Dengue type 4 virus mutants containing deletions in the 3' noncoding region of the RNA genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in rhesus monkeys

Abstract

The dengue type 4 virus (DEN4) genome contains a 384-nucleotide (nt) 3 noncoding sequence in which the last 81 nt, predicted to form a secondary structure, are thought to be essential for virus replication. Immediately upstream of the secondary structure, short RNA sequences that are conserved among mosquito-borne flaviviruses have been identified. A series of deletions that range from 30 to 262 nt were introduced into this upstream region of full-length DEN4 cDNA to create viable deletion mutants, some of which might prove to be useful for inclusion in a live attenuated virus vaccine. When studied by an infectious-center assay, most full-length RNA transcripts of the deletion constructs exhibited reduced infectivity when transfected into simian LLC-MK 2 cells compared with the full-length RNA transcripts of wild-type parental virus. Deletion mutations that extended as far as the 5 boundary of the 3 noncoding region and whose 3 boundary did not extend beyond the last 113 nt of the 3 end were viable. With the exception of mutant 3d 303-183, which contained a deletion of nt 303 to 183 from the 3 terminus, deletion mutants produced plaques that appeared late on simian LLC-MK 2 cells or exhibited a small-plaque morphology on mosquito C6/36 cells compared with the wild-type virus. These mutants also replicated less efficiently and attained a lower titer in LLC-MK 2 cells than parental wild-type virus. Significantly, mutant 3d 303-183 grew to a high titer and was least restricted in growth. Mutant 3d 303-183 and four other moderately to severely restricted mutants were selected for evaluation of infectivity and immunogenicity in rhesus monkeys. There was a suggestion that occurrence and duration of viremia were reduced for some of the deletion mutants compared with the wild-type virus. However, more convincing evidence for attenuation of some of the mutants was provided by an analysis of antibody response to infection. Mutant 3d 303-183 induced an antibody response equivalent to that stimulated by wild-type virus, whereas other mutants induced low to moderate levels of antibodies, as measured by radioimmunoprecipitation and virus neutralization. The immunogenicity of these 3 DEN4 deletion mutants in monkeys appeared to correlate with their efficiency of growth in simian LLC-MK 2 cells. One or more mutants described in this paper may prove to be useful for immunization of humans against disease caused by dengue virus.

Flavivirus reverse genetic systems, construction techniques and applications: A historical perspective

“…DENV-2 viruses containing single point substitution in the 5 0 UTR exhibited reduced replicative fitness in cellulo and reduced neurovirulence for newborn mice (Leardkamolkarn et al, 2010). This observation was confirmed for several other flaviviruses when deletions were engineered into the 3 0 and 5 0 UTR causing attenuation in cell culture and in animals (Blaney et al, 2010(Blaney et al, , 2008Cahour et al, 1995;Mandl et al, 1998;Men et al, 1996;Zeng et al, 1998). Van Slyke et al (2012) engineered point mutations in the WNV NS5 gene which encodes the RNA polymerase (Van Slyke et al, 2012).…”

“…By introducing mutations or constructing intratypic/heterotypic chimeric viruses, numerous virulence determinants have been identified and analyzed to understand the mechanistic basis behind these altered phenotypes (reviews (Hurrelbrink and McMinn, 2003;Mandl, 2005)). Blaney et al (2008Blaney et al ( , 2010, Cahour et al (1995), de Wispelaere and Yang (2012), Huang et al (2005), Hung et al (2004), Leardkamolkarn et al (2010), Matusan et al (2001), Men et al (1996), Modis et al (2004) and Zeng et al (1998); JEV Arroyo et al (2001), Liang et al (2009), Lin et al (2006, Liu et al (2004), Monath et al (2002), Sumiyoshi et al (1995) and Guirakhoo et al (2000Guirakhoo et al ( , 2001Guirakhoo et al ( , 2006, Monath et al (1999Monath et al ( , 2001Monath et al ( , 2003Monath et al ( , 2006, Pugachev et al (2004) and Van Der Most et al (2000); DENV Bray and Lai (1991), Bray et al (1996Bray et al ( , 1998, Caufour et al (2001), Chen et al (1995), Guirakhoo et al (2000Guirakhoo et al ( , 2001Guirakhoo et al ( , 2006 Antivirals DENV Qing et al (2010) and Yang et al (2014) Using an infectious clone of WNV, Pijlman et al (2008) showed that all members of...…”

Emergency Services of Viral RNAs: Repair and Remodeling

“…Various deletions and insertions in the 3= UTR as well as complete deletion of the poly(A) tail may not kill alphaviruses (457)(458)(459), flaviviruses (460)(461)(462), coronaviruses (463)(464)(465), and various plant viruses (466)(467)(468)(469). The repair of the damaged genomes may again involve different mechanisms, including RNA recombination, the use of the viral RdRP-or host-dependent polyadenylation activities, and perhaps some others.…”

Core protein-mediated 5′–3′ annealing of the West Nile virus genomic RNA in vitro

“…1). Mutations disrupting complementarity in any of these elements abrogate viral replication in subgenomic replicon model systems (Alvarez et al, 2008(Alvarez et al, , 2005bBredenbeek et al, 2003;Corver et al, 2003;Friebe and Harris, 2010;Friebe et al, 2011;Khromykh et al, 2001;Lo et al, 2003;Men et al, 1996;Villordo et al, 2010;Zhang et al, 2008), while compensatory mutations rescue viability (Alvarez et al, 2008(Alvarez et al, , 2005bFriebe and Harris, 2010;Friebe et al, 2011;Khromykh et al, 2001;Lo et al, 2003;Zhang et al, 2008).…”