RNase III cleavage demonstrates a long range RNA: RNA duplex element flanking the hepatitis C virus internal ribosome entry site

Here, we show that Escherichia coli Ribonuclease III cleaves specifically the RNA genome of hepatitis C virus (HCV) within the first 570 nt with similar efficiency within two sequences which are ∼400 bases apart in the linear HCV map. Demonstrations include determination of the specificity of the cleavage sites at positions C(27) and U(33) in the first (5′) motif and G(439) in the second (3′) motif, complete competition inhibition of 5′ and 3′ HCV RNA cleavages by added double-stranded RNA in a 1:6 to 1:8 weight ratio, respectively, 50% reverse competition inhibition of the RNase III T7 R1.1 mRNA substrate cleavage by HCV RNA at 1:1 molar ratio, and determination of the 5′ phosphate and 3′ hydroxyl end groups of the newly generated termini after cleavage. By comparing the activity and specificity of the commercial RNase III enzyme, used in this study, with the natural E.coli RNase III enzyme, on the natural bacteriophage T7 R1.1 mRNA substrate, we demonstrated that the HCV cuts fall into the category of specific, secondary RNase III cleavages. This reaction identifies regions of unusual RNA structure, and we further showed that blocking or deletion of one of the two RNase III-sensitive sequence motifs impeded cleavage at the other, providing direct evidence that both sequence motifs, besides being far apart in the linear RNA sequence, occur in a single RNA structural motif, which encloses the HCV internal ribosome entry site in a large RNA loop

Characterizing the function and structural organization of the 5′ tRNA-like motif within the hepatitis C virus quasispecies

Hepatitis C virus (HCV) RNA is recognized and cleaved in vitro by RNase P enzyme near the AUG start codon. Because RNase P identifies transfer RNA (tRNA) precursors, it has been proposed that HCV RNA adopts structural similarities to tRNA. Here, we present experimental evidence of RNase P sensitivity conservation in natural RNA variant sequences, including a mutant sequence (A368–G) selected in vitro because it presented changes in the RNA structure of the relevant motif. The variation did not abrogate the original RNase P cleavage, but instead, it allowed a second cleavage at least 10 times more efficient, 4 nt downstream from the original one. The minimal RNA fragment that confers sensitivity to human RNase P enzyme was located between positions 299 and 408 (110 nt). Therefore, most of the tRNA-like domain resides within the viral internal ribosome entry site (IRES) element. In the variant, in which the mutation stabilizes a 4 nt stem–loop, the second cleavage required a shorter (60 nt) substrate, internal to the minimal fragment substrate, conforming a second tRNA-like structure with similarities to a ‘Russian-doll’ toy. This new structure did not impair IRES activity, albeit slightly reduced the efficiency of translation both in vitro and in transfected cells. Conservation of the original tRNA-like conformation together with preservation of IRES activity points to an essential role for this motif. This conservation is compatible with the presence of RNA structures with different complexity around the AUG start codon within a single viral population (quasispecies)

Propidium monoazide RTqPCR assays for the assessment of hepatitis A inactivation and for a better estimation of the health risk of contaminated waters.

The waterborne transmission of hepatitis A virus (HAV), the main cause of acute hepatitis, is well documented. Recently, two ISO proposals for sensitive determination of this pathogen by RTqPCR in water and food have been published (ISO/TS 15216-1 and ISO/TS 15216-2), and could enable the formulation of regulatory standards for viruses in the near future. However, since detected viral genomes do not always correlate with virus infectivity, molecular approaches need to be optimized to better predict infectivity of contaminated samples. Two methods involving the use of propidium monoazide (PMA), with or without Triton X-100, prior to RTqPCR amplification were optimized and adapted to infer the performance of infectious viral inactivation upon two different water treatments: free chlorine and high temperature. Significant correlations between the decrease of genome copies and infectivity were found for both inactivation procedures. The best procedure to infer chlorine inactivation was the PMA-RTqPCR assay, in which 1, 2 or 3-log genome copies reductions corresponded to reductions of infectious viruses of 2.61 ± 0.55, 3.76 ± 0.53 and 4.92 ± 0.76 logs, respectively. For heat-inactivated viruses, the best method was the PMA/Triton-RTqPCR assay, with a 1, 2 or 3-log genome reduction corresponding to reductions of infectious viruses of 2.15 ± 1.31, 2.99 ± 0.79 and 3.83 ± 0.70 logs, respectively. Finally, the level of damaged virions was evaluated in distinct types of water naturally contaminated with HAV. While most HAV genomes quantified in sewage corresponded to undamaged capsids, the analysis of a river water sample indicated that more than 98% of viruses were not infectious. Although the PMA/Triton-RTqPCR assay may still overestimate infectivity, it is more reliable than the RTqPCR alone and it seems to be a rapid and cost-effective method that can be applied on different types of water, and that it undeniably provides a more accurate measure of the health risk associated to contaminated waters

