HCV Phylogenetic Classification

Abstract

HCV’s considerable genetic variability, which exists at various levels across viral populations in individual infected individuals at any given moment and during evolution, is a distinguishing feature of the virus. Because of this, it was discovered in 1993 through phylogenetic analysis of incomplete HCV sequences from several patient isolates worldwide that the virus could be divided into six major genotypes with significant subtypes. Based on a study of full-length ORF sequences, this categorisation was later verified. A seventh significant genotype has been identified, albeit only detected in a few people. An eight genotype has also been recently identified. The number of published ORF sequenced HCV isolates has dramatically increased because of breakthroughs in sequence analysis tools. This chapter seeks to identify the 7 main genotypes and 93 additional subtypes of HCV.

1. Introduction

Much emphasis has been paid to the phylogeny and molecular evolution of HCV over the last two decades. These events have far-reaching ramifications for viral taxonomy and disease epidemiology, as well as pathogen control and tracing. Furthermore, they are critical components in diagnoses, treatment regimen selection, and patient follow-up schedules, as well as vaccine development. In this paper, we outline the current perspective of HCV phylogeny and examine the genesis, distribution, and clinical significance of HCV genotypes. In addition, we describe the available evidence on HCV molecular evolution in the context of host-virus interplay at various biological levels, during the disease course, and after treatment.

The genetic diversity of the hepatitis C virus (HCV) is quite significant, and the variety of HCV genotypes and subtypes is growing. HCV was formerly divided into 7 different genotypes that varied by more than 30% at the nucleotide level [1]. Four individuals from the Indian state of Punjab who were epidemiologically unrelated were recently found to have the unique HCV genotype 8, which forms a different phylogenetic group from previously reported genomes [2]. Subtypes of genotypes having a sequence divergence of less than 15% are further subdivided [1]. HCV genotypes 1, 2, and 3 are present globally, albeit their distribution varies depending on the region [3].

The most common HCV genotype worldwide (46%) is genotype 1. While subtypes 1a and 1b of the HCV virus are more common in North America, Europe, and Australia, subtype 1b infection affects 73% of HCV-infected people in Japan. Regardless of location, persons who inject drugs (PWIDs) have a disproportionately large distribution of genotype 3, the second most common genotype in the world (30%) and is mainly found in South Asia. HCV genotype 4 infections are primarily prevalent in Africa and the Middle East, while genotypes 5 and 6 are only found in Southern Africa and Southeast Asia, respectively [4]. Multiple subtypes make up genotypes 1, 2, 3, 4, and 6, exhibiting a high genetic diversity level. HCV genotype 7a was discovered in a patient from the Democratic Republic of the Congo in 2006. Another patient from the same area was later found to have genotype 7b infection [1, 5]. Only one subtype has been documented for genotype 5 and the recently discovered genotype 8.

It is essential to describe novel subtypes and comprehend the potential effects of novel subtypes on treatment success, given the substantial genetic variety of HCV, both at the genotype and subtype levels. This chapter reviews a thorough investigation of viral diversity and sequence variation across genotypes to uncover uncharacterised subgroups and their impact on treatment outcomes.

2. Confirmed genotypes and subtypes revision

From the 18 mentioned in 2005 (1), there are now more verified genotypes and subtypes: 67 in 2013 (2), 86 in 2017, 90 in May 2019, and 93 in March 2022 (Table 1). The first virtually entire genome sequence of the HCV was published in 1989. Prior to this discovery, HCV had developed and spread unnoticed across the human population for hundreds of years, giving birth to a diverse range of endemic and epidemic isolates capable of causing chronic liver disease. It quickly became apparent that isolates from various people or nations had significant genetic variability [63]. This variation was compiled after thorough research and surveys by organisations from around the world, and variants were assigned as genotypes and subtypes in a consensus classification and nomenclature system. Official standards were also established for the assignment and naming of future variants [6]. Phylogenetic groups distinct from previously described sequences, at least three epidemiologically unrelated isolates, one or more complete coding region sequences, and the exclusion of intergenotypic or intersubtypic recombination, regardless of whether the components were classified, are all requirements for genotype and subtype assignments [1]. Using these criteria validated the identification of six unique genotypes with 18 subtypes. In addition, 58 subtypes were temporarily designated, awaiting the discovery of further isolates or complete sequencing for the coding area. This consensus on nomenclature was mirrored by the creation of several curated databases, including the Los Alamos HCV Sequence Database, the euHCVdb [64], and the Hepatitis Virus Database (http://s2as02.genes.nig.ac.jp/), which organised HCV sequences as they became available and indicated which genotypes and subtypes were confirmed or provisionally assigned. A proposal to standardise the numbering of HCV concerning genotype 1a isolate H77 was made concurrently (AF009606) [65].

