An outbreak of hepatitis E virus genotype 4d caused by consuming undercooked pig liver in a nursing home in Zhejiang Province, China.

Hepatitis E infection is typically caused by contaminated water or food. In July and August 2022, an outbreak of hepatitis E was reported in a nursing home in Zhejiang Province, China. Local authorities and workers took immediate actions to confirm the outbreak, investigated the sources of infection and routes of transmission, took measures to terminate the outbreak, and summarized the lessons learned. An epidemiological investigation was conducted on all individuals in the nursing home, including demographic information, clinical symptoms, history of dietary, water intake and contact. Stool and blood samples were collected from these populations for laboratory examinations. The hygiene environment of the nursing home was also investigated. A case-control study was conducted to identify the risk factors for this outbreak. Of the 722 subjects in the nursing home, 77 were diagnosed with hepatitis E, for an attack rate of 10.66 %. Among them, 18 (23.38 %, 18/77) individuals had symptoms such as jaundice, fever, and loss of appetite and were defined as the population with hepatitis E. The average age of people infected with hepatitis E virus (HEV) was 59.96 years and the attack rate of hepatitis E among women (12.02 %, 59/491) was greater than that among men (7.79 %, 18/231). The rate was the highest among caregivers (22.22 %, 32/144) and lowest among logistics personnel (6.25 %, 2/32); however, these differences were not statistically significant (P > 0.05). Laboratory sequencing results indicated that the genotype of this hepatitis E outbreak was 4d. A case-control study showed that consuming pig liver (odds ratio (OR) = 7.50; 95 % confidence interval [CI]: 3.84-16.14, P < 0.001) and consuming raw fruits and vegetables (OR = 5.92; 95 % CI: 1.74-37.13, P = 0.017) were risk factors for this outbreak of Hepatitis E. Moreover, a monitoring video showed that the canteen personnel did not separate raw and cooked foods, and pig livers were cooked for only 2 min and 10 s. Approximately 1 month after the outbreak, an emergency vaccination for HEV was administered. No new cases were reported after two long incubation periods (approximately 4 months). The outbreak of HEV genotype 4d was likely caused by consuming undercooked pig liver, resulting in an attack rate of 10.66 %. This was related to the rapid stir-frying cooking method and the hygiene habit of not separating raw and cooked foods.

An analysis of select swine feed ingredients and pork products imported into the United States from African swine fever virus affected countries.

The potential for feed ingredients to serve as a vehicle for African swine fever virus (ASFV) introduction to the United States remains a significant concern. It is therefore imperative that channels through which high-risk livestock feeds and feed ingredients are imported into the United States from ASFV-positive countries are identified and considered into the USDA's ASF National Response Framework. The purpose of this study is to demonstrate the use of a novel analytical tool to categorically quantify pork products and potential high-risk feed ingredients that have entered the United States from ASFV-positive countries over a 5-year period (2016-2020). Data for this study were obtained at the United States International Trade Commission Harmonized Tariff Schedule (HTS) website (www.hs.usitc.gov), a publicly available website that provides transaction information on specific trade commodities between the United States and its international trading partners. A total of 29 high-risk pork products or feed ingredients with the potential to be fed to pigs were analysed. High risk products and ingredients were defined as those that previous research has shown to facilitate extended viral survivability, and includes products such as soybean oilcake, distillers grains, pet food, and pork sausage casings. Data were exported into Microsoft Excel and organized into pivot tables to describe the quantity of each product by country of origin and Port of Entry (POE). The analysis focused on the 60 ASFV-positive countries as currently reported by the World Organization for Animal Health (OIE). In 2020, a total of 486,902 metric tons (MT) of these high-risk products were imported into the United States from a total of 19 of the 60 foreign countries currently listed as ASFV positive by the OIE. A majority of imported animal feed ingredients came from India in 2020 (85.8%; 392,243 MT), whereas the majority of pork products and by-products were imported from Poland (21,191 MT, 70.6%). Soybean oilcake from India entered the United States through a total of 15 ports of entry (POEs) in 2020. Of these POEs, a total of five POEs received greater than 91% of all of soy oilcake originating from India, including Baltimore, MD (37.7%); San Francisco, CA (30.1%); Seattle, WA (12.7%); New Orleans, LA (5.9%); and Detroit, MI (5.1%). When examining trends over a 5-year period, a few noteworthy findings include the dramatic rise in soybean oilcake imported from India when compared to China, and a dramatic decrease in the total volume of pork sausage casings imported from China into the United States. To support the risk management of feed imports, our novel approach focuses on seaport of highest risk and quantity of product received. Data provided in this report represent an initial listing of suspect pork products and feed ingredients entering the United States, much of which is destined for animal feed. Specific detailed examples are provided in order to demonstrate the tool's flexible interface, which can be quickly modified to zero-in on specific feed ingredients, countries, or POEs depending on what the user's question may be. To the authors' knowledge, the use of and application of such a tool has not been previously utilized to support ongoing risk mitigation efforts. Potential outlets for future use of the analytical tool will include a more user-friendly and interactive interface providing an inclusive analysis of global livestock feed ingredient sourcing.

