The categorization of HCV isolates into genotypes occurred during the characterization of HCV clones in different laboratories by sequencing. This has led to the establishment of extensive phylogenetic trees, which are usually based upon comparative sequence analysis of subgenomic regions, such as those spanning core, E1, or NS5B (Bukh et al., 1995) (Section 3.2). Given the high sequence diversity of HCV, sequence analysis of these short regions has often provided enough information for definitive genotyping and, in many cases, subtyping as well (Bukh et al., 1993). Although sequencing is the “gold standard” for genotyping and subtyping, several faster methods have been designed to provide the same information. For example, RT/PCR products from different subgenomic regions of the viral genome have been generated using universal primers that presumably permit amplification of viral RNA from all genotypes and subtypes (Ch. 19). Following amplification using universal primers, one approach then uses genotype-specific primers for further (nested) amplification of only targets with the corresponding sequences (Kato et al., 1991; Okamoto et al., 1994, 1996). Genotype-specific core region primers could distinguish between 1a, 1b, 1c, 2a, 2b, and 3a sequences in the nested amplification step (Okamoto et al., 1992c, 1994). Alternatively, the RT/PCR products resulting from amplification using universal primers have been tested for hybridization to genotype-specific probes (Li et al., 1991, 1994; Takada et al., 1993) or have been digested with one or more restriction endonucleases to permit identification of digestion fragments that are genotype specific (Nakao et al., 1991; Murphy, Willems ' Delage, 1994).

The low levels of HCV RNA in the blood requires sensitive template or signal amplification methods for detection. These procedures are highly sensitive and are prone to crossover contamination, especially when the procedures are carried out manually. To limit contamination, laboratories that are going to be doing amplification on a regular basis need to take a number of precautions (Table 18.1). One of the driving forces for automation of the amplification process is to eliminate virtually all contamination by reducing the number of steps that need to be performed manually. Even so, a major source of variability is sample collection, handling, and storage, especially prior to nucleic acid extraction and amplification. For HCV, it is very important to use a dedicated vacutainer tube that remains unopened until received by the molecular diagnostic laboratory. There does not seem to be a difference in HCV RNA levels between serum and plasma samples obtained from the same patient on the same day (Cuypers et al., 1992; Krajden et al., 1996). However, other independent observations have shown that fresh frozen plasma reproducibly yielded almost twofold higher levels of HCV RNA compared with a corresponding serum sample from the same patient (Busch et al., 1992). HCV RNA levels in clinical samples were also sensitive to storage at room temperature as well as to repeated freezing and thawing in some but not all studies (Busch et al., 1992; Wang et al., 1992; Fong et al., 1993; Quan et al., 1993; Halfon et al., 1996).

Since the discovery of hepatitis C virus (HCV) in 1989, there has been an explosion of research and information on the virus. This is evidenced by the more than 20 000 papers on HCV (as of December, 2000), as well as the numerous reviews in journals and edited books. The purpose of this book is to present an overview of the different disciplines that have contributed to an understanding of the virus, its diseases, current and proposed treatments, and much more. Importantly, this book attempts to integrate the various disciplines to provide an overall picture of what has been accomplished in the field, and what major questions still remain.

This book is organized so that the reader will first understand how and why it was so difficult to identify HCV and then how the development of antibody and HCV RNA detection tests have nearly eliminated HCV-associated post-transfusion hepatitis. The book then explores the physical characteristics of the virus, the structure of the genome, polyprotein synthesis, and the proposed replication cycle of HCV. With a broad understanding of the virus, and the ability to screen for both viral antibodies and RNA in blood, considerable work has now been done to elucidate the epidemiology and transmission of HCV. For example, population-based viral antibody surveys have revealed the seroprevalence and burden of infection in different geographic regions of the world, which has been central to the identification of risk factors for transmission and the development of preventative measures to reduce risk.

