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Jogaejeot, seasoned Venerupis philippinarum, is a traditional Korean fermented food, and hepatitis A virus (HAV) can be transmitted through contaminated food, especially bivalve shellfish, causing acute gastroenteritis worldwide. Here, we carried out a phylogenetic analysis to identify and characterize HAV strains in jogaejeot samples associated with hepatitis A (HA) outbreaks in Seoul, South Korea, in 2019. The HAV strains were identified using blast and molecular analysis of the amplified HAV VP1-P2B genome region. The HAV strains identified in the five jogaejeot samples shared at least 99% sequence identity, were all classified as genotype IA and were most closely related to strains that are widespread in East Asia. These results support a link between the consumption of jogaejeot and the HA outbreaks observed in 2019 in Seoul. In addition, they indicate a need for more stringent enforcement of food safety regulations for the shellfish industry, especially against HAV, and the value of widespread vaccination., (© 2021 The Society for Applied Microbiology.)

Aim: To improve the efficacy of intercalating dyes to distinguishing between infectious and inactivated hepatitis A virus (HAV) in food., Methods and Results: Different intercalating dyes were evaluated for the discrimination between infectious and thermally inactivated HAV suspensions combining with the RT-qPCR proposed in the ISO 15216. Among them, PMAxx was the best dye in removing the RT-qPCR signal from inactivated HAV. Applied to lettuce and spinach, PMAxx-Triton pretreatment resulted in complete removal of the RT-qPCR signal from inactivated HAV. Likewise, this study demonstrates that this pretreatment is suitable for the discrimination of inactivated HAV in shellfish without further sample dilution. In mussels and oysters, the developed viability RT-qPCR method reduced the signal of inactivated HAV between 1·7 and 2·2 logs at high inoculation level, and signal was completely removed at low inoculation level., Conclusions: This study showed that the use of PMAxx is an important improvement to assess HAV infectivity by RT-qPCR. It was shown that PMAxx-Triton pretreatment is suitable for the analysis of infectious HAV in complex food samples such as vegetables and shellfish., Significance and Impact of the Study: The PMAxx-Triton pretreatment can be easily incorporated to the ISO norm for infectious virus detection., (© 2017 The Society for Applied Microbiology.)

Aims: The aim of the work was to evaluate the circulation of the viruses and to determine a correlation between faecal indicators and viruses., Methods and Results: Raw wastewater and effluent samples were collected from three wastewater treatment plants, during three sampling periods, and analysed, using cultural and molecular methods, to determine bacteria and virus presence. The results show a removal of bacterial indicators, but a limited reduction of the phages. The viral analysis displays the circulation of cultivable enteroviruses and differences in the seasonal-geographical distribution. Hepatitis A virus was found with only two genotypes: IA-IB. Rotavirus was present in 11.11%, 24.14%, 2.78% of the samples in the 1st, 2nd and 3rd sampling periods; Astrovirus in 33.33%, 6.9%, 25%; Adenovirus in 7.41%, 3.45%, 2.78%; Norovirus in 7.41%, 10.34%, 5.56% respectively. Adenovirus was never identified in plants B and C as Rotavirus in plant C., Conclusions: The presence of faecal indicators was not predictive of the enteric virus presence, whereas a different circulation of Enteroviruses was found in the wastewater treatment plants., Significance and Impact of the Study: The study shows the importance and the usefulness of molecular methods to evaluate the virus circulation and the genetic variability of Enteroviruses.

Aims: Waterborne outbreaks of hepatitis A and Norovirus disease have been reported and associated with contaminated water supply in various countries. However, in Mexico, there are no studies that report HAV and NV presence in water. This study reports the application of ultrafiltration and RT-nested PCR methods to concentrate and identify these viruses., Methods and Results: Forty estuarine water samples were collected from the Huizache Caimanero Lagunary Complex. Samples were concentrated by ultrafiltration system (UFS) and RT-nested PCR was performed for HAV and NV identification. These viruses were found in 80% and 70% of the samples collected respectively and both were present in 57.5%. The DNA sequences analysis showed that 21 estuarine water samples were associated with HAV and 13 with NV. Faecal coliforms were isolated in 48.57% of the samples, while Escherichia coli were found in 34.28%., Conclusions: DNA sequencing showed that the genotype IB for HAV and GII for NV were predominant in México. No significant relationships were detected between indicators and viruses (P < 0.05)., Significance and Impact of the Study: This study shows that the UFS is adequate for viral concentration. This is the first study analysing the genetic sequence of HAV and NV isolated from Mexican estuarine water.