Genotype1	Locus/Isolate(s)2	Accession number(s)	Reference(s)
Genotype 1
1a	HPCPLYPRE, HPCCGAA	M62321, M67463	[7, 8]
1b	HPCJCG, HPCHUMR	D90208, M58335	[9, 10]
1c	HPCCGS, AY051292	D14853, AY051292	[11]
1d	QC103	KJ439768	[12]
1e	148,636	KC248194	[13]
1 g	1804	AM910652	[14]
1 h	EBW443, EBW9	KC248198, KC248199	[13]
1i	QC181	KJ439772	[12]
1j	QC329	KJ439773	[12]
1 l	136,142, EBW424	KC248193, KC248197	[13]
1 m	QC196, QC87	KJ439778, KJ439782	[12]
1n	QC113, QC74	KJ439775, KJ439781	[12]
1o	QC316, DE/17-0414	KJ439779, MH885469	[12]
Genotype 2
2a	HPCPOLP, JFH-1	D00944, AB047639	[15, 16]
2b	HPCJ8G, JPUT971017	D10988, AB030907	[17, 18]
2c	BEBE1	D50409	[19]
2d	QC259	JF735114	[20]
2e	QC64	JF735120	[20]
2f	ZS542, GZ98799	KC844042, KC844050	[21]
2i	D54	DQ155561	[22]
2j	C1799, QC232	HM777358 JF735113	[20]
2 k	VAT96	AB031663	[23]
2 l	MRS89, PTR7904	KC197235, KC197240	[24]
2 m	QC178, BID-G1314	JF735111, JX227967	[20]
2q	963,852	FN666428, FN666429	[25]
2r	QC283	JF735115	[20]
2 t	MRS40	KC197238	[24]
2u	QC182	JF735112	[20]
2v	495-05	MW041295	[26]
Genotype 3
3a	HPCEGS, HPCK3A	D17763, D28917	[27, 28]
3b	HPCFG	D49374	[29]
3d	NE274	KJ470619	[30]
3e	NE145	KJ470618	[30]
3 g	BID-G1243, QC260	JX227954, JF735123	[31]
3 h	QC29	JF735121	[31]
3i	IND-HCV, BID-G1244	FJ407092, JX227955	[32]
3 k	HPCJK049E1, QC105	D63821, JF735122	[31]
Genotype 4
4a	ED43	Y11604	[33]
4b	QC264	FJ462435	[34]
4c	QC381	FJ462436	[34]
4d	03-18, QC382	DQ418786, FJ462437	[34]
4f	IFBT88, PS6	EF589161, EU392175	[35]
4 g	QC193	FJ462432	[34]
4 k	PS3, QC383	EU392173, FJ462438	[34, 35]
4 l	QC274	FJ839870	[34]
4 m	QC249	FJ462433	[34]
4n	QC97	FJ462441	[34]
4o	QC93	FJ462440	[34]
4p	QC139	FJ462431	[34]
4q	QC262	FJ462434	[34]
4r	QC384	FJ462439	[34]
4 s	QC361	JF735136	[36]
4 t	QC155	FJ839869	[34]
4v	CYHCV073, BID-G1248	HQ537009, JX227959	[37]
4w3	P212, P245	FJ025855, FJ025856	[38]
Genotype 5
5a	EUH1480, SA134	Y13184, AF064490	[39]
Genotype 6
6a	EUHK2,6a33	Y12083, AY859526	[40]
6b	Th580	D84262	[41]
6c	Th846	EF424629	[42]
6d	VN235	D84263	[41]
6e	GX004	DQ314805	[43]
6f	C-0044	DQ835760	[44]
6 g	HPCJK046E2	D63822	[45]
6 h	VN004	D84265	[41]
6i	Th602	DQ835770	[44]
6j	Th553	DQ835769	[44]
6 k	VN405	D84264	[41]
6 l	537,796	EF424628	[42]
6 m	B4/92	DQ835767	[44]
6n	KM42, D86/93	DQ278894, DQ835768	[44]
6o	QC227	EF424627	[42]
6p	QC216	EF424626	[42]
6q	QC99	EF424625	[42]
6r	QC245	EU408328	[46]
6 s	QC66	EU408329	[46]
6 t	VT21, D49	EF632071, EU246939	[47, 48]
6u	D83	EU246940	[47]
6v	NK46, KMN-02	EU158186, EU798760	[49]
6w	GZ52557, D140	DQ278892, EU643834	[50]
6xa5	DH012, DH028	EU408330, EU408332	[51]
6xb	TV476, VN110	JX183552, KJ567645	[52, 53]
6xc	TV520	KJ567651	[53]
6xd	L23, L347	KM252789, KM252790	[54]
6xe	DH027, KM98	JX183557, KM252792	[52, 55]
6xf	VN214, TV469	KJ567647, KJ567646	[53]
6xg	KS27, KS81	MH492360, MH492361	[54]
6xh	1350-1	MG879000	[56]
6xi	KM35, YNKH261	JX183549, MZ504973	[52, 57]
6xj	KM45, YNKH298a	DQ2788916, MZ171127	[58, 59]
Genotype 7
7a	QC69	EF108306	[60]
7b	BAK1	KX092342	[61]
Genotype 8
8a	GT8-1	MH590698	[62]

