Inhibition of Respiratory Syncytial Virus Replication by Simultaneous Targeting of mRNA and Genomic RNA Using Dual-Targeting siRNAs.

We attempted to generate siRNAs with two active strands, which can simultaneously knock down the expression of mRNA and viral genomic RNA. In this study, short hairpin RNAs (shRNAs) against N and F genes were used. Expression of F and N mRNA transcripts as well as genomic RNA was determined with relative real-time RT-PCR. The RSV load in infected cell culture supernatant was determined by absolute quantitative real-time PCR. We found that (i) in the presence of shRNA-N, a greater reduction in viral genomic RNA was found; (ii) the level of expression at MOI 0.01 was reduced more than MOI 0.1; (iii) reduction in N transcript was greater than F; and (iv) finally, in combination pre-treatment with two shRNAs, the reduction was not significant as compared to single shRNA transfection. shRNAs also inhibited the production of RSV progeny as shown by viral load in infected HEp-2 cells. (i) Virus load reduction was greater at MOI 0.01 than 0.1 and (ii) significant load reduction was not seen with combination shRNA pre-treatment. The antiviral potency was also confirmed by plaque assay and western blot analysis. Our results provided further evidence that RNAi could be a powerful treatment option against respiratory viruses.

Comparison of neutralizing antibody titers against outbreak-associated measles genotypes (D4, H1 and B3) in Iran.

Despite the accessibility of a promising vaccine, outbreaks of the measles virus (MV) take place even in well-vaccinated populations. D4, H1 and B3 genotypes have been detected regularly in different regions of Iran. These observations highlight the necessity of evaluating the protective efficacy of the vaccine against currently circulating MV genotypes during the elimination phase. A focus reduction neutralization test has been developed to measure the neutralizing antibodies against different genotypes of MV, such as H1, D4, B3 and vaccine strain (A), in children after second doses of measles vaccine. The geometric mean titer (GMT) rates of the sera against D4, H1, B3 and A genotypes were 95.9, 90.5, 32.0 and 76.1, respectively. Low GMTs of antibody against the B3 genotype compared with the other genotypes were indicated. Based on the current study results, the MV antibody titers in the sera of vaccinated cases are sufficient to neutralize all circulating genotypes in Iran; however, neutralizing antibody titers were lower for the B3 genotype than for the H1, D4 and A genotypes. The heterogeneous nature of MV, for instance the nucleotide sequence diversity between different strains, necessitates the evaluation of the protective efficacy of the vaccine against measles B3 genotype in countries where this virus has been the most commonly identified circulating genotype.

Implementation of a national measles elimination program in Iran: Phylogenetic analysis of measles virus strains isolated during 2010-2012 outbreaks.

Measles virus (MV) causes small and large outbreaks in Iran. Molecular assays allow identifying and the sources of measles imported from neighboring countries. We carried out a phylogenetic analysis of measles virus circulating in Iran over the period 2010-2012. Specimens from suspected cases of measles were collected from different regions of Iran. Virus isolation was performed on urine and throat swabs. Partial nucleoprotein gene segments of MV were amplified by RT-PCR. PCR products of 173 samples were sequenced and analyzed. The median age of confirmed cases was 2 years. Among all confirmed cases, 32% had unknown vaccination status, 20% had been vaccinated, and 48% had not been vaccinated. Genotypes B3 and D8 (for the first time), H1 and D4 were detected mainly in unvaccinated toddlers and young children. Genotype B3 became predominant in 2012 and was closely related to African strains. H1 strains were also found in small and large outbreaks during 2012 but were not identical to Iranian H1-2009 strains. A majority of the Iranian D4 strains during 2010-2012 outbreaks were linked to the D4 strain identified in the Pakistan in 2007. We identified a single case in 2010 belonging to D8 genotype with 99.7% identity to Indian isolates. Although the vaccination program is currently good enough to prevent nationwide epidemics and successfully decreased measles incidence in Iran, the fraction of protected individuals in the population was not high enough to prevent continuous introduction of cases from abroad. Due to increasing number of susceptible individuals in some areas, sustained transmission of the newly introduced viral genotype remains possible.