Hepatitis E Outbreak in the Central Part of Italy Sustained by Multiple HEV Genotype 3 Strains, June-December 2019.

In European countries, autochthonous acute hepatitis E cases are caused by Hepatitis E Virus (HEV) genotype 3 and are usually observed as sporadic cases. In mid/late September 2019, a hepatitis E outbreak caused by HEV genotype 3 was recognized by detection of identical/highly similar HEV sequences in some hepatitis E cases from two Italian regions, Abruzzo and Lazio, with most cases from this latter region showing a link with Abruzzo. Overall, 47 cases of HEV infection were finally observed with onsets from 8 June 2019 to 6 December 2019; they represent a marked increase as compared with just a few cases in the same period of time in the past years and in the same areas. HEV sequencing was successful in 35 cases. The phylogenetic analysis of the viral sequences showed 30 of them grouped in three distinct molecular clusters, termed A, B, and C: strains in cluster A and B were of subtype 3e and strains in cluster C were of subtype 3f. No strains detected in Abruzzo in the past years clustered with the strains involved in the present outbreak. The outbreak curve showed partially overlapped temporal distribution of the three clusters. Analysis of collected epidemiological data identified pork products as the most likely source of the outbreak. Overall, the findings suggest that the outbreak might have been caused by newly and almost simultaneously introduced strains not previously circulating in this area, which are possibly harbored by pork products or live animals imported from outside Abruzzo. This possibility deserves further studies in this area in order to monitor the circulation of HEV in human cases as well as in pigs and wild boars.

Presence of hepatitis E virus in commercially available pork products.

An increasing number of hepatitis E virus (HEV) infections in industrialized countries have been foodborne and linked to the consumption of undercooked pork products. To date, data on the prevalence of HEV in pork products sold in the United States is limited and no standard processing method exists for the detection of HEV in foods. In order to develop a processing method for the detection of HEV in pork products, ground pork and pork liver were selected for method development. Murine norovirus (MNV) was used as a process control. A filtration step prior to RNA detection was shown to reduce the level of PCR inhibitors in ground pork and an additional ultracentrifugation process was successful in removing PCR inhibitors in pork liver. MNV RNA was detected in ground pork and liver samples inoculated with 4.7 log10 PFU/g and 3.0 log10 PFU/g, respectively. Using the developed method for viral RNA detection in ground pork and pork liver, 20 packages of ground pork (six 1 g sub-samples per package) and 14 pork livers (four 1 g sub-samples per liver) were screened for the presence of HEV RNA. Fifteen out of 119 (12.6%) ground pork samples tested positive for HEV RNA and 13 out of 20 packages (65%) contained at least one positive sample. Twenty-five of 56 (45%) of pork liver samples were positive for HEV RNA and 6 of 14 livers (43%) had all sub-samples test positive for HEV RNA. Overall, the results indicate ground pork and pig liver as a potential source of HEV.

Detection of Hepatitis E Virus in the Pig Livers and Retail Pork Samples Collected in Selected Cities in China.

