Induction of Hepatitis E Virus Anti-ORF3 Antibodies from Systemic Administration of a Muscle-Specific Adeno-Associated Virus (AAV) Vector.

The hepatitis E virus (HEV) is a major global health problem, leading to large outbreaks in the developing world and chronic infections in the developed world. HEV is a non-enveloped virus, which circulates in the blood in a quasi-enveloped form. The quasi-envelope protects HEV particles from neutralising anti-capsid antibodies in the serum; however, most vaccine approaches are designed to induce an immune response against the HEV capsid. In this study, we explored systemic in vivo administration of a novel synthetic and myotropic Adeno-associated virus vector (AAVMYO3) to express the small HEV phosphoprotein ORF3 (found on quasi-enveloped HEV) in the musculature of mice, resulting in the robust and dose-dependent formation of anti-ORF3 antibodies. Neutralisation assays using the serum of ORF3 AAV-transduced mice showed a modest inhibitory effect on the infection of quasi-enveloped HEV in vivo, comparable to previously characterised anti-ORF3 antibodies used as a control. The novel AAVMYO3 capsid used in this study can serve as a versatile platform for the continued development of vector-based vaccines against HEV and other infectious agents, which could complement traditional vaccines akin to the current positive experience with SARS-CoV-2.

Circular DNA viruses identified in short-finned pilot whale and orca tissue samples.

Members of the Delphinidae family are widely distributed across the world's oceans. We used a viral metagenomic approach to identify viruses in orca (Orcinus orca) and short-finned pilot whale (Globicephala macrorhynchus) muscle, kidney, and liver samples from deceased animals. From orca tissue samples (muscle, kidney, and liver), we identified a novel polyomavirus (Polyomaviridae), three cressdnaviruses, and two genomoviruses (Genomoviridae). In the short-finned pilot whale we were able to identify one genomovirus in a kidney sample. The presence of unclassified cressdnavirus within two samples (muscle and kidney) of the same animal supports the possibility these viruses might be widespread within the animal. The orca polyomavirus identified here is the first of its species and is not closely related to the only other dolphin polyomavirus previously discovered. The identification and verification of these viruses expands the current knowledge of viruses that are associated with the Delphinidae family.

Levels of Zinc Transporters mRNA Depending on Zinc Status and HIV-1 Tat Induced Inflammation in Muscle (Rhabdomyosarcoma) and Monocyte (THP-1) Cell Lines.

Monocytes and muscles demonstrate functionally contrasting behavior under conditions of zinc deficiency with relation to zinc storage system (muscle retain zinc in contrast to monocytes). We aimed to understand the effects of zinc status and HIV-1 Tat mediated inflammation on expression of zinc transporters in these types of cells. Expression of zinc transporters [ZnTs, ZIPs, and metallothionein (MT)] was quantified by qRT-PCR in RD, THP-1 cells separately and in co-cultured THP-1-RD cells. ZnT1 protein expression levels were confirmed by Western blot. Significant increase of MT and ZnT1 mRNA in response to zinc supplementation and decrease during zinc deficiency indicates significance of the genes encoding transporters in maintaining zinc homeostasis in these tissues. In the RD cells ZIP10 exhibited inverse relation to zinc status whereas no correlation was found in the THP-1 cells. Tat-induced inflammation resulted in the significant elevation of MT, IL6, ZIP7, ZIP8, ZIP9 transcripts in the co-cultured RD cells, whereas THP-1 cells demonstrated increased IL-1ß levels and reduced levels of ZIP7 and ZIP14. Zinc status and HIV-1Tat induced inflammation appear to influence differential expression of MT, ZnTs, and ZIPs in the muscle and monocyte cells.

Circulation of Fluorescently Labelled Phage in a Murine Model.

Interactions between bacteriophages and mammals strongly affect possible applications of bacteriophages. This has created a need for tools that facilitate studies of phage circulation and deposition in tissues. Here, we propose red fluorescent protein (RFP)-labelled E. coli lytic phages as a new tool for the investigation of phage interactions with cells and tissues. The interaction of RFP-labelled phages with living eukaryotic cells (macrophages) was visualized after 20 min of co-incubation. RFP-labeled phages were applied in a murine model of phage circulation in vivo. Phages administered by three different routes (intravenously, orally, rectally) were detected through the course of time. The intravenous route of administration was the most efficient for phage delivery to multiple body compartments: 20 min after administration, virions were detected in lymph nodes, lungs, and liver; 30 min after administration, they were detectable in muscles; and 1 h after administration, phages were detected in spleen and lymph nodes. Oral and rectal administration of RFP-labelled phages allowed for their detection in the gastrointestinal (GI) tract only.