The development of a protective HCV vaccine has been among the most diffcult challenges since the discovery of the virus for a number of reasons. First, despite the consistent efforts of many laboratories, there is still no permissive system that consistently replicates HCV at high enough levels to evaluate antibodies that may be neutralizing (Table 11.1 and Ch. 11). Second, with the exception of the chimpanzee, which is expensive, endangered, and diffcult to work with, there are no convenient animal models that are susceptible to HCV and that could be used for candidate vaccine challenge studies (Section 1.2). Third, the genotype, subtype and quasispecies nature of HCV (Weiner et al., 1992; Bukh et al., 1997; Simmonds et al., 1993, 1997) (Section 3.2) may require the construction of polyvalent vaccines that would protect against a large number of closely related epitopes. The fact that the genetic heterogeneity of the virus can change rapidly in an infected individual (Ogata et al., 1991), and that antiviral immunity may select for neutralization escape mutants (within HVR1) (Martell et al., 1992; Weiner et al., 1992; Shimizu et al., 1994) as well as CTL escape mutants (Weiner et al., 1995), may limit the effectiveness and utility of any vaccine. Fourth, it is not clear whether the envelope polypeptides contain all of the antigenic determinants required for effective neutralization (Chien et al., 1993).

Prior to the discovery of HCV, a series of studies in patients with acute NANB PTH (which was mostly caused by HCV) (Hopf et al., 1990; Di Bisceglie et al., 1991; Tremolada et al., 1992; Koretz et al., 1993a,b; Mattsson, Sonnerborg & Weiland, 1993) showed that only 4–13% of 406 patients became symptomatic over an 8–14 year follow-up. Histologically defined cirrhosis was identified in 8–24% of patients, and HCC was observed in 0.7–1.3%. While these studies did not provide a longer follow-up, they did help to establish a link between NANB PTH, cirrhosis, and HCC. Independent observations in HCV-infected patients with chronic hepatitis who were followed for 4–11 years (Roberts, Searle & Cooksley, 1993; Takahashi et al., 1993; Yano et al., 1993) revealed cirrhosis in 8–46% and HCC in 11–19%. In these studies, however, the time and duration of infection were not known. Additional longitudinal studies among patients chronically infected with HCV, where the time of transmission through transfusion was known, estimated that chronic hepatitis developed within 10 years of transfusion, cirrhosis by 21 years, and HCC by 29 years (Kiyosawa et al., 1990). Some patients developed HCC only after 40–50 years of infection. These results were independently confirmed (Tong et al., 1995) and highlight both the protracted asymptomatic phase of infection, lasting for 10–20 years, and the seriousness of the sequelae. By comparison, clinical observations have shown that cirrhosis could appear several years after infection.

The “silent” epidemic of hepatitis C is no longer silent, thanks to the discovery and molecular cloning of the virus, coupled with the development and deployment of assays that specifically detect anti-HCV and HCV RNA in infected patients. These combined approaches have reduced the risk of acquiring HCV from contaminated blood to almost zero. On the clinical level, the challenge is to try to stem the magnitude of the epidemic in the foreseeable future by intensifying community outreach and education programs designed to avoid infection: making the public aware of the remaining major risk factors, including IV drug abuse and the problems of transmission associated with multiple sexual partners. For those already infected, the need for counseling has never been greater, not only for the prevention of transmission but also in making important therapeutic decisions that patients can live with. Fortunately, the use of IFN and ribavirin combination therapy has provided a great deal of hope for chronically infected patients, who otherwise have a very high risk for the development of end-stage liver disease. There is still a long way to go before all patients with chronic HCV infection can be effectively and safely treated. The combination of high throughput screening assays (using individual virus gene products produced by recombinant DNA technology), and the willingness of physicians to introduce some of the best candidates into clinical trials, will invariably lead to an improvement in the therapeutic tools available and the outcome of infection.

