propidium-monoazide and Hepatitis-A

Transmitted through the fecal-oral route, the hepatitis A virus (HAV) is acquired primarily through close personal contact and foodborne transmission. HAV detection in food is mainly carried out by quantitative RT-PCR (RT-qPCR). The discrimination of infectious and inactivated viruses remains a key obstacle when using RT-qPCR to quantify enteric viruses in food samples. Initially, viability dyes, propidium monoazide (PMA) and ethidium monoazide (EMA), were evaluated for the detection and quantification of infectious HAV in lettuce wash water. Results showed that PMA combined with 0.5% Triton X-100 (Triton) was the best pretreatment to assess HAV infectivity and completely eliminated the signal of thermally inactivated HAV in lettuce wash water. This procedure was further evaluated in artificially inoculated foods (at concentrations of ca. 6×10(4), 6×10(3) and 6×10(2)TCID50) including lettuce, parsley, spinach, cockles and coquina clams. The PMA-0.5% Triton pretreatment reduced the signal of thermally inactivated HAV between 0.5 and 2 logs, in lettuce and spinach concentrates. Moreover, this pretreatment reduced the signal of inactivated HAV by more than 1.5 logs, in parsley and ten-fold diluted shellfish samples inoculated at the lowest concentration. Overall, this pretreatment (50 μM PMA-0.5% Triton) significantly reduced the detection of thermally inactivated HAV, depending on the initial virus concentration and the food matrix.

Topics: Animals; Azides; Bivalvia; Cell Line; Food Microbiology; Hepatitis A; Hepatitis A virus; Hot Temperature; Indicators and Reagents; Microbial Viability; Octoxynol; Propidium; Real-Time Polymerase Chain Reaction; RNA, Viral; Vegetables; Virus Inactivation

The discrimination of infectious and inactivated viruses remains a key obstacle when using quantitative RT-PCR (RT-qPCR) to quantify enteric viruses. In this study, propidium monoazide (PMA) and RNase pretreatments were evaluated for the detection and quantification of infectious hepatitis A virus (HAV). For thermally inactivated HAV, PMA treatment was more effective than RNase treatment for differentiating infectious and inactivated viruses, with HAV titers reduced by more than 2.4 log(10) units. Results showed that combining 50 μM of PMA and RT-qPCR selectively quantify infectious HAV in media suspensions. Therefore, PMA treatment previous to RT-qPCR detection is a promising alternative to assess HAV infectivity.

Topics: Animals; Azides; Cell Line; Hepatitis A; Hepatitis A virus; Hot Temperature; Humans; Hypochlorous Acid; Macaca mulatta; Propidium; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleases; RNA, Viral; Virus Inactivation