Stability of African Swine Fever Virus in Carcasses of Domestic Pigs and Wild Boar Experimentally Infected with the ASFV "Estonia 2014" Isolate.

Europe is currently experiencing a long-lasting African swine fever (ASF) epidemic, both in domestic pigs and wild boar. There is great concern that carcasses of infected wild boar may act as long-term virus reservoirs in the environment. We evaluated the tenacity of ASF virus (ASFV) in tissues and body fluids from experimentally infected domestic pigs and wild boar, which were stored on different matrices and at different temperatures. Samples were analysed at regular intervals for viral genome and infectious virus. ASFV was most stable in spleen or muscles stored at -20 °C and in blood stored at 4 °C. In bones stored at -20 °C, infectious virus was detected for up to three months, and at 4 °C for up to one month, while at room temperature (RT), no infectious virus could be recovered after one week. Skin stored at -20 °C, 4 °C and RT remained infectious for up to three, six and three months, respectively. In urine and faeces, no infectious virus was recovered after one week, irrespective of the matrix. In conclusion, tissues and organs from decomposing carcasses that persist in the environment for a long time can be a source of infection for several months, especially at low temperatures.

Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants.

Adeno-associated virus (AAV) forms the basis for several commercial gene therapy products and for countless gene transfer vectors derived from natural or synthetic viral isolates that are under intense preclinical evaluation. Here, we report a versatile pipeline that enables the direct side-by-side comparison of pre-selected AAV capsids in high-throughput and in the same animal, by combining DNA/RNA barcoding with multiplexed next-generation sequencing. For validation, we create three independent libraries comprising 183 different AAV variants including widely used benchmarks and screened them in all major tissues in adult mice. Thereby, we discover a peptide-displaying AAV9 mutant called AAVMYO that exhibits superior efficiency and specificity in the musculature including skeletal muscle, heart and diaphragm following peripheral delivery, and that holds great potential for muscle gene therapy. Our comprehensive methodology is compatible with any capsids, targets and species, and will thus facilitate and accelerate the stratification of optimal AAV vectors for human gene therapy.

Peripheral ProBDNF Delivered by an AAV Vector to the Muscle Triggers Depression-Like Behaviours in Mice.

Major depression is a leading cause of morbidity and disease burden in modern society. Current drug treatment is only effective in a fraction of patients as underlying mechanisms of depression are not fully understood. ProBDNF, a precursor of brain-derived neurotrophic factor (BDNF), and its receptor p75NTR are highly upregulated in patients with major depression and in animal models of depression induced by chronic stress. Here, we hypothesise that proBDNF may be a pathogenic factor triggering depression. C57BL/6 mice were injected in the bilateral gluteus maximus muscle with AAV-proBDNF or AAV-EGFP. Four weeks after the injection, AAV-proBDNF injected animals developed depression-like behaviours, which were evident for 4-8 weeks and then returned to the control level after 12 weeks. In the second experiment, mice were divided into three groups; one group was treated with sheep anti-proBDNF antibody after AAV-proBDNF injection whereas the other two groups received PBS injection after the AAV-proBDNF or AAV-EGFP delivery. The group that was injected with AAV-proBDNF showed a time-dependent increase in immobility time in the tail suspension test and forced swim test, reduced sucrose consumption and decreased grooming time after sucrose spraying. Treatment with sheep anti-proBDNF antibody alleviated the depressive-like symptoms. Peripheral AAV-proBDNF delivery also resulted in a reduction of density and length of dendritic spines in the dentate gyrus and amygdala. Thus, we conclude that peripheral proBDNF is a primary pathogenic factor triggering depression-like behavioural changes in mice likely by reducing dendritic spine plasticity.

Arboviruses and Muscle Disorders: From Disease to Cell Biology.

Infections due to arboviruses (arthropod-borne viruses) have dramatically increased worldwide during the last few years. In humans, symptoms associated with acute infection of most arboviruses are often described as "dengue-like syndrome", including fever, rash, conjunctivitis, arthralgia, and muscular symptoms such as myalgia, myositis, or rhabdomyolysis. In some cases, muscular symptoms may persist over months, especially following flavivirus and alphavirus infections. However, in humans the cellular targets of infection in muscle have been rarely identified. Animal models provide insights to elucidate pathological mechanisms through studying viral tropism, viral-induced inflammation, or potential viral persistence in the muscle compartment. The tropism of arboviruses for muscle cells as well as the viral-induced cytopathic effect and cellular alterations can be confirmed in vitro using cellular models. This review describes the link between muscle alterations and arbovirus infection, and the underlying mechanisms.

