Genomic investigation of Salmonella enterica Serovar Welikade from a pediatric diarrhea case first time in Shanghai, China.

BACKGROUND: Salmonella, an important foodborne pathogen, was estimated to be responsible for 95.1 million cases and 50,771 deaths worldwide. Sixteen serovars were responsible for approximately 80% of Salmonella infections in humans in China, and infections caused by a few uncommon serovars have been reported in recent years, though not with S. Welikade. This study reports the first clinical case caused by S. Welikade in China and places Chinese S. Welikade isolates in the context of global isolates via genomic analysis. For comparison, S. Welikade isolates were also screened in the Chinese Local Surveillance System for Salmonella (CLSSS). The minimum inhibitory concentrations (MICs) of 28 antimicrobial agents were determined using the broth microdilution method. The isolates were sequenced on an Illumina platform to identify antimicrobial resistance genes, virulence genes, and phylogenetic relationships. RESULTS: The S. Welikade isolate (Sal097) was isolated from a two-year-old boy with acute gastroenteritis in 2021. Along with the other two isolates found in CLSSS, the three Chinese isolates were susceptible to all the examined antimicrobial agents, and their sequence types (STs) were ST5123 (n = 2) and ST3774 (n = 1). Single nucleotide polymorphism (SNP)-based phylogenetic analysis revealed that global S. Welikade strains can be divided into four groups, and these three Chinese isolates were assigned to B (n = 2; Sal097 and XXB1016) and C (n = 1; XXB700). In Group B, the two Chinese ST5123 isolates were closely clustered with three UK ST5123 isolates. In Group C, the Chinese isolate was closely related to the other 12 ST3774 isolates. The number of virulence genes in the S. Welikade isolates ranged from 59 to 152. The galF gene was only present in Group A, the pipB2 gene was only absent from Group A, the avrA gene was only absent from Group B, and the allB, sseK1, sspH2, STM0287, and tlde1 were found only within Group C and D isolates. There were 15 loci unique to the Sal097 isolate. CONCLUSION: This study is the first to characterize and investigate clinical S. Welikade isolates in China. Responsible for a pediatric case of gastroenteritis in 2021, the clinical isolate harbored no antimicrobial resistance and belonged to phylogenetic Group B of global S. Welikade genomes.

In vitro digestion of SARS-CoV-2 contaminated berries reveals high inactivation of infectious virus during gastrointestinal passage.

IMPORTANCE: During the pandemic, news outlets occasionally reported on the detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA on various foods, raising concerns over contaminated foods initiating infections. Coronavirus disease 2019 (COVID-19) patients often experience gastrointestinal symptoms and shed SARS-CoV-2 RNA in their feces. In addition, active virus replication in the gastrointestinal tract was shown; however, infectious viruses were rarely detected in feces. We previously showed that SARS-CoV-2 remained infectious on frozen berries for at least a month. Here, in vitro digestion models showed that SARS-CoV-2 on berries exhibits minimal inactivation at the oral phase and the virus may escape gastric inactivation early during feeding. However, high intestinal inactivation of the virus on berries suggested that SARS-CoV-2 was less likely to initiate infection in the small intestine. In contrast, the oral cavity is a potential site where infection might be initiated, providing more input for the gastrointestinal tract. High intestinal inactivation might explain the difficulty of detecting infectious SARS-CoV-2 in feces but not of virus RNA.

Hepatitis A Virus and Hepatitis E Virus as Food- and Waterborne Pathogens-Transmission Routes and Methods for Detection in Food.

Foodborne viruses are an important threat to food safety and public health. Globally, there are approximately 5 million cases of acute viral hepatitis due to hepatitis A virus (HAV) and hepatitis E virus (HEV) every year. HAV is responsible for numerous food-related viral outbreaks worldwide, while HEV is an emerging pathogen with a global health burden. The reported HEV cases in Europe have increased tenfold in the last 20 years due to its zoonotic transmission through the consumption of infected meat or meat products. HEV is considered the most common cause of acute viral hepatitis worldwide currently. This review focuses on the latest findings on the foodborne transmission routes of HAV and HEV and the methods for their detection in different food matrices.

Investigation of SARS-CoV-2 Detection Method Applicability and Virus Occurrence in Food and Food Packaging.

