Anatomy of an HCV infection

Hepatitis C researcher Rowena Bull and her colleagues have provided an intimate account of the surprising behaviour of the hepatitis C virus during the first 100 days of infection. Their findings point towards new strategies to prevent chronic HCV infection, cirrhosis and liver cancer.

At least 250,000 Australians are chronically infected by hepatitis C virus (HCV). They are hosts to a molecular shape-shifter - a single-stranded RNA virus with an inherent capacity to mutate rapidly and present a constantly shifting target to the human immune system.

Dr Rowena Bull, of the University of NSW’s (UNSW’s) School of Medical Sciences, has taken on some of the unanswered questions about a virus that still heads the rollcall of notifiable new infections in Australia.

How does a virus that mutates rapidly and randomly maintain its ability to infect new hosts? And, post-infection, how does it evade the immune system’s multipronged defensive responses to establish a chronic infection in the host’s liver cells?

The other side of the infection equation is how individuals differ in their immune response to the virus - some rapidly clear an initial HCV infection, while others become chronically infected.

Bull says answers to these questions will inform efforts to develop a vaccine to protect against hepatitis C infection.

Some individuals who become chronically infected with HCV remain asymptomatic and lead normal lives, untroubled by the virus. But nearly half will develop cirrhosis and around 1 in 100 will develop liver cancer.

HCV in the prison population

Bull and UNSW colleague Dr Fabio Luciani led a study, published in 2011, that traced the early phases of new infections in four NSW prisoners. HCV infections are common in the prison population because prisoners share needles to inject drugs.

Dr Andrew Lloyd, at UNSW, established a monitoring program in a cohort of 600 NSW prisoners. This helped Bull and colleagues identify four subjects with newly acquired HCV infections.

“All of the recruits in the cohort admit to using drugs and to actively injecting while they are in prison,” she said. “Dr Lloyd screens the high-risk subjects every three months and medium-risk subjects every six months - basically, we wait for individuals to become infected, then study them intensively.”

Bull said they tracked the virus’s evolution from blood samples taken from four newly infected individuals over the course of several months - two cleared the infection and two developed chronic HCV infections.

Focusing on hypervariable regions

Bull says HCV’s single-stranded RNA genome, of around 9.5 kilobases, evolves rapidly in the absence of a template strand to maintain its fidelity as it replicates in its host’s hepatocytes.

There are seven basic HCV genotypes in circulation globally, which differ by ~30% at the nucleotide level. This effectively renders them distinct species.

HCV packages its RNA genome into a capsid of two envelope proteins - Env1 and Env2.

“The protein envelope is quite rigid,” said Bull, “and both Env proteins undergo post-translational modification by glycosylation. These glycosylation sites remain fairly consistent between variants of both proteins, despite variation within and near the receptor-binding domains.”

In the absence of vaccine-induced herd immunity, HCV is virtually free to explore the extensive landscape of structures available for escaping its host’s immune response. The search is on for highly conserved domains that could be targeted by a vaccine.

Hypervariable domains, which may serve to decoy antibodies away from conserved receptor-binding sites, provide a means to track the virus’s evolution.

The work Bull and colleagues conducted in prisoners revealed how HCV undergoes two genetic bottlenecks in the first 100 days post-infection, both driven by the host’s immune response.

They synthesised near full-length HCV cDNAs from frequently taken samples of viral RNA. After amplifying the whole genome in two or three fragments, the resulting DNA was sequenced with Murdoch University’s 454 next-generation pyrosequencer in Perth.

“The advantages of next-generation 454 sequencing are that it’s fast, requires less manpower and can tell you about all the variants within a sample from a single individual,” Bull said. “Sanger sequencing is much more labour-intensive, although it has a much lower error rate.

“We actually sequence the whole genome of each strain, but we then focus on the hypervariable region because it produces a strong signature when the genomes of the different strains are compared. We look at the sequences that are changing the most, because they’re most likely to be involved in the transition from the acute to chronic phase of infection.”

By taking frequent samples, including a sample from each subject prior to the appearance of serum antibodies to HCV, Bull has recorded genomic changes in the virus throughout its pas-de-deux with the immune system.

Immune-escape mutations

Little is known about the phenotypic characteristics of founder viruses in HCV infections.

However, a recent analysis of HCV variants that emerged in a transplant patient who contracted HCV from a donor liver suggested successful strains have an increased ability to enter hepatocytes. These variants also exhibited lower neutralising affinities in the face of the host’s antibody response after seroconversion.

One of the prisoners Bull and colleagues studied, subject 23_Ch, was infected with at least two founder viruses that differed by only three nucleotides in their Env protein-coding regions. The variable loci, at residues 443 and 446, lie close to the CD81 receptor binding domain - the CD81 receptor interacts with the HCV Env2 protein.

As infection progressed, the HCV variant 23BF outcompeted rival variant 23AF, suggesting 23BF possessed superior infectivity during the acute phase of infection.

“The ability of any variant to persist in its host is probably due in part to chance mutations that allow the virus to stay a step ahead of the immune response and generate the correct immune-escape mutations,” said Bull.

