Xin-Wei Wang

Mechanisms of Inactivation of Hepatitis A Virus by Chlorine

ABSTRACT The study was intended to investigate the feasibility of reverse transcription-PCR (RT-PCR) for evaluation of the efficacy of inactivation of viruses in water and to elucidate the mechanisms of inactivation of hepatitis A virus (HAV) by chlorine. Cell culture, enzyme-linked immunosorbent assay, and long-overlap RT-PCR were used to detect the infectivity, antigenicity, and entire genome of HAV inactivated or destroyed by chlorine. The cell culture results revealed the complete inactivation of infectivity after 30 min of exposure to 10 or 20 mg of chlorine per liter and the highest level of sensitivity in the 5′ nontranslated regions (5′NTR), inactivation of which took as much time as the inactivation of infectivity of HAV by chlorine. However, antigenicity was not completely destroyed under these conditions. Some fractions in the coding region were resistant to chlorine. To determine the specific region of the 5′NTR lost, three segments of primers were redesigned to monitor the region from bp 1 to 1023 across the entire genome. It was shown that the sequence from bp 1 to 671 was the region most sensitive to chlorine. The results suggested that the inactivation of HAV by chlorine was due to the loss of the 5′NTR. It is believed that PCR can be used to assess the efficacy of disinfection of HAV by chlorine as well as to research the mechanisms of inactivation of viruses by disinfectants.

A new and simple method for concentration of enteric viruses from water.

A new type of electropositive filter media particle was tested to adsorb bacteriophage f2 and enteric viruses from tap water. 3 x nutrient broth (pH 7.2) was used to elute the adsorbed viruses, and the eluate was reconcentrated by polyethylene glycol (Mw 6000) precipitation with a final concentration of 10% (wt./vol.). The adsorption of bacteriophage was reliable and efficient, and not affected by the pH value, temperature, turbidity and organic materials in water. This method gave a recovery of Polio 1 virus 96.0% for small-volume tap water; 88.7% for large-volume water; and gave a comparable recovery of HAV, Coxsackie B3 and Echo 7 from tap water. The concentration method need not acidify virus-containing water, add exogenous multivalent cation salts, or require expensive equipment.

Effects of chlorine and chlorine dioxide on human rotavirus infectivity and genome stability

Despite the health risks posed by waterborne human rotavirus (HRV), little information is available concerning the effectiveness of chlorine or chlorine dioxide (ClO2), two common disinfectants of public water sources, against HRV and their effects on its genome remain poorly understood. This study investigated the effects of chlorine and ClO2 on purified HRV by using cell culture and RT-PCR to assess virus infectivity and genetic integrity, respectively. The disinfection efficacy of ClO2 was found to be higher than that of chlorine. According to the efficiency factor Hom model, Ct value (mg/L min) ranges required for a 4-log reduction of HRV at 20 °C by chlorine and ClO2 were 5.55-5.59 and 1.21-2.47 mg/L min, respectively. Detection of the 11 HRV genome segments revealed that damage to the 1227-2354 bp of the VP4 gene was associated with the disappearance of viral infectivity by chlorine. However, no complete accordance between culturing and RT-PCR assays was observed after treatment of HRV with ClO2. These results collectively indicate that the current practice of chlorine disinfection may be inadequate to manage the risk of waterborne HRV infection, and offer the potential to monitor the infectivity of HRV adapting PCR-based protocols in chlorine disinfection.

Study on the resistance of severe acute respiratory syndrome-associated coronavirus

Abstract In this study, the persistence of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) was observed in feces, urine and water. In addition, the inactivation of SARS-CoV in wastewater with sodium hypochlorite and chlorine dioxide was also studied. In vitro experiments demonstrated that the virus could only persist for 2 days in hospital wastewater, domestic sewage and dechlorinated tap water, while 3 days in feces, 14 days in PBS and 17 days in urine at 20°C. However, at 4°C, the SARS-CoV could persist for 14 days in wastewater and at least 17 days in feces or urine. SARS-CoV is more susceptible to disinfectants than Escherichia coli and f2 phage. Free chlorine was found to inactivate SARS-CoV better than chlorine dioxide. Free residue chlorine over 0.5mg/L for chlorine or 2.19mg/L for chlorine dioxide in wastewater ensures complete inactivation of SARS-CoV while it does not inactivate completely E. coli and f2 phage.

Concentration and detection of SARS coronavirus in sewage from Xiao Tang Shan Hospital and the 309th Hospital.