Research background: While it is clear that SARS CoV-2 coronavirus is the primary respiratory virus, there are no entirely clarified ways of transmission. Foodborne transmission has remained an unexplained path. Therefore, the goals of this paper are to examine and present an assessment of the most appropriate of the four selected kits for RNA extraction for the testing and detection of SARS-CoV-2 on food packaging surfaces, food surfaces, and in food. This will enable to indicate the possibility of infection through contact or direct food consumption. Experimental approach: Finding the best technique is vital as RNA extraction is one of the essential elements in detecting SARS-CoV-2. This was achieved through an experiment with four commercial kits following the original manufacturers' protocols, and with a modification of the original protocols that included the use of ethanol and isopropanol. The selected kit was used for RNA extraction from the swabs of packaging surfaces, food surface, and ready-to-eat food samples. The coronavirus was then identified using real-time reverse transcription-polymerase chain reaction (RT-PCR) assays to determine whether the SARS-CoV-2 virus or viral particles are present in the food chain with the overall purpose of demonstrating the possibility that food can contribute as a vehicle for the transmission of the virus. Results and conclusions: The findings of this investigation made the most effective extraction kit and protocol stand out. The results of the applicability of the kit indicated a significant share of positive samples of viral SARS-CoV-2 virus particles on surfaces from the environment where infected persons with 'silent' COVID-19 infection, with mild symptoms or no symptoms, were present. However, according to the findings of the second part of the study, the virus was not detected on the examined samples of food packaging surfaces, food surfaces, and food. Novelty and scientific contribution: The presented results distinguished one of the most suitable protocols for isolating RNA from environmental surface samples. The main contribution of the study is in the presentation of the results, that is, the examination of samples that are primarily related to the food chain, food packaging, food surfaces, and ready-to-eat food. The results of this study could also be helpful for further determination of the potential of food as a vector for the transmission of coronaviruses.

Persistence and survival of Cryptosporidium parvum oocysts on lamb's lettuce leaves during plant growth and in washing conditions of minimally-processed salads.

Cryptosporidium is the causative agent of cryptosporidiosis, which results, among others, in profuse diarrhoea. Transmission to humans occurs via the faecal-oral route directly by contact with infected hosts or indirectly by waterborne or foodborne routes. For the latter, parasite transmission is closely linked to the oocyst's ability to persist and survive in food matrices. In this study, we evaluated the persistence and survival of Cryptosporidium oocysts in lamb's lettuce: i) during plant growth and ii) in conditions mimicking the industrial washing process applied in minimally-processed vegetables (MPV). Results show that oocysts persisted during the growth of lamb's lettuce, i.e. two months from the 2-leaf stage until the 8-leaf harvest time (-0.89 Log10 of oocysts). However, their survival decreased from as early as one week (-0.61 Log10), and only 6 % of oocysts remained infective at the time of harvest. The washing process had a limited effect on parasite load (<0.5 Log10) and no effect on survival; chlorination of washing water did not improve the efficiency (removal and inactivation) of the process. The ability of C. parvum to persist and survive throughout the food chain may drive its transmission to humans through MPV products. Appropriate management measures should be implemented at each operational level to limit contamination and ensure food safety of fresh produce.

SARS-CoV-2 Remains Infectious on Refrigerated Deli Food, Meats, and Fresh Produce for up to 21 Days.

SARS-CoV-2, the virus that causes COVID-19, has been detected on foods and food packaging and the virus can infect oral cavity and intestinal cells, suggesting that infection could potentially occur following ingestion of virus-contaminated foods. To determine the relative risk of infection from different types of foods, we assessed survival of SARS-CoV-2 on refrigerated ready-to-eat deli items, fresh produce, and meats (including seafood). Deli items and meats with high protein, fat, and moisture maintained infectivity of SARS-CoV-2 for up to 21 days. However, processed meat, such as salami, and some fresh produce exhibited antiviral effects. SARS-CoV-2 also remained infectious in ground beef cooked rare or medium, but not well-done. Although infectious SARS-CoV-2 was inactivated on the foods over time, viral RNA was not degraded in similar trends, regardless of food type; thus, PCR-based assays for detection of pathogens on foods only indicate the presence of viral RNA, but do not correlate with presence or quantity of infectious virus. The survival and high recovery of SARS-CoV-2 on certain foods support the possibility that food contaminated with SARS-CoV-2 could potentially be a source of infection, highlighting the importance of proper food handling and cooking to inactivate any contaminating virus prior to consumption.

