Hypervariable region 1 (HVR1) of hepatitis C virus (HCV) E2 envelope glycoprotein has been implicated in virus neutralization and persistence. We deleted HVR1 from JFH1-based HCV recombinants expressing Core/E1/E2/p7/NS2 of genotypes 1 to 6, previously found to grow efficiently in human hepatoma Huh7.5 cells. The 2a ⌬HVR1 , 5a ⌬HVR1 , and 6a ⌬HVR1 Core-NS2 recombinants retained viability in Huh7.5 cells, whereas 1a ⌬HVR1 , 1b ⌬HVR1 , 2b ⌬HVR1 , 3a ⌬HVR1 , and 4a ⌬HVR1 recombinants were severely attenuated. However, except for recombinant 4a ⌬HVR1 , viruses eventually spread, and reverse genetics studies revealed adaptive envelope mutations that rescued the infectivity of 1a ⌬HVR1 , 1b ⌬HVR1 , 2b ⌬HVR1 , and 3a ⌬HVR1 recombinants. Thus, HVR1 might have distinct functional roles for different HCV isolates. Ultracentrifugation studies showed that deletion of HVR1 did not alter HCV RNA density distribution, whereas infectious particle density changed from a range of 1.0 to 1.1 g/ml to a single peak at ϳ1.1 g/ml, suggesting that HVR1 was critical for low-density HCV particle infectivity. Using chronic-phase HCV patient sera, we found three distinct neutralization profiles for the original viruses with these genotypes. In contrast, all HVR1-deleted viruses were highly sensitive with similar neutralization profiles. In vivo relevance for the role of HVR1 in protecting HCV from neutralization was demonstrated by ex vivo neutralization of 2a and 2a ⌬HVR1 produced in human liver chimeric mice. Due to the high density and neutralization susceptibility of HVR1-deleted viruses, we investigated whether a correlation existed between density and neutralization susceptibility for the original viruses with genotypes 1 to 6. Only the 2a virus displayed such a correlation. Our findings indicate that HVR1 of HCV shields important conserved neutralization epitopes with implications for viral persistence, immunotherapy, and vaccine development.

To facilitate genotype-specific high-throughput studies of hepatitis C virus (HCV), we have developed reporter viruses using JFH1-based recombinants expressing core-nonstructural protein 2 (NS2) of genotype 1 to 7 prototype isolates. We introduced enhanced green fluorescent protein (EGFP) into NS5A domain III of the genotype 2a virus J6/JFH1 [2a(J6)]. During Huh7.5 cell culture adaptation, 2a(J6)-EGFP acquired a 40-amino-acid (aa) (⌬40) or 25-aa (⌬25) deletion in NS5A domain II, rescuing the impairment of viral assembly caused by the EGFP insertion. ⌬40 conferred efficient growth characteristics to 2a(J6) tagged with EGFP, DsRed-Express2, mCherry, or Renilla luciferase (RLuc), yielding peak supernatant infectivity titers of 4 to 5 log 10 focus-forming units (FFU)/ml. 2a(J6) with ⌬40 or ⌬25 was fully viable in Huh7.5 cells. In human liver chimeric mice, 2a(J6)-EGFP⌬40 acquired various deletions in EGFP, while 2a(J6)⌬40 did not show an impaired viability. We further developed panels of JFH1-based genotype 1 to 7 core-NS2 recombinants expressing EGFP-or RLuc-NS5A⌬40 fusion proteins. In cell culture, the different EGFP recombinants showed growth characteristics comparable to those of the nontagged recombinants, with peak infectivity titers of 4 to 5 log 10 FFU/ml. RLuc recombinants showed slightly less efficient growth characteristics, with peak infectivity titers up to 10-fold lower. Overall, the EGFP and RLuc recombinants were genetically stable after one viral passage. The usefulness of these reporter viruses for high-throughput fluorescence-and luminescence-based studies of HCV-receptor interactions and serum-neutralizing antibodies was demonstrated. Finally, using RLuc viruses, we showed that the genotype-specific core-NS2 sequence did not influence the response to alfa-2b interferon (IFN-alfa-2b) and that genotype 1 to 7 viruses all responded to treatment with p7 ion channel inhibitors.Hepatitis C virus (HCV) is a small, enveloped virus classified as a member of the family Flaviviridae. Its single-stranded RNA genome contains one long open reading frame (ORF) encoding structural proteins (core and envelope glycoproteins E1 and E2), p7, forming a putative ion channel, and nonstructural protein 2 (NS2), NS3, NS4A, NS4B, NS5A, and NS5B (8). The NS5A phosphoprotein consists of 3 domains (47). While NS5A domains I and II are important for replication (46,47), domain III has been shown to be important for viral assembly (5,28,45). Due to significant genetic heterogeneity, HCV was classified into 7 major genotypes and numerous subtypes that differ in ϳ30% and ϳ20% of their sequences, respectively (42). Genotypes also differ biologically (34) and in their rates of response to interferon (IFN)-based combination therapy and specific antivirals (27, 36).HCV poses a major socioeconomic burden, with approximately 180 million chronically infected individuals worldwide who are at risk of developing liver cirrhosis and hepatocellular carcinoma (15, 41). Treatment options are limited; combination therapy with IFN-alfa-2 and ri...

Internal transcribed spacers (ITS) and the 5.8S ribosomal gene of 21 Naegleria fowleri strains and eight other species including Naegleria gruberi were sequenced. The results showed that this region can help differentiate between and within species. The phylogeny of Naegleria spp. deduced from the ITS and the 5.8S gene produced four major lineages, fowleri-lovaniensis, galeacystis-italica-clarki-gruberi-australiensis, andersoni-jamiesoni, and pussardi, that fit perfectly with those inferred from the 18S rRNA gene analysis. The N. gruberi isolate, NG260, was closely related to Naegleria pussardi. The other N. gruberi isolates branched together with Naegleria australiensis in another lineage. The ITS and 5.8S results for N. fowleri were congruent with those previously deduced by RAPD analysis. The phylogenetic analysis inferred from ITS and RAPD data revealed two major groups. The French Cattenom and Chooz and South Pacific strains constituted the first group. The second group encompassed the strains corresponding to the Euro-American and Widespread RAPD variants and shared the same substitution in the 5.8S gene. In addition, it was possible to define species specific primers in ITS regions to rapidly identify N. fowleri.