Hepatitis E virus (HEV) is a biological hazard that must be controlled and is a recognized etiological agent in viral hepatitis. This is a zoonotic virus and can be transmitted through the fecal-oral route. The pig is an important reservoir host of HEV, and is a source of contamination for the consumer after the consumption of raw or undercooked pork products. When detected, the most prevalent genotype of HEV in China is genotype 4 (denoted as HEV-4). To ensure the safety of this food of animal origin, we undertook a survey of HEV contamination in pig livers and pork samples available for sale, in retail outlets in selected cities in China. Viral RNA was purified from samples collected by lysing in Trizol followed by purification using trichloromethane and virus RNA extract kit. An additional step was applied to improve the recovery rate by adding RNase OUT when extracting virus RNA from pig livers, and the RNA productions were washed in 75% (v/v) ethanol to remove inhibitors. In total, 158 pig livers and 80 pork samples were procured and analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). After purification of total RNA from all samples taken and analyzed by RT-qPCR, a single pig liver was positive by this method for HEV. The positive rate was calculated as 0.63%. In this study, a single positive sample was detected. Considering the dietary habits of Chinese people, pork is a popular food that on occasion may be contaminated with HEV, thereby posing a threat to consumer health. Ongoing surveillance is required to assess the risk to human health arising from HEV-contaminated pork being offered for sale, at retail outlets, especially in the areas of China where pig production is practiced.

A plate of viruses: Viral metagenomics of supermarket chicken, pork and beef from Brazil.

A viral metagenomics study was conducted in beef, pork, and chicken sold in supermarkets from Southern Brazil. From chicken, six distinct gyroviruses (GyV) were detected, including GyV3 and GyV6, which for the first time were detected in samples from avian species, plus a novel smacovirus species and two highly divergent circular Rep-encoding ssDNA (CRESS-DNA) viruses. From pork, genomes of numerous anelloviruses, porcine parvovirus 5 (PPV5) and 6 (PPV6), two new genomoviruses and two new CRESS-DNA viruses were found. Finally, two new CRESS-DNA genomes were recovered from beef. Although none of these viruses have history of transmission to humans, the findings reported here reveal that such agents are inevitably consumed in diets that include these types of meat.

High Predictive Power of Meat Juice Serology on the Presence of Hepatitis E Virus in Slaughter Pigs.

Hepatitis E virus (HEV) as a zoonotic agent can be responsible for an acute hepatitis in humans, which is usually self-limiting. Progression toward a chronic stage is possible, especially in immunocompromised patients. In the past decade, the number of hepatitis E cases in humans in Germany has increased enormously to 3491 cases in 2018. Domestic pigs have been identified as a main animal reservoir and the consumption of raw and undercooked pork products, that is, livers or liver products, meat or meat products, is known as a potential risk of foodborne HEV infection. The aim of this study was to determine whether serological tests are appropriate to predict the occurrence of HEV in the liver and muscle of domestic pigs in Germany. In 2018, samples of meat juice, liver, and ham muscle were collected from 250 fattening pigs at an abattoir in North West Germany. Samples were analyzed for the presence of HEV antibodies using enzyme-linked immunosorbent assay respectively for the presence of HEV RNA using real-time reverse transcription-polymerase chain reaction. In total, 62% (155/250) of the meat juice samples were positive for HEV antibodies at a single animal basis. At herd level, 72% (18/25) of the herds were seropositive. The HEV prevalence in the liver was 17.2% (43/250). Each positive liver sample originated from seropositive herds respectively from HEV seropositive pigs. This study demonstrates for the first time the significant correlation between a positive HEV serology and the occurrence of HEV RNA in the liver of slaughter pigs (χ2 = 31.83; p < 0.001), highlighting the significant predictive power of positive serological results on the occurrence of HEV RNA in the liver.

Assessing the risk of ASFV entry into Japan through pork products illegally brought in by air passengers from China and fed to pigs in Japan.