Aims: A one-year survey was conducted to examine hepatitis A virus (HAV) prevalence, distribution of genotypes and their relationship to bacterial indicators in raw and treated sewage samples., Methods and Results: Fifty sewage samples (raw = 25 and treated = 25) were collected twice monthly from one sewage treatment plant in Rio de Janeiro. Virus concentration was performed by adsorption to an electronegative membrane followed by ultrafiltration. Viral RNA was detected by nested reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR and positive products were directly sequenced. Total and faecal coliform concentrations were also determined. By nested RT-PCR, HAV RNA was detected in 16/50 (32%) and eight (16%) of them were found in treated sewage samples. By real-time PCR, HAV RNA was detected in 46/50 (92%) samples and 24 were from treated sewage. Phylogenetic analyses classified nine isolates (56%) as subgenotype IA and seven (44%) as IB., Conclusions: Real-time PCR was more sensitive than nested RT-PCR; the presence of subgenotypes IA and IB was described and bacterial indicators cannot be used to predict HAV presence in sewage., Significance and Impact of the Study: These results demonstrated that HAV still remains in the environment after sewage treatment and could play an important role in maintaining the endemicity of HAV infection.

Background: The genetic relatedness of hepatitis A virus (HAV) isolates was determined to identify possible infection sources for case patients in the Sentinel Counties Study of Acute Viral Hepatitis., Methods: A 315-nucleotide segment of the VP1-P2 region of the HAV genome was amplified and sequenced from serum of case patients and analyzed together with risk-factor data., Results: Of 508 HAV-RNA-positive case patients, 449 (88.4%) were interviewed, and 255 (50.1%) reported >/=1 risk factor. Some 123 unique nucleotide sequence patterns (UNSPs) were identified--77 (62.6%) from only 1 case patient and the rest in 2-99 persons. Among international travelers, a single person was more often infected with a single type of UNSP (17/54 [31.5%]), compared with other case patients (48/393 [12.2%]; P<.001). UNSPs from travelers to Mexico (33/37 [89.2%]) clustered with those from Hispanic children (47/49 [95.9%]). Of 119 men who had sex with men, 96 (80.7%) had the same or similar UNSPs, which were also found in 37 men and 10 women with no identified infection source., Conclusion: HAV is often transmitted within networks of persons with similar risk factors, which may be the infection source for others in the community.

Aims: A novel integrated cell culture/strand-specific reverse transcriptase-polymerase chain reaction (RT-PCR) assay was established for detection of infectious hepatitis A virus (HAV)., Methods and Results: The specificity of tagged RT-PCR was assessed using HAV genomic positive-strand RNA extracted from HAV virions as reference. Water samples artificially contaminated with infectious or formalin-inactivated HAV were subjected to integrated cell culture (ICC)/RT-PCR and ICC/strand-specific RT-PCR assays respectively. The tagged RT-PCR had high specificity for HAV negative-strand RNA. By demonstrating the formation of negative-strand RNA replicative intermediate, ICC/strand-specific RT-PCR can distinguish between infectious and non-infectious HAV. The described method detected infectious HAV at inoculation level of 10(0) TCID50 per flask within 4 days., Conclusions: The ICC/strand-specific RT-PCR is a novel, rapid, sensitive and reliable method for detection of infectious HAV., Significance and Impact of the Study: Coupled with a suitable virus concentration and purification system, ICC/strand-specific RT-PCR will provide a novel and rapid method for detection of infectious HAV in clinical, environmental and food samples. This assay may be used as an alternative method to test the effective inactivation of inactivated virus vaccines. It may also be adapted to assess the efficacy of disinfection of HAV and enteric viruses in foods and water.