Scotomas in molecular virology and epidemiology of hepatitis C virus

Table 1 Summary of in vitro and in vivo models for hepatitis C virus

In vitro and in vivo models	Established year	Advantages	Deficiencies
In vitro
Cultivation of HCV	1993-1999	Achieved cultivation of HCV in human foetal liver cells, human hepatocytes or PBMC. Illustrated HCV is quite species selective and has a narrow range of hosts	Requires specific cellular factors to support viral lifecycle. Primary human and chimpanzee hepatocytes or highly differentiated cells dependent. Most of them have yielded limited success. Poor reproducibility and low levels of HCV replication
HCV replicon	1995-2000	Provided a cell-based model for the study on HCV genome replication
HCV VLP	1998-1999	Rare evidence to support that HCV structural proteins core, E1, and E2 could form VLP
HCVpp	2003	Provided a convenient and feasible tool for studies on viral entry, HCV receptor, neutralizing antibody, etc.
HCVcc	2005	A break through in production of infectious hepatitis C virus in tissue culture
In vivo
Chimpanzee	1979	The only recognized animal model for HCV study, played a critical role in HCV discovery and play an essential role in defining the natural history of HCV	Chimpanzees differ from humans in their course of infection, that chronic carriers do not develop cirrhosis or fibrosis, limited availability, cost performance, and public resistance
Tree shrew	1998	Might be a succedaneum for chimpanzees	Persistent HCV infection could not be established and only 25% of infected animals developed transient or intermittent viremia. Germ line was not available to a small animal model
Chimeric human liver mouse	2001	Exhibited prolonged infection with high viral titers following inoculation with HCV isolated from human serum. HCV can be transmitted horizontally. Drug evaluation	Since the mice were immunodeficient, they were not appropriate models to study HCV pathogenesis
Genetically humanized mouse	2011	Represents the first immunocompetent mice model for HCV study. Allows for the studies of HCV coreceptor biology in vivo	Operation is difficult

HCVpp: Hepatitis C virus (HCV) pseudotyped viral particles; VLP: Virus like particle; HCVcc: Cell culture derived HCV.

Table 2 Summary of the properties of hepatitis C virus structural proteins

	Core	E1	E2	p7
Genome location	342-914	915-1490	1491-2579	2580-2769
Translation processing site	Rough ER
Amino acid composition	191	192	363	63
Molecular weight (kDa)	21-23	33-35	70-72	7
Glycosylation	No	Yes	Yes	No
Cleavage	ER signal peptidase and SPP
Crystal structure	Not available			Revealed
Functional unit	Dimer	Heterodimer?		Hexamer
Common function	Viral particle formation. Core, E1 and E2, together with p7 and NS2, are required for virus assembly (assembly module)
Unique function	Capsid protein, viral particle formation, viral genome recognizing and packaging. Interacts with cLDs in early viral particle formation process. Counters host antiviral factors and involves pathogenesis	Envelope glycoproteins, interact with SRB1, CD81, CLDN1, OCLN, etc. to trigger viral entry. Promote fusion with the endosomal membrane. Counter host immune response via hypervariable regions		Viroporin. Has key roles in organizing the virus assembly complex. p7-NS2 complex interacts with the NS3-4A enzyme to retrieve core protein from cLDs to form viral particle
Major scotomas	How do the core form the viral capsid? The signals and processes that mediate RNA packaging are largely unknown. What impeded us to resolve the structure of the viral glycoproteins? What is the real process in HCV entry? How are these receptors and co-receptors temporally and spatially used to ensure the early infection processes?

Start co-ordinates based on H77 (accession number, NC_004102). SRB1: Scavenger receptor class B member 1; CD81: Tetraspanin CD81; CLDN1: The tight junction protein claudin 1; OCLN: The tight junction protein occludin; cLDs: Cytosolic lipid droplets; ER: Endoplasmic reticulum; SPP: Signal peptidase and signal peptide peptidase; HCV: Hepatitis C virus. The molecular weights of E1 and E2 refer to the glycosylated forms.

