuated virus strain of the porcine reproductive andrespiratory syndrome virus (North American type)


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Abstract

Primary genome structures of 3 variants of the NADC-8 North American virulent strain of porcine reproductive
and respiratory syndrome virus (PRRSV) were compared for the purpose of detecting any potential genetic virulence
determinants of genus Arterivirus. Apart from the virulent variant, we also investigated the attenuated variant,
obtained after 251 passages in cell culture, and the intermediate variant isolated from a pig after a partial
reversion of the attenuated virus. The attenuated variant genome acquired a 3-nucleotide deletion and 50 mutations
versus its virulent precursor. A comparison of the attenuated and intermediary virus variants denoted 8 nucleotide
mutations entailing substitutions of 6 amino acids in 3 open reading frames (ORF1a, ORF1b and ORF6). A
32-clone library was constructed in the pACYC177 plasm id vector, which comprised full-size copies of the genome
of the NADC-8 attenuated variant strain (251), virus PPCC, for the purpose of experimentally verifying the functional
role of the obtained mutations. Full-size analogues (+-chain of RNA) of the viral genome, comprising the
CAP-structures and polyadenylated ones were obtained in vitro on the basis of the cloned DNA. Seven of the 8
analyzed clones of the viral genome were infected and their insertion into the MARC-145 cell resulted in obtaining
of infectious PRRSVs. Four of the constructed recombinant viruses had delayed growth parameters, and 3 of them
were similar to the parental strain. The described technology (inverse genetics) would make it possible to introduce
changes into the viral genome in applied and fundamental research of Arteriviruses.

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Copyright (c) 2004 Grebennikova T.V., Zaberezhny A.D., Musiyenko M.I., Mengeling W., Lager K., Aliper T.I., Nepoklonov E.A.

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