Muscle destruction caused by coxsackievirus A10 in gerbils: Construction of a novel animal model for antiviral evaluation.

The morbidity and mortality of coxsackievirus A10 (CVA10)-associated hand, foot, and mouth disease (HFMD) have been increasing in recent years, while few studies on the vaccine and animal model of CVA10 have been reported. Here, we first established a CVA10-infected gerbil model and employed it to evaluate the immunoprotective effect of an inactivated CVA10 vaccine. The results showed that gerbils up to the age of 14 days were fully susceptible to CVA10, and all died within five days post-infection by intraperitoneal inoculation. Lethargy, wasting, hind-limb paralysis, and even death could be observed in the CVA10-infected gerbils. Pathological examination suggested that CVA10 has a strong tropism toward muscle tissue, and muscle bundle fracture and muscular fibers necrosis were observed in the limb muscles. Additionally, active immunization results showed that gerbils immunized with the inactivated CVA10 vaccine were 100 % protected from lethal CVA10 challenge. The antisera from vaccinated gerbils also showed high neutralizing titers against CVA10. Based on these results, the CVA10-infected gerbil model was a suitable tool for analyzing the pathogenesis of CVA10 and assessing the protective efficacy of CVA10 candidate vaccines.

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.

Detection of Hepatitis E Virus in Livers and Muscle Tissues of Wild Boars in Italy.

In industrialized countries, hepatitis E is now recognized as an emerging zoonosis. Autochthonous cases have been increased over recent years in Europe and are mainly associated with HEV-3 infections. Pigs and wild boars are considered the main reservoirs of the zoonotic HEV-3 and HEV-4 genotypes. Over the past decade, the number of wild boars has drastically increased in Europe. Due to habitats closer to humans and domestic animals, the role of wild boar as a reservoir of the zoonotic HEV is considered to be an emerging issue. In this study, we investigated the presence of HEV RNA by a real-time RT-PCR assay in paired liver and muscle samples collected from 196 wild boars (Sus scrofa) hunted in the two areas of Central and Southern Italy. Twenty animals (10.2%) were HEV RNA positive in livers, 11 of which were also positive in muscles. The ORF2 and ORF1 partial viral sequences were obtained for nine paired livers and muscles, and when aligned were identical to each other. Phylogenetic analyses confirmed detection of different HEV-3 subtypes: 3c, 3f, 3i and some that were not assigned to any subtypes that have so far been identified. Results need further investigation because they are based on analyses of sequences of short genome regions. Nevertheless, we observed that the same strains were circulating in the wild boar populations from the two investigated areas, confirming persistence of the same HEV strains in the wild boar population over time.

Analysis of a novel RNA virus in a wild northern white-breasted hedgehog (Erinaceus roumanicus).

Tombusviruses are generally considered plant viruses. A novel tombus-/carmotetravirus-like RNA virus was identified in a faecal sample and blood and muscle tissues from a wild northern white-breasted hedgehog (Erinaceus roumanicus). The complete genome of the virus, called H14-hedgehog/2015/HUN (GenBank accession number MN044446), is 4,118 nucleotides in length with a readthrough stop codon of type/group 1 in ORF1 and lacks a poly(A) tract at the 3' end. The predicted ORF1-RT (RdRp) and the capsid proteins had low (31-33%) amino acid sequence identity to unclassified tombus-/noda-like viruses (Hubei tombus-like virus 12 and Beihai noda-like virus 10), respectively, discovered recently in invertebrate animals. An in vivo experimental plant inoculation study showed that an in vitro-transcribed H14-hedgehog/2015/HUN viral RNA did not replicate in Nicotiana benthamiana, Chenopodium quinoa, or Chenopodium murale, the most susceptible hosts for plant-origin tombusviruses.

High Amplification of the Antiviral Activity of Curcumin through Transformation into Carbon Quantum Dots.

