Differential dynamics of hepatic protein expressions with long-term cultivated hepatitis C virus infection.

BACKGROUND: The liver maintains blood chemical homeostasis by active uptake and secretion through endocytosis, exocytosis, and intracellular trafficking between the plasma and intracellular membranes. Hepatitis C virus (HCV) infection affects the host membrane architecture and might thus impair the regulation of the cellular transportation machinery. Additionally, the hepatic expressions of differential protein dynamics with long-term HCV infection remain fully recover. METHODS: In this study, comparative proteomic analysis was performed in HCV-infected and mock-control Huh7 cells according to the viral dynamics of exponential, plateau, declined, and silencing phases at the acute stage, and the chronic stage. The proteins with <0.8-fold and ≥1.25-fold changes in expression were analyzed using functional pathway clustering prediction. RESULTS: The combined experimental repetitions identified full-spectrum cellular proteins in each of 5 sample sets from acute exponential, plateau, declined, and silencing phases, and the chronic stage. The clustering results revealed that HCV infection might differentiate regulatory pathways involving extracellular exosome, cadherin, melanosome, and RNA binding. Overall host proteins in HCV-infected cells exhibited kinetic pattern 1, in which cellular expression was downregulated from the acute exponential to plateau phases, reached a nadir, and was then elevated at the chronic stage. The proteins involved in the membrane-budding pathway exhibited kinetic pattern 2, in which their expressions were distinctly downregulated at the chronic stage. CONCLUSION: The current comparative proteomics revealed the differential regulatory effects of HCV infection on host intracellular transport functional pathways, which might contribute to the pathogenic mechanisms of HCV in hepatocytes that sustain long-term infection.

Viral dynamics of persistent hepatitis C virus infection in high-sensitive reporter cells resemble patient's viremia.

BACKGROUND: Hepatitis C virus (HCV) infection has a high persistence rate in patients. Although immune cells play a central role in determining the outcomes of HCV infection, the liver is crucial in controlling HCV activity from acute to chronic stages. This investigation grew HCV from a long-term cell culture, and provided an experimental model for studies on HCV persistence in hepatocytes. METHODS: Huh7.5 cells implanted with the NS3/4 protease-based secreted alkaline phosphatase (SEAP) reporter were infected with JFH-1 HCV (moiety of infection = 0.01) and incubated for over 130 days. RESULTS: The viral activity was obtained by sampling supernatant continuously for SEAP activity measurement. Combined with extracellular and intracellular HCV-RNAs and viral infectivity assays, the experimental results exhibited in vitro viral dynamics resembling the patients' viremia pattern from acute to chronic infections. The HCV in acute infection comprised exponential accumulation (week 1), plateau (week 2), declining production (weeks 3-4) and silencing (weeks 5-14) phases, and were then reactivated at the onset of chronic infection (after week 15). The HCV-infected cells grew more slowly than the mock controls, and exhibited a prominent decrease of cell growth rate and increase of early apoptosis in the declining-to-silencing phase transition, suggesting that fitness selection might occur as the infected cells moved across the boundary of active to occult viral activity. CONCLUSION: Cultivated HCV in the highly sensitive NS3/4-based SEAP reporter cells could establish persistence, which might mimic the viral dynamics from acute to chronic infections in hepatitis C patients.

Determination of IgG avidity in BALB/c mice experimentally infected with Toxocara canis / Determinação da avidez de IgG em camundongos BALB/c experimentalmente infectados com Toxocara canis

Toxocariasis is a zoonotic disease in that IgM titers can remain high for long periods making difficult to determine the stage of the disease. The aim of this study is to investigate the applicability of indirect ELISA, associated with urea, to discriminate between the acute and chronic toxocariasis. IgG avidity was evaluated in 25 BALB/c mice experimentally infected with 1000 Toxocara canis eggs. Blood samples were collected, and sera treated with 6 M urea and assayed by ELISA every two weeks. The percent IgG avidity was determined using the mean absorbance of sera treated with urea, divided by the mean absorbance of untreated sera. In the first 15 days post-inoculation, was observed a low percentage, between 7.25 and 27.5%, IgG avidity, characteristic of an acute infection. After 60 days of infection, all the mice showed between 31.4 and 58% IgG avidity, indicating a chronic infection.

A toxocaríase é uma zoonose na qual os títulos de IgM podem permanecer elevados por longos períodos, tornando difícil a determinação do estágio em que a doença se encontra. O objetivo deste estudo foi investigar a aplicabilidade de um teste indireto de ELISA, associado com ureia, para fazer a discriminação entre as fases aguda e crônica da toxocaríase. A avidez de IgG foi avaliada em 25 camundongos BALB/c experimentalmente infectados com 1000 ovos embrionados de Toxocara canis. A cada duas semanas, amostras de sangue foram coletadas, o soro tratado com ureia 6M e realizado o ensaio pela técnica de ELISA. O percentual de avidez de IgG foi determinado, usando-se a média das absorbâncias dos soros tratados com ureia dividida pela média das absorbâncias dos soros não tratados. Nos primeiros 15 dias pós-inoculação, foi observado um baixo percentual de avidez de IgG, entre 7,25 e 27,5%, característico da fase aguda da infecção. Após 60 dias de infecção, todos apresentaram avidez de IgG entre 31,4 e 58%, indicando a fase crônica da infecção.