Inactivation of hepatitis A virus and human norovirus in clams subjected to heat treatment

Bivalve mollusk contamination by enteric viruses, especially human noroviruses (HuNoV) and hepatitis A virus (HAV), is a problem with health and economic implications. The aim of the study was the evaluation of the effect of heat treatment in clams (Tawera gayi) experimentally contaminated with HuNoV using a PMA-viability RTqPCR assay to minimize measurement of non-infectious viruses, and used HAV as a model to estimate infectivity loss. Spiked clams were immersed in water at 90°C to ensure that internal meat temperature was maintained above 90°C for at least 5 min. The treatment resulted in >3.89 ± 0.24 log10 TCID50/g reduction of infectious HAV, confirming inactivation. For HuNoV, RTqPCR assays showed log10 reductions of 2.96 ± 0.79 and 2.56 ± 0.56, for GI and GII, respectively, and the use of PMA resulted in an additional log10 reduction for GII, providing a better correlation with risk reduction. In the absence of a cell culture system which could be used to determine HuNoV infectivity reduction, a performance criteria based on PMA-RTqPCR log reduction could be used to evaluate food product safety. According to data from this study, heat treatments of clams which cause reductions >3.5 log10 for GII as measured by PMA-RTqPCR assay may be regarded as an acceptable inactivation treatment, and could be set as a performance criterion to test the effectiveness of other time-temperature inactivation processes

Advances for the Hepatitis A virus antigen production using a virus strain with codon frequency optimization adjustments in specific locations

The available cell-adapted hepatitis A virus (HAV) strains show a very slow replication phenotype hampering the affordable production of antigen. A fast-growing strain characterized by the occurrence of mutations in the internal ribosome entry site (IRES), combined with changes in the codon composition has been selected in our laboratory. A characterization of the IRES activity of this fast-growing strain (HM175-HP; HP) vs. its parental strain (HM175; L0) was assessed in two cell substrates used in vaccine production (MRC-5 and Vero cells) compared with the FRhK-4 cell line in which its selection was performed. The HP-derived IRES was significantly more active than the L0-derived IRES in all cells tested and both IRES were more active in the FRhK-4 cells. The translation efficiency of the HP-derived IRES was also much higher than the L0-derived IRES, particularly, in genes with a HP codon usage background. These results correlated with a higher virus production in a shorter time for the HP strain compared to the L0 strain in any of the three cell lines tested, and of both strains in the FRhK-4 cells compared to Vero and MRC-5 cells. The addition of wortmannin resulted in the increase of infectious viruses and antigen in the supernatant of FRhK-4 infected cells, independently of the strain. Finally, the replication of both strains in a clone of FRhK-4 cells adapted to grow with synthetic sera was optimal and again the HP strain showed higher yields

Summary of secondary structure probing of HCV 331–390 (WT and A368G) RNAs

<p><b>Copyright information:</b></p><p>Taken from "Characterizing the function and structural organization of the 5′ tRNA-like motif within the hepatitis C virus quasispecies"</p><p>Nucleic Acids Research 2005;33(5):1487-1502.</p><p>Published online 8 Mar 2005</p><p>PMCID:PMC1062876.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Sensitivity to various RNases is indicated on the predicted RNA structure. HCV WT structure was forced for base pairing between CU355–356 and AG366–367. HCV A368G was predicted by RNA structure 3.5 program () (only one structure was predicted). Arrows indicate cleavages by single-strand-specific RNases T1 and A, whereas triangles show cleavage by double-strand-specific RNase V1. Nucleotide numbering is used as in (5′–3′ orientation)

