Cell Culture Systems and Drug Targets for Hepatitis A Virus Infection.

Hepatitis A virus (HAV) infection is one of the major causes of acute hepatitis, and this infection occasionally causes acute liver failure. HAV infection is associated with HAV-contaminated food and water as well as sexual transmission among men who have sex with men. Although an HAV vaccine has been developed, outbreaks of hepatitis A and life-threatening severe HAV infections are still observed worldwide. Therefore, an improved HAV vaccine and anti-HAV drugs for severe hepatitis A should be developed. Here, we reviewed cell culture systems for HAV infection, and other issues. This review may help with improving the HAV vaccine and developing anti-HAV drugs.

Superinfection of hepatitis A virus in hepatocytes infected with hepatitis B virus.

Hepatitis A virus (HAV) infection is a major cause of acute hepatitis including acute liver failure. Hepatitis B infection (HBV) occurs worldwide, with the highest rates in Asian and African countries, and there are several reports that HAV infection may have a more severe clinical course in patients with chronic HBV infection. We previously demonstrated that Japanese miso extracts have inhibitory effects on HAV replication. In the present study, we examined the replication of HAV and HBV in a hepatocyte superinfection model and the inhibitory effects of Japanese miso extracts on both viruses. According to the results, HAV infection inhibited HBV replication in superinfected hepatocytes, and Japanese rice-koji miso extracts had inhibitory effects on HAV replication. Our findings provide useful information for clinicians in managing HAV infection in patients with chronic HBV infection.

Development of a fast and efficient method for hepatitis A virus concentration from green onion.

Hepatitis A virus (HAV) can cause serious liver disease and even death. HAV outbreaks are associated with the consumption of raw or minimally processed produce, making it a major public health concern. Infections have occurred despite the fact that effective HAV vaccine has been available. Development of a rapid and sensitive HAV detection method is necessary for an investigation of an HAV outbreak. Detection of HAV is complicated by the lack of a reliable culture method. In addition, due to the low infectious dose of HAV, these methods must be very sensitive. Current methods rely on efficient sample preparation and concentration steps followed by sensitive molecular detection techniques. Using green onions which was involved in most recent HAV outbreaks as a representative produce, a method of capturing virus particles was developed using carboxyl-derivatized magnetic beads in this study. Carboxyl beads, like antibody-coated beads or cationic beads, detect HAV at a level as low as 100 pfu/25g of green onions. RNA from virus concentrated in this manner can be released by heat-shock (98°C 5min) for molecular detection without sacrificing sensitivity. Bypassing the RNA extraction procedure saves time and removes multiple manipulation steps, which makes large scale HAV screening possible. In addition, the inclusion of beef extract and pectinase rather than NP40 in the elution buffer improved the HAV liberation from the food matrix over current methods by nearly 10 fold. The method proposed in this study provides a promising tool to improve food risk assessment and protect public health.

Reduction of Enteric Viruses by Blueberry Juice and Blueberry Proanthocyanidins.

Blueberry and blueberry extracts are known for their health benefits and antimicrobial properties. Natural therapeutic or preventive options to decrease the incidences of foodborne viral illnesses are becoming popular and being researched. This study aimed to determine the antiviral effects of blueberry juice (BJ) and blueberry proanthocyanidins (BB-PAC, B-type PAC structurally different from A-type PAC found in cranberries) against the infectivity of hepatitis A virus (HAV) and human norovirus surrogates (feline calicivirus (FCV-F9) and murine norovirus (MNV-1)) at 37 °C over 24 h using standard plaque assays. Viruses at ~5 log PFU/ml were mixed with equal volumes of BJ (pH 2.8), neutralized BJ (pH 7.0), BB-PAC (1, 2, 4, and 10 mg/ml), malic acid (pH 3.0), or phosphate-buffered saline (pH 7.2) and incubated over 24 h at 37 °C. Each experiment was carried out in duplicate and replicated thrice. FCV-F9 titers were found to be reduced to undetectable levels with 1 and 2 mg/ml BB-PAC after 5 min, with 0.5 mg/ml BB-PAC after 1-h, and with BJ after 3-h. MNV-1 titers were reduced to undetectable levels after 3 h with 1, 2, and 5 mg/ml BB-PAC and after 6 h with BJ. HAV titers were reduced to undetectable levels after 30 min with 2 and 5 mg/ml BB-PAC, after 3 h with 1 mg/ml BB-PAC, and by ~2 log PFU/ml with BJ after 24-h. BB-PAC shows preventive potential against infection by the tested enteric viruses in a dose- and time-dependent manner, although further in vitro studies in model food systems and in vivo studies using animal models are warranted.

Antiviral effects of grape seed extract against feline calicivirus, murine norovirus, and hepatitis A virus in model food systems and under gastric conditions.

Grape seed extract (GSE) has antiviral activities against hepatitis A virus (HAV) and human norovirus surrogates (feline calicivirus (FCV-F9) and murine norovirus (MNV-1)). The objectives of this study were to determine (1) time and dose-dependence of GSE against FCV-F9, MNV-1, and HAV at room temperature (RT) and 37 °C over 24 h; and (2) GSE effects in model foods (apple juice (AJ) and 2% milk) and simulated gastric conditions at 37 °C. Viruses at ∼5 log PFU/ml were treated with 0.5-8 mg/ml GSE prepared in water, AJ, milk or gastric juices, or water over 24 h at RT or 37 °C. Infectivity of triplicate treatments was evaluated using plaque assays. GSE effects increased with time and concentration. GSE at 1 mg/ml in AJ reduced MNV-1 to undetectable levels after 1 h and by 1 log in milk after 24 h. GSE at 1 and 2 mg/ml in AJ reduced HAV to undetectable levels after 1 h, while 2 and 4 mg/ml GSE in milk caused ∼1 log reduction after 24 h. GSE at 2 mg/ml in intestinal fluid reduced FCV-F9, MNV-1 and HAV to undetectable levels after 6 h. GSE appears to be a suitable natural option for foodborne viral reduction.