Table 1.

Confirmed HCV genotypes and subtypes (march, 2022) (adapted from Simmonds et al. [6]).

¹

Gene/subtype names that have been unanimously suggested. Two sequences of an HCV genotype have been listed where more than one is available, with the sequences prioritised by (i) publication date, or (ii) submission date when unpublished.

²

Locus (or isolate name if locus is the same as the accession number).

³

Previously described as 4b [38].

⁴

A chimpanzee infected experimentally with (human-derived) isolate SA13 had its sequence taken from the acute phase plasma.

⁵

Previously described as 6u [47].

⁶

Previously described as 6 k [58].

An alignment of these genotypes/subtypes is be found at http://hcv.lanl.gov/content/sequence/NEWALIGN/align.html.

SSEv1.1 [66] and Muscle v3.8.31 [67] were used to align with unique HCV entire or nearly complete coding area sequences from NCBI Genome (969 sequences, http://www.ncbi.nlm.nih.gov/genome) and the Los Alamos HCV sequence database (1364 sequences >8000 nt from http://hcv.lanl.gov/content/index). Seven significant phylogenetic groups corresponding to genotypes 1 through 8 are revealed by phylogenetic analysis of sequences that comprise >95% of the coding area (Figure 1). 100% of bootstrap replications support clustering of the constituent subtypes within these genotypes.

Figure 1.

The typical full coding area sequences of HCV are arranged in this phylogenetic tree. As proposed in the 2005 consensus proposal (1), for an HCV isolate to be considered as a new confirmed genotype or subtype, a complete coding region sequence that: (a) forms a distinct phylogenetic group from previously described sequences, (b) is represented by at least three epidemiologically unrelated isolates, and (c) does not represent a recombinant between other genotypes or subtypes should be obtained.

According to the consensus criteria, confirmed subtypes (indicated by a letter after the genotype) need sequence data from at least two other isolates in core/E1 (>90% of the sequence corresponding to positions 869 to 1292 of the H77 reference sequence [accession number AF009606] numbered according to reference [65]) and NS5B (>90% of pos (Table 1) [6].

Analysis of the many possible subtypes that have been sequenced (Figure 2) lends credence to using a 15% criterion throughout the coding area. Except for the distances of 14 and 14.2% between JX227963 and two subtype 4 g sequences, this shows significant and regular gaps in the pairwise distances within and between each genotype’s subtype distribution, which were dispersed as follows.: genotype 1: 12.9–17.0%, genotype 2: 13.1–17.6%, genotype 3: 12.5–19.6%, genotype 4: 12.7–15.3%, and genotype 6: 9.9–14.9% (with the exception of the 13.1–13.7% between EU246931 and three subtype 6e sequences). Therefore, a substantial distinction between isolates that differ by 13% throughout their full coding area sequences (members of the same subtype) and those that differ by >15% can be determined for all genotypes with very few exceptions (different genotypes or subtypes). Sequences that are not currently represented by three or more independent isolates of recognised HCV subtypes but are different from any of those subtypes are included in this chapter. It is uncertain if the reported outliers result from different epidemiological histories or technological issues [1].

Figure 2.