But once a variant establishes a chronic infection, it persists because some as-yet unidentified factor down-regulates the host’s immune response - in particular, cytotoxic T cells (CD8(+) T-lymphocytes) no longer kill virus-infected hepatocytes.

“We need to look at how CD4 and CD8 lymphocytes work together with neutralising antibodies during HCV infection, and which of the factors contribute to persistence of the virus in some subjects and clearance in others,” Bull said.

“We particularly want to see how mutations emerge in chronic infections and whether the selection of persistent variants of the virus is driven by CD8 T-lymphocytes or by neutralising antibodies.”

Genetic bottlenecks

Bull says the current dogma is that neutralising antibodies aren’t important in the early phase of infection and, ultimately, to whether the virus is cleared or establishes a chronic infection.

Testing this proposition is problematic in the absence of an animal model for human HCV infection - HCV also infects chimpanzees, but most rapidly clear the infection.

Bull says the value of their project was the access they had to a prospective cohort of subjects who were at high risk of HCV infection. This enabled the identification of subjects within the first few days or weeks of their new HCV infection. This had been challenging in past research because the majority of HCV infections are asymptomatic and it can be years or decades before an individual presents clinically with hepatitis.

Detailed analysis of the pattern and kinetics of viral evolution revealed that early primary HCV infections involve at least two significant genetic bottleneck events, when variation in founder virus strains is tightly constrained by the host’s immune response.

The first occurs during or immediately after infection - just one or two founder viruses from the potentially large range of variants circulating in the general population are involved in establishing the initial infection. The rest presumably to not get to replicate.

A successful founding strain - or strains - then replicates within its new host, spawning new variants as replication errors accumulate across the genome.

Around 100 days post-infection, the new variants are subjected to a selective sweep as they come under fire from the immune system, and another bottleneck occurs. In some cases there are no survivors and the infected subject clears the infection. In others, one or more strains survive the elimination process and go on to establish a chronic infection.

A rapid evolution

Subject 23_Ch had two founder strains when tested, whereas the other three subjects were colonised by a single strain - and two cleared the infection.

The presence of a single founder virus in three of four intravenous-drug user-transmitted HCV subjects is comparable to studies in HIV which have shown that the majority of subjects with mucosal transmission had a single founder virus.

However, in contrast to HCV, HIV infection resulting from intravenous drug use typically involves multiple virus strains - although five appears to be the limit.

Moreover, studies of HIV Env sequences in 20 heterosexual transmission pairs revealed that the founder variant(s) in the recipient comprised a small fraction of the viral variants in the donor.

From a limited sample number, HCV appears to have the same potential for more than one variant to establish an initial infection.

However, it remains unclear whether the transmission bottleneck is attributable to a lower number of variants being transferred between hosts or is the result of early evolutionary events where a larger number of strains are rapidly eliminated due to varying fitness constraints.

Given that the two founder viruses identified in subject 23_Ch were very similar, Bull and colleagues think it plausible that a single founder virus rapidly evolved in the first few days post-infection.

The novel finding that a genetic bottleneck occurred during the acute phase of infection in subjects studied, regardless of the long-term consequences of infection, is driving Bull and colleagues to better understand the selective pressures imposed by the host’s immune response early in HCV infections.

The aim is to identify factors that drive the evolution of HCV towards chronicity in the hope that this may lead to new vaccines or antiviral therapies that could eliminate ‘escape’ variants before they can establish a chronic infection. The group is about to publish a paper on how the immune system reacts to HCV infection.

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Hepatitis C facts

In the mid-1970s, US health authorities became alarmed as increasing numbers of US patients who had undergone blood transfusions developed chronic hepatitis infections. After excluding the hepatitis A and hepatitis B viruses as culprits, researchers in the Department of Transfusion Medicine at the National Institutes of Health gave the new disease a temporary name: hepatitis non-A, non-B.

By the time the mystery hepatitis virus was identified and described in 1989, a pandemic of hepatitis C virus (HCV) was underway in Western nations, predominantly among recreational drug users who had shared needles.

In 2010, around 250,000 Australians were living with chronic HCV infections and around 9700 new cases were estimated to be occurring each year.

Because infection tends to be asymptomatic in its early years, many more infections remain undiagnosed.

Advances in detection, diagnosis and treatment have since reduced the rate of transfusion-acquired infection to near zero, but HCV infections remain the most common notifiable diseases in Australia. There is still no vaccine.

According to a hepatitis C fact sheet published by Hepatitis NSW, for every 100 patients who have had the disease for 20 years but remain untreated, 45 will escape serious liver damage, 47 will develop progressive mild-to-moderate liver damage, seven will develop cirrhosis and one will develop liver cancer or suffer liver failure requiring a transplant.

Current antiviral therapies for chronic infection include pegylated interferon and ribovarin, but the success of these therapies in clearing the infection appears to depend on the patient’s genotype. New treatment strategies, such as Gilead’s sofosbivir, that offer higher clearance rates and are predicted to have little opportunity for drug resistance to develop are on the horizon.

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