It is demonstrated that carbon quantum dots derived from curcumin (Cur-CQDs) through one-step dry heating are effective antiviral agents against enterovirus 71 (EV71). The surface properties of Cur-CQDs, as well as their antiviral activity, are highly dependent on the heating temperature during synthesis. The one-step heating of curcumin at 180 °C preserves many of the moieties of polymeric curcumin on the surfaces of the as-synthesized Cur-CQDs, resulting in superior antiviral characteristics. It is proposed that curcumin undergoes a series of structural changes through dehydration, polymerization, and carbonization to form core-shell CQDs whose surfaces remain a pyrolytic curcumin-like polymer, boosting the antiviral activity. The results reveal that curcumin possesses insignificant inhibitory activity against EV71 infection in RD cells [half-maximal effective concentration (EC50 ) >200 µg mL-1 ] but exhibits high cytotoxicity toward RD cells (half-maximal cytotoxic concentration (CC50 ) <13 µg mL-1 ). The EC50 (0.2 µg mL-1 ) and CC50 (452.2 µg mL-1 ) of Cur-CQDs are >1000-fold lower and >34-fold higher, respectively, than those of curcumin, demonstrating their far superior antiviral capabilities and high biocompatibility. In vivo, intraperitoneal administration of Cur-CQDs significantly decreases mortality and provides protection against virus-induced hind-limb paralysis in new-born mice challenged with a lethal dose of EV71.

Diverse picornaviruses are prevalent among free-living and laboratory rats (Rattus norvegicus) in Hungary and can cause disseminated infections.

In this study, the full length genomes of three phylogenetically distant picornaviruses (family Picornaviridae) belonging to the genus Rosavirus (rat08/rRoB/HUN, MN116648), Kobuvirus (rat08/rAiA/HUN, MN116647), and Cardiovirus (rat08/rCaB/HUN, MN116646) were obtained from a single faecal sample of a free-living Norway rat (Rattus norvegicus) in Hungary using viral metagenomics and RT-PCR/Sanger sequencing. The acquired complete genomes were in silico analyzed in detail revealing the presence of a second minor open reading frame encoding an alternative Leader peptide (L*) in rat08/rCaB/HUN and a ca. 222 nt-long sequence repeat with compact secondary RNA structure in the 3' UTR of rat08/rRoB/HUN. The studied rat picornaviruses were frequently detectable by RT-PCR with relatively high viral loads ranged between 8.99E+02 and 1.29E+06 copies/ml in rat faecal samples collected from five geographically distant locations throughout Hungary. The VP1 sequence-based phylogenetic analyses show the presence of multiple, mostly location-specific lineages for all three picornaviruses. Rat rosavirus and rat cardiovirus were identified in spleen while rat cardiovirus was also detected in liver, muscle and kidney samples with variable copy numbers (6.42E+01-1.90E+05 copies/µg total RNA) suggesting extra-intestinal dissemination. Both viruses were also prevalent (70.0% and 18.2%) among two populations of laboratory rats ("Wistar-type" and "hooded-type") held in different, isolated laboratory animal units.

Lycorine Derivative LY-55 Inhibits EV71 and CVA16 Replication Through Downregulating Autophagy.

Hand, foot, and mouth disease (HFMD) is a global health concern, especially in the Asia-Pacific region. HFMD caused by Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) infection is usually self-limited but occasionally leads to severe pulmonary edema, neurological complications, and even death. Unfortunately, no effective drugs are currently available in clinical practice for the prevention and treatment of HFMD. Thus, anti-HFMD drugs must be urgently developed. A previous study had reported that lycorine could inhibit EV71 replication. In the present study, we found that LY-55, a lycorine derivative, inhibited the replication of EV71 and CVA16 in vitro and provided partial protection to mice from EV71 infection, as indicated by the decreased viral load and protein expression levels in muscles, clinical scores, and increased survival rates of infected mice. Mechanistically, LY-55 was not directly viricidal. Instead, the LY-55-mediated inhibition of EV71 and CVA16 was found to be mechanistically possible, at least in part, through downregulating autophagy, which plays an important role for EV71 and CVA16 replication. These findings suggest that LY-55 could be a potential lead or supplement for the development of anti-HFMD agents in the future.

Macropinocytosis dependent entry of Chikungunya virus into human muscle cells.