A risk assessment was conducted to assess the risk of ASFV entry into Japan through pork products illegally brought in by air passengers from China and fed to pigs in Japan. Scenario tree modelling was used with the following entry and exposure pathway considered to be the most likely route of ASF entry: an ASFV infected pork product is illegally brought into Japan by air travellers from China; this pork product is then used in a restaurant where scrap waste is recycled for animal feed and subsequently fed to pigs without being heat-treated. Input parameter values were based on surveys conducted by the authors, scientific data gathered from the literature and official data published by government agencies. The annual probability of ASFV entry into Japan via this pathway was predicted to be 0.20 (90% prediction interval: 0.00-0.90). The wide prediction interval was mainly caused by the uncertainty regarding the dose response relation of ASFV, followed by the probability of an ASF infected pig dying on affected farms, the loading of ASFV in an infected pig and the probability of an illegally imported pork product being heat-treated in China and used in restaurants. The results of scenario analysis revealed that the annual probability of ASFV entry into Japan will increase with an increase in the number of ASF affected farms in China. The probability of ASFV entry will increase substantially even if only a small proportion of Ecofeed is not heat-treated during the production process. The probability will decrease if an increased proportion of farms that feed swill apply heat-treatment before feeding swill to their pigs. These findings indicate that stringent application of heat-treatment of Ecofeed and swill is key to protecting the Japanese pig industry from the introduction of ASFV.

Swine hepatitis E virus: Cross-species infection, pork safety and chronic infection.

Swine hepatitis E virus (swine HEV) belongs to the species Orthohepevirus A within the genus Orthohepevirus in the family Hepeviridae. Four different genotypes of swine HEV within the species Orthohepevirus A have been identified so far from domesticated and wild swine population: genotypes 3 (HEV-3) and 4 (HEV-4) swine HEVs are zoonotic and infect humans, whereas HEV-5 and HEV-6 are only identified from swine. As a zoonotic agent, swine HEV is an emerging public health concern in many industrialized countries. Pigs are natural reservoir for HEV, consumption of raw or undercooked pork is an important route of foodborne HEV transmission. Occupational risks such as direct contact with infected pigs also increase the risk of HEV transmission in humans. Cross-species infection of HEV-3 and HEV-4 have been documented under experimental and natural conditions. Both swine HEV-3 and swine HEV-4 infect non-human primates, the surrogates of man. Swine HEV, predominantly HEV-3, can establish chronic infection in immunocompromised patients especially in solid organ transplant recipients. The zoonotic HEV-3, and to lesser extent HEV-4, have also been shown to cause neurological diseases and kidney injury. In this review, we focus on the epidemiology of swine HEV, host and viral determinants influencing cross-species HEV infection, zoonotic infection and its associated pork safety concern, as well as swine HEV-associated chronic infection and neurological diseases.

Isolation and application of bacteriophages alone or in combination with nisin against planktonic and biofilm cells of Staphylococcus aureus.

Staphylococcus aureus is a notorious foodborne pathogen since it has ability to produce variety of toxins including heat-stable enterotoxin, form biofilm, and acquire resistance to antibiotics. Biocontrol of foodborne pathogens by lytic bacteriophages garners increasing interest from both researchers and food industry. In the present study, 29 phages against S. aureus were successfully isolated from chicken, pork, and fish. Characterization of the isolates revealed that phage SA46-CTH2 belonging to Podoviridae family had a number of features suitable for food industry applications such as wide host range, short latent period, large burst size, high stress tolerance, and a genome free of virulence genes. Furthermore, phage SA46-CTH2 alone or in combination with nisin exhibited great efficacy in reducing planktonic and biofilm cells of S. aureus at various conditions tested. The combination of phage SA46-CTH2 and nisin was also found to be able to inhibit the regrowth of S. aureus at both 37 and 24 °C.Key points• A total of 29 S. aureus phages were successfully isolated from fish, pork, and chicken products. • Phage SA46-CTH2 was characterized by host range, morphology, and genome sequencing. • SA46-CTH2 significantly reduced both planktonic and biofilm cells of S. aureus. • Combination of SA46-CTH2 and nisin inhibited the regrowth of S. aureus.

Development and evaluation of a time-saving RT-qRPA method for the detection of genotype 4 HEV presence in raw pork liver.