Estimating the burden and modeling mitigation strategies of pork-related hepatitis E virus foodborne transmission in representative European countries.

Hepatitis E virus (HEV) is an emerging zoonotic pathogen posing global health burden, and the concerns in Europe are tremendously growing. Pigs serve as a main reservoir, contributing to pork-related foodborne transmission. In this study, we aim to specifically simulate this foodborne transmission route and to assess potential interventions. We firstly established a dose-response relationship between the risk of transmission to human and the amount of ingested viruses. We further estimated the incidence of HEV infection specifically attributed to pork-related foodborne transmission in four representative European countries. Finally, we demonstrated a proof-of-concept of mitigating HEV transmission by implementing vaccination in human and pig populations. Our modeling approach bears essential implications for better understanding the transmission of pork-related foodborne HEV and for developing mitigation strategies.

Survival of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and Herpes Simplex Virus 1 (HSV-1) on Foods Stored at Refrigerated Temperature.

Outbreaks of coronavirus infectious disease 2019 (COVID-19) in meat processing plants and media reports of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection on foods have raised concerns of a public health risk from contaminated foods. We used herpes simplex virus 1, a non-Biosafety Level 3 (non-BSL3) enveloped virus, as a surrogate to develop and validate methods before assessing the survival of infectious SARS-CoV-2 on foods. Several food types, including chicken, seafood, and produce, were held at 4 °C and assessed for infectious virus survival (herpes simplex virus 1 (HSV-1) and SARS-CoV-2) at 0 h, 1 h, and 24 h post-inoculation (hpi) by plaque assay. At all three time points, recovery of SARS-CoV-2 was similar from chicken, salmon, shrimp, and spinach, ranging from 3.4 to 4.3 log PFU/mL. However, initial (0 h) virus recovery from apples and mushrooms was significantly lower than that from poultry and seafood, and infectious virus decreased over time, with recovery from mushrooms becoming undetectable by 24 hpi. Comparing infectious virus titers with viral genome copies confirmed that PCR-based tests only indicate presence of viral nucleic acid, which does not necessarily correlate with the quantity of infectious virus. The survival and high recovery of SARS-CoV-2 on certain foods highlight the importance of safe food handling practices in mitigating any public health concerns related to potentially contaminated foods.

Predominance of Human Bocavirus Genotypes 1 and 2 in Oysters in Thailand.

Human bocavirus (HBoV) has been recognized as an important pathogen that causes respiratory infection and acute gastroenteritis in young children worldwide. HBoV is most likely transmitted by the respiratory route and by fecal-oral transmission. Recently, HBoV has been detected in several types of environmental water and in bivalve shellfish. However, study of the existence of HBoV in oysters is still undocumented in Thailand. In this study, 144 oyster samples collected from different markets in Chiang Mai, Thailand, in 2017 and 2018 were investigated for the presence of HBoV by nested PCR and sequencing. HBoV was detected in 11 out of 144 samples (7.6%). Nine HBoV-positive samples (81.8%) were identified as genotype 1 (HBoV1) and two (18.2%) as HBoV2. A monthly investigation of HBoV in oyster samples from July 2017 to June 2018 showed that HBoV was sporadically detected in particular months spanning the rainy and colder season, with a peak in January. This study demonstrates the presence and genotype diversity of HBoV in oyster samples in Thailand. The findings contribute to evaluating the risk of foodborne transmission of HBoV and to monitoring outbreaks of HBoV in Thailand and in other countries. IMPORTANCE Human bocavirus is recognized as an important cause of respiratory infection and of acute gastroenteritis in children worldwide. Human bocavirus has been widely detected in many clinical specimens, as well as in several types of environmental samples. Most previous studies describe the incidence of bocavirus infection in humans, whereas few data are available for the occurrence of human bocavirus in food materials, particularly that in bivalve shellfish. Our findings provide evidence for the existence and prevalence of human bocavirus in oysters, suggesting that further monitoring of the potential risk of food- and waterborne transmission of this virus to humans should be undertaken.

Examining the persistence of human Coronavirus 229E on fresh produce.