The establishment of tissue culture systems that consistently support high levels of HCV replication is a priority not only for elucidating the replication cycle of the virus but also for screening of putative antiviral compounds. Although many groups have reported virus production in various tissue culture systems (Table 11.1), the low levels of virus produced over time has necessitated using RT/PCR for detection (Ch. 12). Given that HCV is closely related to flaviviruses (Miller & Purcell, 1990) (Table 2.1), it has been assumed that the replication cycle of HCV, like that of flaviviruses, would contain a minus strand intermediate (Chambers et al., 1990; Monath, 1990). The finding of HCV minus strand RNA in tissue culture cells (Table 11.1) and in liver samples (Fong et al., 1991b; Gerber et al., 1992; Negro et al., 1998) from infected patients is consistent with a role for minus strand in the replication cycle, although this has yet to be formally proven. From a technical perspective, detection of minus strand RNA is problematic at best, since it involves using strand-specific primers, which may also randomly prime on the viral plus strand (or on cellular mRNAs), resulting in false-positive results. In tissue culture systems that depend upon infection of susceptible cells with virus from an infectious serum, it is not always clear whether the RT/PCR results reflect de novo production of RNA and/or detection of RNA extracted from virus particles that simply adhered to the tissue culture cells.

The chapters of this book have provided a multi- and interdisciplinary outline of HCV. However, there is still so much more work that has to be done before the virus, its biology, and associated diseases are understood well enough to generate effective vaccines and new potent therapeutics. To this end, a sampling of outstanding questions in the field are presented below.

What are some of the implications of the physical heterogeneity of hepatitis C?

Various studies have shown the density of HCV to vary from 1.03 to 1.20 g/ml (Bradley et al., 1991; Carrick et al., 1992; Miyamoto et al., 1992; Hijikata et al., 1993b; Thomssen et al., 1993; Kanto et al., 1994; Choo et al., 1995; Nakajima et al., 1996; Yoshikura et al., 1996). Although this may result from differences in the types or quantity of viral polypeptide that make up different virus particles, it seems more likely that this physical heterogeneity may be contributed by the association of virus particles with different types and amounts of host cell and serum components. The findings that LDLs and immunoglobulins are associated with HCV particles of different densities, and that virus from different densities also differ in infectivity, suggest that the host–virus relationship that develops after infection depends, in part, upon the physical composition of the virus particles. For example, complexing of virus particles with immunoglobulins may limit virus spread and infection of susceptible hepatocytes following a bout of hepatitis.

The narrow host range of HCV infection has limited animal studies to chimpanzees (Section 1.2), even though numerous attempts have been made experimentally to infect a wide variety of both laboratory and wild animals (reviewed in Bradley, 2000), including several primates other than chimpanzees (Abe et al., 1993). The success in transmitting HBV to primates other than chimpanzees prompted additional attempts with HCV. In this regard, several reports showed successful transmission of HCV to tamarins (Dienes, Feinstone & Popper, 1980; Feinstone et al., 1981; Watanabe et al., 1987), although their lower susceptibility to the virus, combined with the variable incubation periods of infection in different animals (Feinstone et al., 1981), has made this primate model less attractive than the chimpanzee for further development and application. Recently, HCV has been successfully transmitted to tree shrews, which are plentiful and much less expensive than chimpanzees (Xie et al., 1998). Although transient viremia, seroconversion to anti-HCV, and mild hepatitis were observed in many tree shrews, it was not clear whether the liver disease was associated with HCV infection. Much more work needs to be done on this interesting model to assess its future utility properly. As outlined in Sections 1.2 and 1.4, chimpanzees are readily infected with HCV, and their use was central to the discovery of this virus.