Hepatitis E virus (HEV) is a zoonotic pathogen spreading worldwide. Pig was known as its first and main animal reservoir. In China, pork consumption is very large and the risk of potential HEV contamination should not be underestimated. The present study aims to develop a quantitative real-time reverse transcription combining recombinase polymerase amplification assay (RT-qRPA) for the rapid detection of HEV RNA presence in raw pork liver on the Jinzhou markets in China. Methods: the specific primers and probes for RT-qRPA assay were designed targeting the ORF2/3 conserved region in genotype 4 swine HEV isolate (accession no. DQ279091.2) according to the TwistDx manual instructions. The specificity, sensitivity and reproducibility evaluations of the RT-qRPA method were subsequently conducted in assessing agreement with the standard RT-qPCR method. Results: the qRPA method step exhibited the obvious time-saving advantage which worked under the isothermal condition at 39 °C within about 30 min to complete the run while the compared standard qPCR method in the same cycle took almost 60 min to do. Both methods could exclusively detect the HEV genome equivalents from the quantified HEV-VLPs spiked samples. And both methods shared the same limit of detection (LOD) that was estimated at 1.25 × 103 genome equivalents copies/g spiked sample by the probit analysis. The recovery rate of HEV-VLPs reached a range of 9.56-14.65% by the RT-qRPA method which was higher than that of 1.34-2.34% by the standard RT-qPCR method. The detected HEV RNA positive rate in the field was 1.8% (1 out of 55) by both methods under Cohen's kappa statistic accessing with perfect agreement (κ = 1.00, p < 0.0005). The viral load in positive sample detected by the RT-qRPA method was estimated at 2.2125 × 105 genome copies/g pork liver sample. Conclusions, the present reported RT-qRPA method mainly targeting genotype 4 HEV is a rapid and reliable method. Its time-saving quality offers a promising for the development of a portable tool used in the routine monitoring of HEV contamination in the field.

Quantitative risk assessment of African swine fever virus introduction to Japan via pork products brought in air passengers' luggage.

The spread of African swine fever (ASF) has reached pandemic levels over the last decade, and outbreaks of this disease in China, Mongolia, Vietnam and Cambodia in 2018 and 2019 could accelerate its transmission to neighbouring Asian territories. Thus, the risk that the ASF virus (ASFV) will be introduced to disease-free territories increases each year. Since Japan is an island nation, the most likely way in which ASFV would be introduced is via pork products brought in air passengers' luggage (PPAP). Therefore, in the present study, we assessed the risk of ASFV introduction to Japan via PPAP. For the purposes of this analysis, we considered 214 international commercial flights travelling from 47 origin territories to 31 destination airports as potential routes of ASFV introduction via PPAP. The risk was estimated quantitatively through a stochastic model that considered the volume of air passengers' luggage, the amount of confiscated pork products that were carried in air passengers' luggage and the disease status of the origin territory. The overall mean annual probability of ASFV introduction to Japan via PPAP was found to be 0.941 [95% confidence interval (CI), 0.661-1.000], which approximately corresponds to one introduction every 1.06 years. At the origin territory level, Mongolia was led as the highest risk territory, with a risk of 0.864 (95% CI, 0.434-1.000), followed by China (0.697; 0.223-0.999), Vietnam (0.662; 0.196-0.998) and the Russian Federation (0.136; 0.018-0.401). At the destination airport level, Narita International Airport had the highest risk (0.905; 0.537-1.000), followed by Kansai International Airport (0.496; 0.109-0.961), Tokyo International Airport (0.389; 0.072-0.879) and Chubu Centrair International Airport (0.338; 0.058-0.816). This information will help improve risk management activities and monitoring systems to prevent the introduction of ASFV to Japan.

Risk of African swine fever virus introduction into the United States through smuggling of pork in air passenger luggage.

African swine fever causes substantial economic losses in the swine industry in affected countries. Traditionally confined to Africa with only occasional incursions into other regions, ASF began spreading into Caucasian countries and Eastern Europe in 2007, followed by Western Europe and Asia in 2018. Such a dramatic change in the global epidemiology of ASF has resulted in concerns that the disease may continue to spread into disease-free regions such as the US. In this study, we estimated the risk of introduction of ASF virus into the US through smuggling of pork in air passenger luggage. Results suggest that the mean risk of ASFV introduction into the US via this route has increased by 183.33% from the risk estimated before the disease had spread into Western Europe or Asia. Most of the risk (67.68%) was associated with flights originating from China and Hong Kong, followed by the Russian Federation (26.92%). Five US airports accounted for >90% of the risk. Results here will help to inform decisions related to the design of ASF virus surveillance strategies in the US.

Hepatitis E in pig-derived food products in Cape Town, South Africa, 2014.