Inactivation of Hepatitis A Virus and Human Norovirus in Clams Subjected to Heat Treatment

Bivalve mollusk contamination by enteric viruses, especially human noroviruses (HuNoV) and hepatitis A virus (HAV), is a problem with health and economic implications. The aim of the study was the evaluation of the effect of heat treatment in clams (Tawera gayi) experimentally contaminated with HuNoV using a PMA-viability RTqPCR assay to minimize measurement of non-infectious viruses, and used HAV as a model to estimate infectivity loss. Spiked clams were immersed in water at 90°C to ensure that internal meat temperature was maintained above 90°C for at least 5 min. The treatment resulted in &gt;3.89 ± 0.24 log10TCID50/g reduction of infectious HAV, confirming inactivation. For HuNoV, RTqPCR assays showed log10reductions of 2.96 ± 0.79 and 2.56 ± 0.56, for GI and GII, respectively, and the use of PMA resulted in an additional log10reduction for GII, providing a better correlation with risk reduction. In the absence of a cell culture system which could be used to determine HuNoV infectivity reduction, a performance criteria based on PMA-RTqPCR log reduction could be used to evaluate food product safety. According to data from this study, heat treatments of clams which cause reductions &gt;3.5 log10for GII as measured by PMA-RTqPCR assay may be regarded as an acceptable inactivation treatment, and could be set as a performance criterion to test the effectiveness of other time-temperature inactivation processes

Diagram of genotype 1b HCV IRES RNA secondary structure (sequence 2–418 used in this work) modified from ()

<p><b>Copyright information:</b></p><p>Taken from "Characterizing the function and structural organization of the 5′ tRNA-like motif within the hepatitis C virus quasispecies"</p><p>Nucleic Acids Research 2005;33(5):1487-1502.</p><p>Published online 8 Mar 2005</p><p>PMCID:PMC1062876.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Substitutions C204U and A214U in the WT sequence are indicated; the A368G sequence only differs from the WT sequence by the A368G mutation. A double arrow indicates RNase P cleavage site (361–363). A line between nucleotides 339 and 361 depicts the hybridization site of a DNA primer used for selection in . Structural sub-domains are indicated from I to IV

Propidium monoazide RTqPCR assays for the assessment of hepatitis A inactivation and for a better estimation of the health risk of contaminated waters.

The waterborne transmission of hepatitis A virus (HAV), the main cause of acute hepatitis, is well documented. Recently, two ISO proposals for sensitive determination of this pathogen by RTqPCR in water and food have been published (ISO/TS 15216-1 and ISO/TS 15216-2), and could enable the formulation of regulatory standards for viruses in the near future. However, since detected viral genomes do not always correlate with virus infectivity, molecular approaches need to be optimized to better predict infectivity of contaminated samples. Two methods involving the use of propidium monoazide (PMA), with or without Triton X-100, prior to RTqPCR amplification were optimized and adapted to infer the performance of infectious viral inactivation upon two different water treatments: free chlorine and high temperature. Significant correlations between the decrease of genome copies and infectivity were found for both inactivation procedures. The best procedure to infer chlorine inactivation was the PMA-RTqPCR assay, in which 1, 2 or 3-log genome copies reductions corresponded to reductions of infectious viruses of 2.61 ± 0.55, 3.76 ± 0.53 and 4.92 ± 0.76 logs, respectively. For heat-inactivated viruses, the best method was the PMA/Triton-RTqPCR assay, with a 1, 2 or 3-log genome reduction corresponding to reductions of infectious viruses of 2.15 ± 1.31, 2.99 ± 0.79 and 3.83 ± 0.70 logs, respectively. Finally, the level of damaged virions was evaluated in distinct types of water naturally contaminated with HAV. While most HAV genomes quantified in sewage corresponded to undamaged capsids, the analysis of a river water sample indicated that more than 98% of viruses were not infectious. Although the PMA/Triton-RTqPCR assay may still overestimate infectivity, it is more reliable than the RTqPCR alone and it seems to be a rapid and cost-effective method that can be applied on different types of water, and that it undeniably provides a more accurate measure of the health risk associated to contaminated waters