Physical characteristics of the virus

Prior to the discovery of HCV, chimpanzee studies showed that a NANB agent passed through a filter that excluded agents greater than 80 nm in size, suggesting that the responsible pathogen was probably a virus (Bradley et al., 1985; He et al., 1987). In addition, the sensitivity of the pathogen to chloroform indicated that the NANB agent was probably enveloped (Bradley et al., 1983; Feinstone et al., 1983). Molecular cloning and sequencing of the HCV genome further supported these findings (Choo et al., 1989). When virus particles were characterized by banding on sucrose density gradients, infectious sera contained HCV with densities less than 1.06 g/ml while noninfectious sera contained particles banded at approximately 1.12–1.17 g/ml (Miyamoto et al., 1992; Hijikata et al., 1993c). Although both peaks contained detectable HCV RNA, it was subsequently shown that the lighter peak was associated with low density lipoproteins (LDLs) (Miyamoto et al., 1992; Thomssen, Bonk & Thiel, 1993), while the fractions with higher density were associated with immunoglobulins (Hijikata et al., 1993c; Choo et al., 1995). This was verified in experiments showing that the lighter peak was immunoprecipitated with anti-apolipoprotein sera, while the heavier peak was immunoprecipitated with anti-immunoglobulin (Yoshikura et al., 1996). Some of the material in the heavier fractions may also contain nucleocapsids (Kaito et al., 1994; Kanto et al., 1994), which may arise directly from the lysis of hepatocytes supporting virus replication during a bout of liver disease.

Epidemiological investigations of HCV actually began with the realization that there was still a high frequency of PTH after exclusion of HAV, HBV (Prince et al., 1974; Alter et al., 1975), and other viral and toxic causes of hepatitis (Reesink & van der Poel, 1989). In the 1970s and early 1980s, the frequency of NANB PTH in different countries ranged from 2–11 per 1000 transfusions in the Netherlands and 3 per 1000 in England to 10–28 per 1000 in the United States and 23 per 1000 in Italy (Reesink & van der Poel, 1989). Among patients receiving transfusions, more than 80% of PTH was designated as NANB PTH, with 10–20% symptomatic in the acute phase. Additional observations showed about half of those with NANB PTH developed chronic hepatitis, and up to 20% of those with chronic hepatitis went on to develop cirrhosis. Cirrhosis was also shown to be an important risk factor for the appearance of HCC (Dienstag, 1983; Reesink & van der Poel, 1989). Among people with NANB PTH, more than 40% had a history of intravenous drug abuse and 5–10% had a history of possible or known occupational exposure to blood (mostly among health care providers). In addition to PTH, NANBH was also reported in up to 40% of those with acute sporadic hepatitis in the United States in which there was no history of blood transfusion (Alter et al., 1982).

INTRODUCTION

Each year diarrhoeal diseases contribute to the deaths of more than 2 million people in developing countries, most of whom are children. Enteropathogenic Escherichia coli (EPEC) is an important cause of diarrhoea in young children and makes a major contribution to infant morbidity and mortality in the developing world. A striking feature of EPEC diarrhoea is the age-dependent susceptibility of patients. Infections occur primarily in children less than 2 years of age and symptoms are usually acute but may be very severe and protracted (Nataro and Kaper, 1998). Patients routinely experience profuse watery diarrhoea, but vomiting and low grade fever are also common symptoms. EPEC rarely causes diarrhoea in adults and in volunteers only at very high doses (Donnenberg et al., 1993; Tacket et al., 2000)

EPEC is principally a pathogen of the small bowel and one of several gastrointestinal pathogens of humans and animals able to cause distinctive lesions in the gut, termed attaching and effacing (A/E) lesions. This group of A/E pathogens includes the closely related human pathogen enterohaemorrhagic E. coli (EHEC) (see Chapter 9), and the animal pathogens, rabbit enteropathogenic E. coli (REPEC) and Citrobacter rodentium (Nataro and Kaper, 1998). The remarkable histopathology of A/E lesions can be observed in intestinal biopsies from patients infected with EPEC and other host species. The lesions are characterized by localized destruction of intestinal microvilli, intimate attachment of the bacteria to the host cell surface and the formation of pedestal-like structures underneath tightly adherent bacteria (Fig. 12.1) (Frankel et al., 1998; Vallance and Finlay, 2000).

INTRODUCTION

Haemophilus influenzae is a Gram-negative bacillus that causes significant human disease. This organism is classified on the basis of the presence and serological specificity (serotypes a through f) of its polysaccharide capsule. Systemic infections such as bacteraemia, meningitis, septic arthritis and pneumonia in young children are caused primarily by H. influenzae possessing the type b capsule (Hib), while respiratory infections such as pneumonia in patients with chronic pulmonary disease, otitis media, sinusitis, and bronchitis are caused primarily by non-encapsulated strains (the socalled non-typeable H. influenzae).

