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Six variants of Japanese encephalitis (JE) virus strain P3 were selected for resistance to binding to mouse brain membrane receptor preparations (MRP). All but one of these MRP escape (MRP R ) variants were significantly attenuated in mice for both neuroinvasiveness (>200-fold) and neurovirulence (>500-fold) compared to their parent virus. Attenuated mouse brain MRP R variants...
Six passages of the mosquito-borne flavivirus yellow fever (YF) wild-type strain Asibi in HeLa cells attenuated the virus for monkeys and newborn mice and resulted in loss of mosquito competence. Attenuation after the passage in HeLa cells was not unique to YF virus strain Asibi as demonstrated by the HeLa passage attenuation of wild-type YF virus strain French viscerotropic virus and YF vaccine virus...
The identification of variants that are unable to bind membrane receptor preparations (MRPs) has previously been shown to select attenuated yellow fever and Japanese encephalitis viruses. In this study, this methodology has been extended to the tick-borne serocomplex of flaviviruses. Langat (LGT) virus strain TP21 was bound to mouse or human brain MRPs and viruses that escaped binding were isolated...
Bovine parainfluenza virus type 3 (bPIV3) is under development as a live virus vaccine vector. The RNA genome of a recombinant bPIV3 harbored four nucleotide changes, one of which resulted in a mutation of the viral polymerase (A. A. Haller et al., 2000, J. Virol. 74, 11626–11635). The contribution of this conservative amino acid substitution (I1103V) in the polymerase to the temperature-sensitive...
The replication dynamics of simian immunodeficiency virus (SIVmac32H-C8), attenuated through discrete genetic disruption of the nef gene, were compared with the wild-type parental clone (SIVmac32H-J5) using quantitative molecular methods. The primary viraemia of both infections were similar during the first week, but peaked on Day 10 at higher levels for wild-type virus. Viral RNA levels differed...
In a short time, West Nile virus has developed into a nationwide health and veterinary problem. The high virulence of the circulating virus and related lineage 1 WN strains hinders development of an attenuated live vaccine. We describe an attenuated WN isolate, WN1415, which is a molecularly cloned descendant of the WN prototype B956 strain. The parent virus belongs to lineage 2, members of which...
Transposon linker insertion mutagenesis of a full-length infectious clone (IC) (pBIC) of the pathogenic classical swine fever virus (CSFV) strain Brescia was used to identify genetic determinants of CSFV virulence and host range. Here, we characterize a virus mutant, RB-C22v, possessing a 19-residue insertion at the carboxyl terminus of E1 glycoprotein. Although RB-C22v exhibited normal growth characteristics...
Classical swine fever virus (CSFV) E2 glycoprotein contains a discrete epitope (TAVSPTTLR, residues 829–837 of CSFV polyprotein) recognized by monoclonal antibody (mAb) WH303, used to differentiate CSFV from related ruminant pestiviruses, Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus (BDV), that infect swine without causing disease. Progressive mutations were introduced into mAb WH303...
Transmissible gastroenteritis virus (TGEV) isolates that have been adapted to passage in cell culture maintain their infectivity in vitro but may lose their pathogenicity in vivo. To better understand the genomic mechanisms for viral attenuation, we sequenced the complete genomes of two virulent TGEV strains and their attenuated counterparts: virulent TGEV Miller M6 and attenuated TGEV Miller M60...
West Nile virus (WNV) infections in vertebrates are generally acute but persistent infections have been observed. To investigate the ability of WNV to produce persistent infections, we forced subgenomic WNV replicons to replicate within a cell without causing cell death. Detailed analyses of these cell-adapted genomes revealed mutations within the nonstructural protein genes NS2A (D73H, M108K), NS3...
We have previously reported [Risatti, G.R., Borca, M.V., Kutish, G.F., Lu, Z., Holinka, L.G., French, R.A., Tulman, E.R., Rock, D.L. 2005a. The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine. J. Virol. 79, 3787–3796] that chimeric virus 319.1v containing the E2 glycoprotein gene from Classical Swine Fever Virus (CSFV) vaccine strain CS with the genetic background...
E rns glycoprotein, along with E 1 and E 2 , is one of the three envelope glycoproteins of classical swine fever virus (CSFV). E rns is a heavily glycosylated protein involved in several functions, including virus attachment and entry to target cells, production of neutralizing antibodies, and virulence. The role of added glycans to CSFV strain Brescia E rns ...
Two genetically distinct infectious recombinant virus clones (pMLV, constructed from Ingelvac® PRRS MLV and pMN184, constructed from virulent strain MN184) were developed to study attenuation of contemporary porcine reproductive and respiratory syndrome virus (PRRSV) strain MN184. Two reciprocal chimeric clones (pMLVORF1/MN184 and pMN184ORF1/MLV) were then constructed, such that the 5′UTR/ORF1 of...
To identify genetic changes that lead to the attenuation of measles virus (MV), a strain of MV that is pathogenic in rhesus macaques was adapted to grow in Vero cells, Vero/hSLAM cells and, to simulate the process used to derive live attenuated vaccines, in primary chicken embryo fibroblasts (CEF). Comparison of the complete genomic sequences of the pathogenic wild-type (Davis87-wt) and four cell...
We report here the discovery of an attenuation mechanism of classic swine fever virus (CSFV) induced by introduction of a continuous 12-nt (CUUUUUUCUUUU) insertion in viral 3′ UTR. The 12-nt insertion sequence was first found in one attenuated vaccine strain HCLV (Hog Cholera Lapinized Virus) which did not exist in other CSFV strains. To address the function of the 12-nt insertion in viral replication...
Comparative sequence analysis of a series of strains of equine arteritis virus (EAV) of defined virulence for horses, ranging from the horse-adapted virulent Bucyrus (VB) strain to a fully attenuated vaccine strain derived from it, identified 13 amino acid substitutions associated with attenuation. These include 4 substitutions in the replicase proteins and 9 in the structural proteins. Using reverse...
Tick-borne flaviviruses are maintained in nature in an enzootic cycle involving a tick vector and a vertebrate host. Thus, the virus replicates in two disparate hosts, each providing selective pressures that can influence virus replication and pathogenicity. To identify viral determinants associated with replication in the individual hosts, plaque purified Langat virus (TP21pp) was adapted to growth...
Until recently strategies for controlling Classical Swine Fever Virus (CSFV) involve either prophylactic vaccination or non-vaccination with elimination of infected herds depending on the epidemiological situation of the affected geographical area. Marker vaccines allowing distinction between naturally infected from vaccinated swine could complement “stamping out” measures. Here we developed a double...
E1, along with E rns and E2 is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). Previously we showed that glycosylation status of virulent CSFV strain Brescia E2 or E rns affects virus virulence. Here, the three putative glycosylation sites of E1 were serially removed by means of site directed mutagenesis of a CSFV Brescia infectious clone (BICv) and their...
North American eastern equine encephalitis virus (NA-EEEV) strains cause high mortality in humans, whereas South American strains (SA-EEEV) are typically avirulent. To clarify mechanisms of SA-EEEV attenuation, we compared mouse-attenuated BeAr436087 SA-EEEV, considered an EEEV vaccine candidate, with mouse-virulent NA-EEEV strain, FL93-939. Although attenuated, BeAr436087 initially replicated more...
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