Distribution of p-distances between sequences with entire coding regions. Using SSE, the frequency of p-distances within and between genotypes was determined. With the exception of subtypes 1a, 1b, and 2b, where 20 random sequences were employed [66], intra-genotypes pairwise distances were determined for all accessible full coding area sequences. Frequencies were adjusted to get the maximum frequency down to under 300 for p-distances >0.15 (corresponding to a percent difference of 15%). The frequencies scaled as above, and distances between genotypes were determined using one or two samples of each confirmed and unassigned subtype (adapted from [1]).

The eight genotypes that have been confirmed (described in Figure 1) consist of 93 established subtypes, 13 subtypes that have been allocated tentatively, and 47 subtypes that have not yet been assigned [68]. These tables are available on the ICTV website at http://talk.ictvonline.org/links/hcv/hcv-classification.html and will be updated on a regular basis by the authors with data from other resources, such as typing tools, HCV databases (http://hcv.lanl.gov/;http://euhcvdb.ibcp.fr/euHCVdb/), and subtyping tools (e.g., On the ICTV Website and at http://hcv.lanl.gov/content/sequence/NEWALIGN/align.html.

A few variations with contradictory assignments were discovered during the production of these tables. Isolates P026, P212, and P245 (FJ025854-6) are classified as subtype 4b [38], although their full coding region sequences only share 85% similarity with isolate Z1 (U10235, L16677), which is provisionally designated as 4b [69] and more closely linked to isolate QC264’s core/E1 (FJ46243516 [34]). A third isolate (P213, GU049362) has the NS5B sequence for the same new subtype that P212 and P245 belong to, making this verified subtype 4w. Despite being represented by a single nucleotide that varies from all other genotype 4 sequences by more than 17.5%, isolate P026 is presently unassigned.

Similar to this, isolates KM45 and KM41 (DQ278891,3) have been identified as subtype 6 k [58]. However, they vary from each other and isolate VN405 (D84264) by 6.7 and > 17%, respectively, in the entire coding area sequence, leaving them to be a genotype 6 undefined subtype. Subtype 6u has been assigned to two different groups of isolates: EU408330-2 [51] and EU246940 [47]. The latter was submitted to GenBank first and is represented by NS5B sequences from two additional isolates; as a result, it was given subtype 6u, whereas EU408330, EU408331, and EU408332 were given the subtype 6xa designation.

Finally, Smith et al. [1] analysis of sequence divergence and phylogenetic groupings opines that several isolates [52] classified as “subtype k-related” (TV257, KM35, QC273, TV476), “subtype l-related” (L349, TV533), “intermediate between subtypes 6m and 6n” (DH027), or “intermediate among subtypes 6j and 6i” (QC271) in their GenBank accessions should be regarded as unassigned novel subtypes.

2.1 Further levels of taxonomy

There are challenges in imposing a discrete categorisation method on a complicated taxonomy when defining this taxonomic difference between viral genotypes and subtypes. There are probably many taxonomic hierarchies, particularly for genotypes 3 and 6. For instance, a clade formed by many genotype 6 isolates with subtypes 6 k and 6 l [52]. These sequences and subtypes 6 m and 6n are part of a higher-level clade, while these subtypes and subtypes 6i and 6j are part of another grouping (Figure 1). The discontinuous distribution of p-distances across full coding area sequences (Figure 2), which consists of three practically overlapping ranges (approximately 15–20%, 20–25%, and 25–30%), reflects these evolutionary hierarchies.

2.2 Anticipated developments

The approach for categorising variations into genotypes and subtypes has proven unexpectedly reliable despite the increased amount and diversity of HCV sequences. The partitioning of the seven verified genotypes into subtypes that differ across a whole coding area sequence by >15% represents a natural break in the distribution of sequence distances, and the seven confirmed genotypes show significant bootstrap support (Figure 2). There are still some questions regarding the endemic region of genotype 5, which is represented by a single subtype that has been isolated in Europe, Brazil, North Africa, and South Africa, and genotype 7, which has been isolated from a Congolese immigrant. We may also expect to find more HCV-like viruses in the genus Hepacivirus [70, 71, 72, 73], as well as variations that are more genetically related to HCV than the non-primate Hepacivirus that appears to be an endemic infection of horses globally [70].

3. Conclusion

This chapter established that there are 8 genotypes of HCV, and 93 subtypes. Of the 8 genotypes, 7 were highlighted in this chapter. The chapter also attempted to link HCV genotypes to their endemicity. Utilising the phylogenetic classification of HCV can provide a greater insight into eradicating the virus by 2030.

Acknowledgments

We thank Dr. Donald B. Smith of the University of The University of Edinburgh for granting us permission to use the figures and tables in his earlier published paper.