Use of NS-4 peptides to identify type-specific antibody to hepatitis C virus genotypes 1, 2, 3, 4, 5 and 6

Abstract

The 5' end of the NS-4 protein of different genotypes of hepatitis C virus (HCV) is highly variable in nucleotide and inferred amino acid sequence, with frequent predicted amino acid substitutions between all six of the major HCV genotypes described to date. This region has been shown to be antigenic by epitope mapping, and elicits antibody in HCV-infected individuals with a detectable type-specific component. We have used this sequence data to specify branched peptides for an indirect binding/competition assay to detect typespecific antibody to each major genotype. A total of 183 out of 210 samples (87%) from blood donors and patients with chronic hepatitis C infected with genotypes 1 to 6 showed detectable type-specific antibody to NS-4 peptides that in almost all cases (> 97 %) corresponded to the genotype detected by a PCR typing method. These findings demonstrate the existence of major antigenic differences between genotypes of HCV, and indicate how infection with different variants of HCV may be detected by a serological test.

Complete nucleotide sequence of a type 4 hepatitis C virus variant, the predominant genotype in the Middle East.

“…Phylogenetic analysis of the complete genomic sequence of ED43 revealed a similar relationship to other genotypes as found previously from sequence comparisons of this variant in subgenomic regions such as NS5 and NS4 (Bhattacherjee et al, 1995 ;Simmonds et al, 1993 b) (Fig. 3).…”

“…Complete nucleotide sequences have been determined for at least 26 HCV genomes representing genotypes 1a, 1b, 1c, 2a, 2b, 2c, 3a and 3b and the putative genotypes 10a and 11a. However, genotype 4 has, to date, only been defined by analysis of sequences representing the 5hNCR, core, E1, NS4 and NS5 regions (Simmonds et al, a, b, 1994Bhattacherjee et al, 1995), and additional sequence at the 3hNCR beyond the poly(U) tract has been determined from the particular sample used in the present study (Kolykhalov et al, 1996). To enable a more complete comparison of type 4 with other variants of HCV we have determined the sequence for the ORF and 3hNCR, to the poly(U) tract, of HCV isolate ED43, genotype 4, from an Egyptian residing in Saudi Arabia, which represents the first complete sequence for this genotype.…”

“…The majority of HCV isolates so far studied can be classified into six major groups (Simmonds et al, 1994 b) designated genotypes 1-6, with subdivisions in each (subtypes a, b, c, etc.). A further three genotypes (7, 8 and 9) have been proposed based on partial sequences at the 5h and 3h ends of the genomes of isolates from Vietnam andThailand (Tokita et al, 1994, 1995), and recently another two genotypes (10a and 11a) have been suggested based on comparisons of complete sequences of isolates from Indonesia with those of types 1, 2 and 3 (Tokita et al, 1996) Fragments a-d were produced using primers previously described (Simmonds et al, a, b, 1994Bhattacherjee et al, 1995), fragments e-r were generated using ED43-specific primers and primers based on consensus sequences determined from aligning sequences from the other published genotypes (see Table 1 for accession numbers) and fragments s and t were produced using only ED43-specific primers. The location of each fragment is shown relative to its position with HCV-1 (genotype 1a ; acc.…”

Variation of the hepatitis C virus 5' non-coding region: implications for secondary structure, virus detection and typing

“…Nucleotide sequences of the HCV 5'NCR and corresponding sequences from coding regions of the genome were obtained from published sources (Chan et al, 1992;Bukh et al, 1992Bukh et al, a, 1993Bukh et al, , 1994Stuyver et al, 1993Stuyver et al, , 1994Simmonds et al, 1993a, b, c;Honda et al, 1993;Okamoto et al, 1993Okamoto et al, , 1994Tokita et al, 1994b;van Doorn, 1994;Bhattacherjee et al, 1995), from GenBank (accession nos D13406, D10074, M96362, U01214, D10934, X65924, D10075, D 10077, D 17763, D00830, D16433, D16434, D28917, D11443, X76918, Z36522-Z36527, M86779, M86765, L12353-L12355 and L29585), and new sequence information for viruses from a variety of HCV infected individuals. RNA was extracted from sera, reverse transcribed and amplified by PCR using 5'NCR specific primers 939, 209, 940 and 211 as described previously (Chan et al, 1992).…”

“…However, sequence analysis is impractical for large-scale genotyping of clinical specimens and several indirect methods have been developed. Virus genotype can be predicted from the serological response of infected individuals to the NS4 protein as measured by reactivity to type-specific peptides (Simmonds et al, 1993c;Tsukiyama Kohara et al, 1993;Bhattacherjee et al, 1995). However, such assays do not identify virus subtypes, and are less reliable for multiply exposed patients, such as haemophiliacs, or in the context of a deficient humoral response.…”