BACKGROUND: Hepatitis E virus (HEV) genotypes 3 and 4 are zoonoses, with domestic pigs being the most important reservoir. A high anti- HEV IgG seroprevalence of 26 - 28% has been found in humans in Cape Town, South Africa (SA). Studies in industrialised countries have indicated a high prevalence of HEV in pigs and their associated food products. OBJECTIVES: To determine whether HEV could be found in pig-derived food products in Cape Town. METHODS: Pork-containing food products were purchased from supermarkets and butcheries around the Cape Town metropolitan area. HEV detection by polymerase chain reaction (PCR) was performed, and an amplified viral genome fragment was sequenced from positive samples. Phylogenetic analysis was done on the sequenced fragment. RESULTS: HEV was detected by PCR in 2/144 food samples - both were liver spread samples. One genome fragment sequence was obtained, which was closely related to HEV sequences obtained from humans in Cape Town. CONCLUSIONS: HEV can be found in pork-containing meat products available for sale in Cape Town, suggesting that these products could be a potential source of HEV transmission in our geographical area. Meat of pig origin should be thoroughly cooked before being consumed.

African Swine Fever Virus in Pork Brought into South Korea by Travelers from China, August 2018.

We tested samples of pork products confiscated from travelers to South Korea for African swine fever virus (ASFV). We detected ASFV in 4 food items confiscated from travelers from Shenyang, China, in August 2018. Surveillance of pork products at country entry points is needed to mitigate the risk for ASFV introduction.

Detection of Hepatitis E Virus in Raw Pork and Pig Viscera As Food in Hebei Province of China.

Hepatitis E virus (HEV) is an important human pathogen with pigs serving as the main natural animal reservoir. In China, pork is the most popular meat, while pig viscera are also widely consumed. The aim of this study was to evaluate the prevalence of HEV among pigs at slaughter, and assess the presence of HEV in raw pork and pig viscera as food. Samples of pig blood, raw pork, liver, kidney, and blood curd were collected from slaughterhouse or (and) retail market. Anti-HEV antibody in serum samples was detected using enzyme-linked immunosorbent assay based on an ORF2 antigen sandwich kit. HEV RNA was tested by reverse transcription nested polymerase chain reaction (RT-nested PCR) and the viral load was further assessed using quantitative real-time PCR. The final amplicons of RT-nested PCR were sequenced and undergone phylogenetic analysis. Prevalence of antibodies to HEV was 90.4% (104/115) in pigs at slaughtered level, and one serum sample was HEV RNA positive (0.9%, 1/115). HEV RNA was detected in liver, kidney, and blood samples with positivity of 6.1% (7/114), 3.1% (4/129), and 1.2% (2/170) respectively with viral loads ranged 102.4-104.4 (2.4Log-4.4Log) genome equivalents per gram, but not in pork. The HEV RNA prevalence in both liver and kidney were statistically higher than in pork. Phylogenetic analysis showed that all obtained sequences belonged to HEV genotype 4, which were divided into subtypes 4a, 4b, 4d, and 4i, highly identical to the known human and swine HEV sequences in China. The results indicate that raw pig viscera are more likely to harbor HEV than pork, suggesting a higher transmission risk related to consuming pig organs.

Familial Hepatitis E Outbreak Linked to Wild Boar Meat Consumption.

An HIV-infected patient was diagnosed with acute hepatitis E infection in our hospital. An epidemiological inquiry was performed to collect demographic, food and animal exposure variables in order to identify the potential route of transmission. The patient reported that his family traditionally hunted wild boar for food. All family members were analysed for hepatitis E virus infection. Additionally, route of transmission by wild boar meat consumption and prevalence of HEV infection among wild boar from the same hunting area were investigated. In all-family members (n = 8), HEV-RNA was amplified. Two wild boar meat slices consumed was analysed, showing the presence of HEV. The virus isolated was consistent with genotype 3, revealing 100% homology between family members and meat. Additionally, we tested nine wild boar hunted in the same hunting area. All of them were RNA-HEV positive, isolating the same HEV genotype 3 viral strain. We demonstrated by phylogenetic analysis zoonotic transmission of HEV by wild boar meat consumption. The prevalence of HEV infection among wild boar found in our study suggests that this species is an important route of transmission to human.