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Early infection events highlight the limited transmissibility of hepatitis C virus in vitro

 

Meredith LW, Harris HJ, Wilson GK, Fletcher NF, Balfe P and McKeating JA.
Hepatology, 58:1074-80

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Synopsis:

Hepatitis C virus (HCV) poses a global health problem, with over 170 million chronically infected individuals at risk of developing progressive liver disease. The ability of a virus to spread within a host is a key determinant of its persistence and virulence. HCV can transmit in vitro by cell-free particle diffusion or via contact(s) between infected and naïve hepatocytes. However, limited information is available on the relative efficiency of these routes, our aim is to develop physiologically relevant assays to quantify these processes. To do this, we developed a single-cycle infectious co-culture assay which allowed us to measure the earliest events of viral transmission. A comparison of cell-free and cell-to-cell virus spread demonstrated relatively poor transmission rates, with 10-50 infected producer cells required to infect a single naïve target cell. We found HCV strain J6/JFH to be 10-fold more efficient at spreading via the cell-to-cell route than cell-free, whereas SA13/JFH and HK6/JFH strains showed comparable rates of infection via both routes. The key rate-limiting step in the initiation of transmission appears to be SR-BI localization. Importantly, the level of infectious virus released from cells did not predict the ability of a virus to spread in vitro highlighting the importance of studying cell-associated viruses. These studies demonstrate the relatively poor infectivity of HCV and highlight differences between strains in their efficiency and preferred route of transmission that may inform future therapeutic strategies that target virus entry.


Abstract:

Background and aims: Hepatitis C virus (HCV) poses a global health problem, with over 170 million chronically infected individuals at risk of developing progressive liver disease. The ability of a virus to spread within a host is a key determinant of its persistence and virulence. HCV can transmit in vitro by cell-free particle diffusion or via contact(s) between infected and naïve hepatocytes. However, limited information is available on the relative efficiency of these routes, our aim is to develop physiologically relevant assays to quantify these processes.
Methods: We developed a single cycle infection assay to measure HCV transmission rates.
Results: We compared HCV spread in proliferating and arrested cell systems and demonstrated a significant reduction in cell-to-cell infection of arrested target cells. Comparison of cell-free and cell-to-cell virus spread demonstrated relatively poor transmission rates, with 10-50 infected producer cells required to infect a single naïve target cell. We found HCV strain J6/JFH to be 10-fold more efficient at spreading via the cell-to-cell route than cell-free, whereas SA13/JFH and HK6/JFH strains showed comparable rates of infection via both routes. Importantly, the level of infectious virus released from cells did not predict the ability of a virus to spread in vitro highlighting the importance of studying cell-associated viruses.
Conclusions: These studies demonstrate the relatively poor infectivity of HCV and highlight differences between strains in their efficiency and preferred route of transmission that may inform future therapeutic strategies that target virus entry.


figure 1

Figure 1. Co-culture virus transmission assay.

HCV infected Huh-7.5 “producer” cells, labelled with CMFDA, are seeded at a 1:1 ratio with naive, uninfected Huh7.5 “target” cells. Cells are incubated for 2h to allow cell-junction formation and anti-HCV or irrelevant Ig (100µg/mL) added to neutralize extracellular virus. Cultures are incubated for a further 24h, the supernatant collected and infectious virus titer enumerated by inoculating naive Huh-7.5 cells. Cells were trypsinized, fixed and stained for the presence of viral encoded NS5A and analysed by flow cytometry to define the total number of infected target cells. Cell-cell or nAb resistant virus spread is defined as the number of newly infected target cells when 100% of extracellular virus is neutralized..


figure 2

Figure 2. Single-cycle transmission assay reveals differences in cell-to-cell and cell-free HCV transmission rates.

(A) CMFDA labelled Huh-7.5 cells infected with the indicated HCV strains were co-cultured with naive targets in the presence of anti-HCV Ig or irrelevant Ig, transmission was stopped at defined intervals by adding anti-CD81 and cell-cell and cell-free infection events determined. Data is presented as the number of newly infected targets per 105 infected producers.
(B) Rates of total, cell-cell or cell-free transmission and extracellular virus production were calculated over the first 4h of co- culturing infected and naïve cells. (T-test, ns = non-significant, *** = p<0.0001).
$ represents the mean number of infected targets/105 producers/h and
† represents the infectivity of extracellular virus per 105 producers/h



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