Abstract The transmission of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is associated with close contact to SARS patients and droplet secretions of those patients. The finding of positive RT-PCR results from stools of SARS patients suggests that stools of SARS patients or sewage containing stools of patients could transmit SARS-CoV. We used a novel style of electropositive filter media particle to concentrate the SARS-CoV from the sewage of two hospitals receiving SARS patients in Beijing. We also used cell culture, RT-PCR and gene sequencing to detect and identify the viruses from sewage. No infectious SARS-CoV contamination was found in any of the samples collected, but the nucleic acid of SARS-CoV could be detected in the sewage from the two hospitals before disinfection. While the RNA was only detected in three samples from the 309th Hospital, the others were negative after disinfection. These findings provide strong evidence that SARS-CoV can be excreted through the stool/urine of patients into sewage system, thus making the sewage system a possible route of transmission.

Seroprevalence and evolutionary dynamics of genotype 4 hepatitis E virus in Shandong Province,China

AIM To investigate the seroprevalence and evolutionary dynamics of hepatitis E virus (HEV) and assess the ancestor of HEVs in Chinas Shandong Province. METHODS A total of 2028 serum, 60 fecal and 82 bile samples were collected from the general human population, patients and swine, respectively. This seroepidemiological study was conducted using an immunnosorbent assay and HEV RNA was detected by the reverse transcription-nested polymerase chain reaction (RT-nPCR) method. Complete genome sequences of the prevalent strains (CH-YT-HEV01, CH-YT-HEV02 and CH-YT-sHEV01) were determined, and the sequences were analyzed phylogenetically. In addition, the evolutionary dynamics of three HEV isolates were determined using the framework of coalescent analysis in the program package BEAST, and the time of the most recent common ancestors (TMRCAs) of China-indigenous genotype 4 HEV isolates was calculated. RESULTS The overall viral burden in the general human population was 0.1%, and the positive rates of anti-HEV IgG and IgM in the serum specimens were 25.1% (509/2028) and 2.3% (51/2028), respectively. In addition, IgG positivity increased with age. The phylogenetic analysis based on the full-length nucleotide sequences showed that the strain CH-YT-HEV02 was directly related to CH-YT-sHEV01 with a 94% identity, suggesting that they were involved in cross-species transmission. The isolate CH-YT-HEV01 was close to HB-3 and CHN-SD-sHEV with a bootstrap value of 100%, sharing a 96.1%-96.4% identity with each other. Surprisingly, the HB-3 strain was a representative strain prevalent in swine in Hubei, and the isolate CHN-SD-sHEV was obtained from swine in Shandong in a previous report. TMRCA for the clade of CH-YT-HEV01 and HB-3 was 2003, which was consistent with the TMRCA for the clade of CHN-SD-sHEV and HB-3, and they were both earlier than the TMRCA for the clade of CH-YT-HEV01 and CHN-SD-sHEV (2004). CONCLUSION The strains CH-YT-HEV01, CHN-SD-sHEV and HB-3 are involved in trans-regional transmission, and the ancestors of HEVs in Shandong come from Hubei Province.

Antiviral effects of aqueous extract from Spatholobus suberectus Dunn. against coxsackievirus B3 in mice

ObjectiveTo investigate the antiviral effects of the aqueous extract of Spatholobus suberectus Dunn. (A.E.), a Chinese medicinal herb, against coxsackievirus B3 (CVB3).MethodsThe antiviral effects of A.E. against CVB3 in vitro (primarily cultured myocardial cells) and in vivo (BALB/c mice) were determined. Serum pharmacological method was also adopted by in vitro experiments. The effects of A.E. inhibiting the CVB3 mRNA expression were compared by RT-PCR in mice in vivo.ResultsA.E. exhibited obvious antiviral: effects in vivo, and serum samples obtained from the rats with oral administration of A.E. (10 μg/mL, 5 μg/mL), reduced the virus titers in the infected myocardial cells (3.00±0.70, 3.55±0.52, P<0.01). Meanwhile, the viral myocarditis induced by CVB3 was inhibited significantly by A.E., and the 15-day mortality was reduced to 40% and 45% (P<0.01) in mice treated with A.E. at doses of 50 mg/kg and 100 mg/kg, respectively, while the 30-day mortality was decreased to 45% and 50%, respectively (P<0.01). Moreover, the mRNA expression of Coxsackie virus B3 was significantly inhibited by A.E.ConclusionAqueous extract of Spatholobus suberectus Dunn. (A.E.) has inhibitory effect on CVB3 both in vitro and in vivo.