Mlolecular Tool Corresponding With Phenotypie Differenees Of PanAsia-1Strains Of Foot-and-mouth Disease Virus Serotype O In China

Foot-and-mouth disease (FMD), one of serious animal diseases of pigs, cattle, sheep and goats, iscaused by foot-and-mouth disease virus (FMDV). Serotype O is the most prevalent serotype comparedto the other serotypes (A, Asia1) of FMDV in China. To understand genetic variations and phenotypicdifferences at the molecular level, in this study, we selected three virus isolates of the PanAsia-1strainfrom bovine and porcine based on sequence determination and comparison of nucleotides and/ordeduced amino acids homology. The analyses of the complete genome sequences showed: Two bovineisolates in1999and one swine isolate in2008were subdivided into PanAsia-1a and PanAsia-1bsubstrains of the PanAsia-1strain of Middle-East South-Asia (ME-SA) topotype, respectively. Instructural protein, the distinct amino acid residues located at positions136(E-F loop),175(G-H loop)and214(C-terminal) of VP2,174(G-H loop) of VP3,142,152and153(G-H loop) and199(C-terminal) of VP1. The virulence to BHK-21cells and suckling mice of FMDV O/NX/CHA/99(TCID₅0=6.625, LD₅0=7.33) was lower than O/HK/HB/CHA/99(TCID₅0=7.75, LD₅0=8.33) andO/ZheJ/CHA/8/2008(TCID₅0=7.875, LD₅0=8.5). These three viruses differed from their plaquephenotypes on BHK-21cells. O/NX/CHA/99formed small and compact plaques, O/HK/HB/CHA/99formed large and turbid plaques, and O/ZheJ/CHA/8/2008formed large and clear plaques.Concentrations of heparin sodium from0to1mg/ml greatly decreased the plaque formation ofO/NX/CHA/99, but had no effect of O/HK/HB/CHA/99and O/ZheJ/CHA/8/2008on BHK-21cells.Interestingly, O/NX/CHA/99and O/HK/HB/CHA/99produced plaques on CHO-K1cells, and plaqueformation could be inhibited by addition of heparin sodium. However, O/ZheJ/CHA/8/2008can notproduce plaques on CHO-K1cells. These results indicate that O/NX/CHA/99has a high affinity toheparan sulfate (HS) receptor, O/HK/HB/CHA/99can utilize HS as cellular receptors, whereasO/ZheJ/CHA/8/2008propagated in cultured cells independent of HS binding.To identify the key amino acid residues determing the plaque size and the binding ability to HSreceptor, we constructed chimeric full-length cDNA clones and generated chimeric FMDV mutants asdescribed. By using site-directed mutagenesis and exchange-cassette strategy,9,3, and12cDNAsencoding the VP3, VP1, and VP0capsid proteins of an infectious genome-length cDNA copy of HSutilizing-deficient OZK93(rOZK, hs^-) were replaced with appropriate variants and cDNAscorresponding to PanAsia-1strains, producing chimeric FMDV mutants for providing additional insightinto the close relationship among virulence, plaque phenotypes, alternative mechanisms to receptorsusage and amino acid substitutions in capsid proteins of FMDV.25different DNA fragment patternswere designed and stably maintained the sequence of the original rescued viruses, based on T7RNApolymerase in vivo transcription system, respectively. The plaque forming and/or inhibiting assays onBHK-21, CHO-K1, pgsA745(xylosyltransferase I-deficient) and pgsB618(galactosyltransferaseI-deficient) confirmed:1) Chimeric virus containing capsid protein VP1of O/NX/CHA/99formed smalland large blend plaques on BHK-21cells, produced plaques on CHO-K1cells, and grew poorly onpgsA745and pgsB618. Plaque formation was inhibited on CHO-K1cells but had moderate effect on BHK-21cells when heparin sodium was added to diluted virus suspension. The results demonstratedthat the adaptation to HS receptor by this virus appears not to compromise their ability to make use ofintegrins. The capsid protein VP1of O/NX/CHA/99was identified as the critical determinant relevantto FMDV-HS receptor interaction. Residue K83in VP1potentially functions as one of critical aminoacids for HS receptor binding in cultured cells. Q80L change within B-C loop of VP2can lead to loss ofHS receptor binding ability of O/HK/HB/CHA/99.2) Residues79and136were measured in VP2of thePanAsia-1strain as the critical amino acid determinants of plaque phenotypes, which have an influenceon the average size of plaque production.2079H (basic) and2136G (neutral) facilitated the formation ofsmall plaques of rescued FMDV variants.2079Y (neutral) and2136E (acidic) enlarged the plaquesformed by other FMDV mutants. In addition, a single amino acid replacement at residue8(Ser→Ala,hydrophilic, polar→hydrophobic, nonpolar) of VP4can play a helper role in heparin recognition.In conclusion, these studies confirmed the ability of FMDV to bind different classes of receptors isrelated to host tropism. The bovine isolates can infect cells via HS-mediated adsorption, while FMDVisolated from porcine targets susceptible cells via integrins interaction. The efficiency of FMDV-HSreceptor interaction is associated with phenotypic differences on BHK-21cells and virulence. Mutationsat critical amino acid sites (basic-neutral-acidic) in capsid proteins might generate change of plaquephenotypes, of the PanAsia-1strain of FMDV serotype O.