Human coronaviruses (HCoVs) are mainly associated with respiratory infections. However, there is evidence that highly pathogenic HCoVs, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East Respiratory Syndrome (MERS-CoV), infect the gastrointestinal (GI) tract and are shed in the fecal matter of the infected individuals. These observations have raised questions regarding the possibility of fecal-oral route as well as foodborne transmission of SARS-CoV-2 and MERS-CoV. Studies regarding the survival of HCoVs on inanimate surfaces demonstrate that these viruses can remain infectious for hours to days, however, there is limited data regarding the viral survival on fresh produce, which is usually consumed raw or with minimal heat processing. To address this knowledge gap, we examined the persistence of HCoV-229E, as a surrogate for highly pathogenic HCoVs, on the surface of commonly consumed fresh produce, including: apples, tomatoes, cucumbers and lettuce. Herein, we demonstrated that viral infectivity declines within a few hours post-inoculation (p.i) on apples and tomatoes, and no infectious virus was detected at 24h p.i, while the virus persists in infectious form for 72h p.i on cucumbers and lettuce. The stability of viral RNA was examined by droplet-digital RT-PCR (ddRT-PCR), and it was observed that there is no considerable reduction in viral RNA within 72h p.i.

Exposure Profile of Severe Acute Respiratory Syndrome Coronavirus 2 in Canadian Food Sources.

ABSTRACT: A new coronavirus strain known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. This virus is the causative agent for coronavirus disease 2019 (COVID-19) and spreads primarily through human-to-human transmission via infected droplets and aerosols generated by infected persons. Although COVID-19 is a respiratory virus, the potential for transmission of SARS-CoV-2 via food is considered theoretically possible and remains a concern for Canadian consumers. We have conducted an exposure assessment of the likelihood of exposure of SARS-CoV-2 in Canadian food sources at the time of consumption. This article describes the exposure routes considered most relevant in the context of food contamination with SARS-CoV-2, including contaminated food of animal origin, other contaminated fresh foods, fomites, and SARS-CoV-2-contaminated feces. The likelihood of foodborne infection of SARS-CoV-2 via the human digestive tract also was considered. Our analysis indicates that there is no evidence that foodborne transmission of SARS-CoV-2 has occurred, and we consider the likelihood of contracting COVID-19 via food and food packaging in Canada as low to remote. Adherence to safe food practices and cleaning procedures would in any case prevent a potential foodborne infection with SARS-CoV-2.

Occurrence of Clostridioides (Clostridium) difficile in Poultry Giblets at Slaughter and in Retail Pork and Poultry Meat in Southeastern Spain.

ABSTRACT: Raw meat and meat products contaminated with Clostridioides difficile could be a vehicle for spreading community-associated C. difficile infection. This study was conducted to determine the occurrence of C. difficile in pork and poultry meat samples (n = 325) from retail establishments and in edible giblet samples (n = 36) from a poultry processing plant in Murcia (southeastern Spain). C. difficile was isolated after selective enrichment from 2% (6 of 361) of the samples, all of which were from the poultry processing plant. These isolates were recovered from 17% (6 of 36) of the edible chicken giblets, i.e., 28% (5 of 18) of the gizzard samples and 6% (1 of 18) of the liver samples. All six C. difficile isolates were negative for toxin A and B genes by PCR assay. These findings indicate that C. difficile can survive in the gastric acid of the chicken gizzard and could be transmitted to other meat products. However, the very low prevalence of C. difficile in the tested samples indicates that retail meat may not be an important source for transmission of C. difficile to humans.

Exploring the potential of foodborne transmission of respiratory viruses.

The ongoing pandemic involving severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has raised the question whether this virus, which is known to be spread primarily though respiratory droplets, could be spread through the fecal-oral route or via contaminated food. In this article, we present a critical review of the literature exploring the potential foodborne transmission of several respiratory viruses including human coronaviruses, avian influenza virus (AVI), parainfluenza viruses, human respiratory syncytial virus, adenoviruses, rhinoviruses, and Nipah virus. Multiple lines of evidence, including documented expression of receptor proteins on gastrointestinal epithelial cells, in vivo viral replication in gastrointestinal epithelial cell lines, extended fecal shedding of respiratory viruses, and the ability to remain infectious in food environments for extended periods of time raises the theoretical ability of some human respiratory viruses, particularly human coronaviruses and AVI, to spread via food. However, to date, neither epidemiological data nor case reports of clear foodborne transmission of either viruses exist. Thus, foodborne transmission of human respiratory viruses remains only a theoretical possibility.