The organism lives exclusively on human mucosal surfaces, most commonly in the nasopharynx and occasionally on the conjunctivae or in the female genital tract, where it is carried asymptomatically. Respiratory tract colonization rates in children vary between 30% and 100% (Gratten et al., 1989; Trottier et al., 1989; Harabuchi et al., 1994; Faden et al., 1995; Fontanals et al., 2000; St Sauver et al., 2000), with point prevalence rates of about 25% (Bou et al., 2000) to 77–84% (St Sauver et al., 2000), and are higher among children as compared with adults (Turk, 1984).

The pathogenesis of H. influenzae infections is a complex, and incompletely understood, process. Transmission to new hosts is presumed to be via infected respiratory droplets, and the bacterial factors important in successful transmission are poorly defined (Lipsitch and Moxon, 1997). Upon entry into a new host, the organism adheres to respiratory tract structures by means of a number of adhesins that prevent removal of the bacteria by respiratory tract cleansing mechanisms such as mucin trapping and ciliary action.

INTRODUCTION

Microbes such as Neisseria meningitidis (meningococcus) and Haemophilus influenzae that reside in a single niche, the human upper respiratory tract, are particularly adept at genotypic plasticity and generate phenotypic variants at high frequency. They elaborate multiple surface-expressed adhesive ligands, which undergo antigenic and phase variation with remarkable rapidity. Antigenic variation represents primary structural alteration, often arising from genetic rearrangements, and phase variation represents ‘on’ or ‘off’ mode of expression. The evolution of such variation as well as the redundancy in adhesins reflects the polymorphic nature of the host's immune response. The microbial counter-strategies accomplish not only immune evasion but also tissue tropism.

Multiple microbial adhesins may act individually or in concert to interact with target cell molecules, enabling the bacterium primarily to achieve the fundamental requirement of colonization, i.e. adherence to mucosa. Many of the target molecules are involved in normal cell–cell communications or in the reception of hormonal and other signals. As a result, targeting of these signalling molecules additionally allows bacteria to manipulate host cell functions, which may lead to intracellular location, transcytosis or paracytosis across the epithelial barrier. This chapter will outline some general mechanisms of host cell targeting that determine between adhesion and invasion. Further, it will describe several major surface structures of meningococci and their interplay in host cell recognition or evasion. Two specific receptor-targeting mechanisms are explored in detail, since they provide a paradigm to explain epidemiological evidence that implicates hostassociated factors in increased microbial invasion.

INTRODUCTION

Initial interactions between pathogenic bacteria and target cells are crucial events in cell infection. Bacteria have developed various regulatory networks to interact with their hosts. These mechanisms tend to favour bacterial survival and multiplication in infectious sites.

In extracellular bacteria such as Neisseria meningitidis and Neisseria gonorrhoeae, several structures have evolved to permit efficient adhesion to target cells. These two species are exclusive human pathogens. Neisseria meningitidis–host cell interactions alternate between asymptomatic carriage and invasive infections. The organism provokes septicaemia and then may invade the subarachnoidal space and causes meningitis. Other localizations are also observed, such as arthritis. Meningococcal infections occur as sporadic or epidemic cases. Factors that provoke meningococcal epidemics have not been fully elucidated. They are thought to be related to both host and bacteria as well as to the environment. Meningococcal strains involved in epidemics are usually different from those isolated from sporadic cases. Using genetic typing approaches, epidemic clones can be clustered into a few groups (clonal complexes). The percentage of healthy carriers in the general population is about 10%; however, very few individuals develop invasive infections. Moreover, meningococcal strains isolated from carriers are highly heterogeneous and differ genetically from invasive strains.

Bacteria first adhere to the epithelium of the nasopharynx and may then be translocated into the bloodstream. To gain entry into the cerebrospinal fluid (CSF), N. meningitidis has to adhere to endothelial cells and to cross the blood–CSF barrier.