Table 3 Summary of the properties of hepatitis C virus non-structural proteins

	NS2	NS3	NS4A	NS4B	NS5A	NS5B
Genome location	2769-3419	3420-5312	5313-5474	5475-6257	6258-7601	7602-9378
Translation processing site	Rough ER
Amino acid composition	810-1026	1027-1657	1658-1711	1712-1972	1973-2420	2421-3012
Molecular weight (kDa)	21-23	70	8	27	56-58	65-68
Cleavage	Viral cysteine protease NS2-3 and the serine-type protease activity of the viral NS3-NS4A complex
Crystal structure	C-terminal (aa904-1026) was solved	Revealed	Revealed	Not available	Revealed	Revealed
Functional unit	Homodimer	Monomer or oligomer	Monomer	Oligomer	Homodimer	Monomer
Common function	Replication module
Unique function	A metal-dependent proteinase, many functions dependent on the interaction with P7 and NS3. Participation in proteolytic cleavage at the NS2-NS3 junction of the polyprotein. Both the TMDs and protease domain of NS2 are required for the production of virus particles	The DAA targeting protein. NS3 was anchored in ER membrane by cofactor NS4A. NS3-4A complex has serine-type protease activity and NTPase/RNA helicase activities. Nonspecific cleavage of two critical interferon induction proteins: MAVS and TRIF	The central portion of NS4A, residues 21-32, intercalates into NS3 and activates the protease activity by stabilizing this protease subdomain and contributing to the substrate recognition site. The C-terminal acidic portion of NS4A interacts with the NS3 helicase and other HCV proteins and contributes to RNA replication as well as assembly	A master organizer of replication complex formation. NTPase activity? RNA binding?	Produced as multiple phospho-variants. RNA-binding phosphoprotein involved in RNA replication. Phosphorylation of a specific serine residue within the C-terminus by CKIIα is essential for virus assembly. The interaction of NS5A with the cLD-bound core protein is the key steps in HCV assembly	RNA-dependent RNA polymerase
Major scotomas	How HCV particles are organized? What is the accurate duty of each nonstructural protein in viral lifecycle? How do the nonstructural proteins utilize host cellular factors for its own survival? Why HCV lifecycle is tightly associated with components of LDLs and VLDLs?

Start co-ordinates based on H77 (accession number, NC_004102). Aa: Amino acid; TMD: Transmembrane domain; CKII: Casein kinase II; cLD: Cytoplasmic lipid droplet; LDL: Low-density lipoprotein; VLDL: Very-low-density lipoprotein; MAVS: Mitochondrial antiviral signaling protein; TRIF: TIR-domain-containing adaptor inducing interferon; HCV: Hepatitis C virus.

Table 4 Epidemiological features of hepatitis C virus infection

Epidemiological index	Current consensus
Source of infection	Chronic HCV carriers
Route of transmission	HCV transmission occurs primarily through exposure to infected blood. Past: Receiving infected blood or organ transplantation, from accidental exposure to infected blood, and sexual transmission in persons with high risk behaviours. Present: HCV is usually spread by sharing infected needles with a chronic HCV carrier, and some people acquire the infection through nonparenteral means that have not been fully defined.
Susceptible population	General population
Incubation period	Average 6-10 wk
Prevalence and incidence	3% of the world’s population have HCV
Rate of chronic infection	Up to more than 80%
Outcome of chronic infection	10%-20% of chronic HCV carriers may develop into cirrhosis and liver failure. 1%-5% of chronic HCV carriers are associated with the development of hepatocellular carcinoma
Molecular epidemiology	HCV is classified into eleven major genotypes (designated as 1-11), many subtypes (designated a, b, c, etc.), and about 100 different strains (numbered 1, 2, 3, etc.) based on the genomic sequence heterogeneity. Genotypes 1-3 have a worldwide distribution. Types 1a and 1b are the most common, accounting for about 60% of global infections. Type 2 is less frequently represented than type 1. Type 3 is endemic in southeast Asia and is variably distributed in different countries. Genotype 4 is principally found in the Middle East, Egypt, and central Africa. Type 5 is almost exclusively found in South Africa, and genotypes 6-11 are distributed in Asia.
Stability	HCV is inactivated by exposure to lipid solvents or detergents, heating at 60 °C for 10 h or 100 °C for 2 min in aqueous solution, formaldehyde (1:2000) at 37 °C for 72 h, β-propriolactone and UV irradiation.
Vaccine	Not available

HCV: Hepatitis C virus.