Genomic characterization of Salmonella Uzaramo for human invasive infection.

Salmonella is composed of a wide variety of serovars, causing human self-limited gastrointestinal illnesses or invasive infections. Invasive non-typhoidal Salmonella (iNTS) is well documented, with high mortality for children and immunocompromised adults in sub-Saharan Africa and has recently been reported in Southeast Asia. However, iNTS in China remains unknown. In May 2019, a case of invasive infection caused by Salmonella enterica serovar Uzaramo (S. Uzaramo) was reported for the first time in China. Phylogenomic analysis was performed by genomic sequencing the available contextualized isolates, which separated the two Chinese strains into different sublineages. Both phenotypic and genomic characterization demonstrated that the S. Uzaramo isolates showed in general low antimicrobial resistance potential, except one isolated from lake-water in China. Additional comparative genomic analysis and Caenorhabditis elegans killing assays suggested a unique combination of virulence factors, including typhoid toxin and tcf fimbrial adhesin, which might play a role in the invasive infection. This study highlights that the transparency of global surveillance genomic data could accelerate understanding of virulence and antimicrobial resistance makeup of a previously unknown threat.

Presence of toxigenic Clostridioides (Clostridium) difficile in edible bivalve mollusks in Spain.

Clostridioides difficile reservoirs other than humans are becoming increasingly recognized, and the occurrence of the pathogen in shellfish raises concern because spores can survive cooking temperature and edible bivalve mollusks are often consumed raw or poorly cooked. This study was conducted to determine the occurrence of pathogenic C. difficile in retail bivalve mollusks. The microbiological quality of samples was also checked through the isolation of Salmonella spp. and Escherichia coli. We analyzed 129 mollusk samples from different fishmongers and grocery stores in Murcia. C. difficile was isolated from 8.53% (11/129) of the mollusks investigated. Four C. difficile isolates harbored genes for the production of toxin A and B. Salmonella spp. were not isolated from any sample and E. coli was isolated from 1.55% (2/129) of the samples, in both cases in accordance with the current legal requirements for consumption. Our findings indicate that the intake of raw or poorly cooked contaminated bivalve mollusks could be a potential source of C. difficile, leading to a risk for human health.

Salmonella Serotypes: A Novel Measure of Association with Foodborne Transmission.

Most nontyphoidal Salmonella (NTS) illnesses in the United States are thought to be foodborne. However, transmission routes likely vary among the different serotypes. We developed a relative ranking of NTS serotypes according to the strength of their association with foodborne transmission. We used Laboratory-based Enteric Disease Surveillance data to estimate the proportion of infections for each Salmonella serotype reported from 1998 to 2015 and Foodborne Disease Outbreak Surveillance System data to calculate the proportion of foodborne outbreak-associated Salmonella illnesses caused by each serotype. We calculated the ratios of these proportions to create a foodborne relatedness (FBR) measure for each serotype. Of the top 20 serotypes, Saintpaul (2.14), Heidelberg (1.61), and Berta (1.48) had the highest FBR measures; Mississippi (0.01), Bareilly (0.13), and Paratyphi B variant L(+) tartrate(+) (0.20) had the lowest. The FBRs for the three most prevalent serotypes were 1.22 for Enteritidis, 0.77 for Typhimurium, and 1.16 for Newport. This method provides a quantitative approach to estimating the relative differences in the likelihood that an illness caused by a particular serotype was transmitted by food, which may aid in tailoring strategies to prevent Salmonella illnesses and guide future research into serotype-specific source attribution.

Foodborne Transmission of Deformed Wing Virus to Ants (Myrmica rubra).

Virus host shifts occur frequently, but the whole range of host species and the actual transmission pathways are often poorly understood. Deformed wing virus (DWV), an RNA virus described from honeybees (Apis mellifera), has been shown to have a broad host range. Since ants are often scavenging on dead honeybees, foodborne transmission of these viruses may occur. However, the role of the ant Myrmica rubra as an alternative host is not known and foodborne transmission to ants has not been experimentally addressed yet. Here, we show with a 16-week feeding experiment that foodborne transmission enables DWV type-A and -B to infect M. rubra and that these ants may serve as a virus reservoir. However, the titers of both plus- and minus-sense viral RNA strands decreased over time. Since the ants were fed with highly virus-saturated honeybee pupae, this probably resulted in initial viral peaks, then approaching lower equilibrium titers in infected individuals later. Since DWV infections were also found in untreated field-collected M. rubra colonies, our results support the wide host range of DWV and further suggest foodborne transmission as a so far underestimated spread mechanism.