Aqueous Extracts of Hibiscus sabdariffa Calyces Decrease Hepatitis A Virus and Human Norovirus Surrogate Titers.

Hibiscus sabdariffa extract is known to have antioxidant, anti-diabetic, and antimicrobial properties. However, their effects against foodborne viruses are currently unknown. The objective of this study was to determine the antiviral effects of aqueous extracts of H. sabdariffa against human norovirus surrogates (feline calicivirus (FCV-F9) and murine norovirus (MNV-1)) and hepatitis A virus (HAV) at 37 °C over 24 h. Individual viruses (~5 log PFU/ml) were incubated with 40 or 100 mg/ml of aqueous hibiscus extract (HE; pH 3.6), protocatechuic acid (PCA; 3 or 6 mg/ml, pH 3.6), ferulic acid (FA; 0.5 or 1 mg/ml; pH 4.0), malic acid (10 mM; pH 3.0), or phosphate buffered saline (pH 7.2 as control) at 37 °C over 24 h. Each treatment was replicated thrice and plaque assayed in duplicate. FCV-F9 titers were reduced to undetectable levels after 15 min with both 40 and 100 mg/ml HE. MNV-1 was reduced by 1.77 ± 0.10 and 1.88 ± 0.12 log PFU/ml after 6 h with 40 and 100 mg/ml HE, respectively, and to undetectable levels after 24 h by both concentrations. HAV was reduced to undetectable levels by both HE concentrations after 24 h. PCA at 3 mg/ml reduced FCV-F9 titers to undetectable levels after 6 h, MNV-1 by 0.53 ± 0.01 log PFU/ml after 6 h, and caused no significant change in HAV titers. FA reduced FCV-F9 to undetectable levels after 3 h and MNV-1 and HAV after 24 h. Transmission electron microscopy showed no conclusive results. The findings suggest that H. sabdariffa extracts have potential to prevent foodborne viral transmission.

Nested real-time PCR for hepatitis A detection.

AIMS: The hepatitis A virus (HAV) is one of the most important human foodborne pathogens causing a number of worldwide outbreaks each year. The detection of HAV in food samples remains a complex issue, because commonly used detection tools, such as conventional or even real-time PCR assays, are often unable to detect HAV with sufficient sensitivity. The aims of this study were to develop highly sensitive and specific nested real-time PCR (NRT-PCR)-based method for HAV detection in food and to compare it with currently available methods. METHODS AND RESULTS: By combining conventional PCR, nested PCR and real-time PCR techniques, we have developed a specific NRT-PCR assay for the detection of HAV. The procedure involves two consecutive PCRs, the first of which is performed as a conventional RT-PCR using primers specific for HAV 5' noncoding region. The second reaction involves a real-time PCR using a nested primer pair specific for the first PCR product and a TaqMan probe. CONCLUSIONS: We have developed a novel NRT-PCR method capable of detecting as little as 0.2 PFU of HAV, which is significantly more sensitive than any other PCR technique tested in our system. SIGNIFICANCE AND IMPACT OF THE STUDY: NRT-PCR provides a potentially useful method for detecting HAV at extremely low levels, as frequently found in food samples, and can be potentially adopted as a regulatory method to ensure food safety.

Molecular epidemiology of hepatitis A virus in the South-East area of Gyeonggi-do in Korea.

PURPOSE: Hepatitis A virus (HAV) has been a leading cause of acute hepatitis in Korea. The reported genotypes of acute hepatitis A in Korea are the subgenotype IA and IB. The aim of the present study is to investigate HAV genotypes in the south-east area of Gyeonggi-do in Korea. MATERIALS AND METHODS: From June 2004 to June 2006, 46 acute hepatitis A patients were enrolled prospectively. All had sporadic acute hepatitis A patients. All suspected cases of acute hepatitis A were tested for IgM anti-HAV antibodies. We sequenced 168 bp of nucleotides of the putative VP1/P2A junction and determined the HAV genotype with reverse transcriptase polymerase chain reaction. The clinical and laboratory results of all patients were recorded. RESULTS: HAV-ribonucleic acid (RNA) was detected in 41 samples out of 46 samples. Among the 41 samples, 25 (60%) were shown to have subgenotype IIIA and the other 16 (40%) were subgenotype IA. Several amino acid substitutions were found. CONCLUSION: In these HAV sporadic cases, IIIA and IA were identified, and this may reflect co-circulation of various genotypes in Korea. This study provides valuable new data on the genetic distribution of HAV and important information to help design appropriate public health measures.

Green onions: potential mechanism for hepatitis A contamination.

The largest documented foodborne hepatitis A outbreak in U.S. history occurred in November 2003. The source of that outbreak was green onions from a farm in Mexico. Two biomarkers were used to determine ways in which hepatitis A virus (HAV) can contaminate onions. Fluorescent microspheres (1.0 to 10 microm) and HAV vaccine were placed on the soil and the surfaces of pot-grown onions and in the liquid medium of hydroponically cultivated onions. Reverse transcription PCR (RT-PCR) was used to identify HAV RNA. Microspheres were found on the outside and inside of the pot-grown onions for up to 60 days. RT-PCR revealed HAV RNA from the vaccine in well-washed green onions. In the hydroponically grown onions, microspheres were found throughout the onion after only 1 day. RT-PCR also revealed HAV RNA inside the hydroponically grown onions. Both biomarkers support the hypothesis that HAV can contaminate the inside of the growing onion and can be taken up intracellularly through the roots. Once inside, the particles are impossible to remove by cleaning.