Effect of Specimen Type and Processing on the Detection of Clostridioides [Clostridium] difficile in Piglet Fecal Samples.

Subclinical Clostridioides difficile colonization in piglets could be a potential source of this bacterium for community-acquired C. difficile infection. The purposes of this study were to assess the effect of specimen type and processing on C. difficile isolation, culture, and detection by polymerase chain reaction (PCR), and to determine the occurrence of C. difficile in piglets of different ages. We compared different culture procedures-direct plating, ethanol shock, and an enrichment step-to isolate C. difficile from swine feces and rectal swabs. DNA was isolated directly from feces, processed feces, and bacterial isolates to detect the triose phosphate isomerase (tpi) gene and identify the toxins A and B genes. The results show that ethanol shock increased the C. difficile isolation from feces, while it decreased it for rectal swabs, in comparison with direct plating. The use of the enrichment broth gave the highest C. difficile recovery from both types of specimen. Our findings show low sensitivity for tpi gene detection after the DNA extraction directly from feces and an increase in PCR-positive samples when feces were processed before the DNA extraction. The overall prevalence of C. difficile was 16.9% (22/130), of which 100% were found to be toxigenic as assessed by the enrichment culture of fecal samples. The rate of isolation of positive samples decreased with the animal age, regardless of the presence or absence of diarrhea. Our results demonstrate the persistent reservoir of toxigenic C. difficile in fecal samples of piglets and support the impact of specimen processing on its isolation.

Prevention methods of foodborne Chagas disease: Disinfection, heat treatment and quality control by RT-PCR.

The most important mode of transmission causing outbreaks of Chagas disease in the Amazon region is the oral route due to the ingestion of contaminated food. Herein, prevention methods for foodborne diseases caused by Trypanosoma cruzi, namely, sanitization, thermal treatment were investigated and the use of reverse transcription PCR (RT-PCR) amplification for the mRNA-based detection of viable T. cruzi in açai, was developed. Three T. cruzi strains (T. cruzi I, T. cruzi III and Y) were used in the present study. The Amazonian strains T. cruzi I (425) and T. cruzi III (370) showed higher resistance to sodium hypochlorite treatment and heat treatment than the reference strain Y. The blanching of fruits (70 ±â€¯1 °C for 10 s) and pasteurization of juice (82.5 °C for 1 min) efficiently eliminated T. cruzi in food matrices. Additionally, a method that uses RT-PCR amplification of mRNA was developed for the detection of viable T. cruzi in açai, which could play a role in examining food samples, ensuring consumer health, and reducing this foodborne disease.

Quantification and genetic diversity of Hepatitis E virus in wild boar (Sus scrofa) hunted for domestic consumption in Central Italy.

Hepatitis E is an emerging disease in industrialized countries. The food-borne transmission of hepatitis E virus (HEV) is associated principally with products derived from the domestic pig, the wild boar, and deer; however, few quantitative data are available on HEV loads in animals used in food production. This study assessed HEV occurrence, viral load and genetic variability in wild boar hunted for domestic consumption in the district of Viterbo (Central Italy) where high anti-HEV IgG seroprevalence values are reported in humans. A total of 332 liver and 69 intestine samples were obtained from wild boar hunted between 2011 and 2014. The liver tissue in 54 of the animals (16.3%) was HEV-positive. Viral loads in quantifiable liver samples (n = 29) ranged between 3.2 × 102 and 3.8 × 105 genome copies (g.c.)/g with a mean value of 1.85 × 104 g.c./g. A statistically significant positive correlation was found between viral concentration in liver and intestinal tissue, though mean viral load in the intestines was lower (3.13 × 103 g.c./g). Twenty-six samples were characterized molecularly as genotype 3 (G3) and four subtypes (a, c, f and l) were detected. Finally, twelve samples with near identical sequences were identified as G3 but could not be assigned to any of the known subtypes, and could therefore represent a potentially new subtype.