Chikungunya virus (CHIKV) is a re-emerging arbovirus known to cause chronic myalgia and arthralgia with high morbidity. CHIKV is now considered endemic in many countries across Asia and Africa. In this study, the susceptibility of various human, mammalian and mosquito cell lines to CHIKV infection was evaluated. CHIKV infection was found to be cell-type dependent and virus strain-specific. Furthermore, SJCRH30 (human rhabdomyosarcoma cell line) was showed to be highly permissive to CHIKV infection, with maximum production of infectious virions observed at 12 h.p.i. Pre-infection treatment of SJCRH30 with various inhibitors of endocytosis, including monodansylcadaverine (receptor-mediated endocytic inhibitor), dynasore (clathrin-mediated endocytic inhibitor), as well as filipin (caveolin-mediated endocytosis inhibitor), resulted in minimal inhibition of CHIKV infection. In contrast, dose-dependent inhibition of CHIKV infection was observed with the treatment of macropinocytosis inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Furthermore, siRNA-mediated knockdown of sortin nexin 9 (SNX9) a protein involved in macropinosome formation, also resulted in a significant dose-dependent reduction in viral titre. By performing a virus entry assay, CHIKV particles were also observed to colocalize with FITC-dextran, a macropinosome marker. This study shows for the first time, that the infectious entry of CHIKV into human muscle cells is mediated by macropinocytosis. Together, the data from this study may pave the way for the development of specific inhibitors that target the entry process of CHIKV into cells.

Medium optimization and characterization of cell culture system from Penaeus vannamei for adaptation of white spot syndrome virus (WSSV).

The lack of shrimp cell lines and difficulty in establishing shrimp cell culture systems, with an appropriate medium is a major concern in the aquaculture sector. The present study attempts to address this issue by developing an in vitro cell culture system from various tissues (hemocytes, heart, lymphoid tissue, hepatopancreas, gill, eye stalk, and muscle) of Penaeus vannamei (P.vannamei) using commercially available L-15 medium. The cell culture medium was formulated using five different media such as HBSCM-1, HBSCM-2, HBSCM-3, HBSCM-4, and HBSCM-5 containing L-proline and glucose with fetal bovine serum (FBS) supplements. Among the different media used, the HBSCM-5 medium with supplements showed good attachment and proliferation of cells with fibroblast-like, epithelioid, round, and adherent cell morphology in hemocyte culture. The same medium was further screened using different tissues to enhance the cell growth. The hemocytes, heart, and lymphoid tissue cells were passaged five times and maintained up to 20 days. Hepatopancreas and gill cells initially showed good morphological features and survived for more than ten days following subculture cells. Eye stalks and muscle cells perished within five days and did not show any unique morphology. The primary hemocyte cells were subjected to species identification, using cytochrome oxidase subunit I (COI) gene. To assess the primary hemocyte cell culture, cells were used for in vitro propagation of white spot syndrome virus (WSSV) and confirmed by the conventional polymerase chain reaction (PCR). Similarly, the primary cells were treated with bacterial extracellular products (ECPs) from Vibrio parahaemolyticus and Vibrio harveyi, to evaluate the cytotoxicity.

Silencing of HIF-1 in WSSV-infected white shrimp: Effect on viral load and antioxidant enzymes.

Hypoxia inducible factor-1 (HIF-1) is a transcriptional factor that induces genes involved in glucose metabolism. HIF-1 is formed by a regulatory α-subunit (HIF-1α) and a constitutive ß-subunit (HIF-1ß). The white spot syndrome virus (WSSV) induces a shift in glucose metabolism and oxidative stress. HIF-1α is associated with the induction of metabolic changes in tissues of WSSV-infected shrimp. However, the contributions of HIF-1 to viral load and antioxidant responses in WSSV-infected shrimp have been not examined. In this study, the effect of HIF-1 silencing on viral load and the expression and activity of antioxidant enzymes (superoxide dismutase-SOD, glutathione S-transferase-GST, and catalase) along with oxidative damage (lipid peroxidation and protein carbonyl) in tissues of white shrimp infected with the WSSV were studied. The viral load increased in hepatopancreas and muscle after WSSV infection, and the accumulative mortality was of 100% at 72 h post-infection. The expression and activity of SOD, catalase, and GST decreased in each tissue evaluated after WSSV infection. Protein carbonyl concentrations increased in each tissue after WSSV infection, while lipid peroxidation increased in hepatopancreas, but not in muscle. Silencing of HIF-1α decreased the WSSV viral load in hepatopancreas and muscle of infected shrimp along with shrimp mortality. Silencing of HIF-1α ameliorated the antioxidant response in a tissue-specific manner, which translated to a decrease in oxidative damage. These results suggest that HIF-1 is essential for restoring the antioxidant response, which counters the oxidative injury associated with WSSV infection.

Plasmid pcDNA3.1-s11 constructed based on the S11 segment of grass carp reovirus as DNA vaccine provides immune protection.

Although some commercial vaccines against grass carp reovirus (GCRV) are available, given the many varieties of GCRV and limited types of vaccines, the disease caused by GCRV remains a major problem, which leads to economic losses in grass carp aquaculture. A reovirus strain (GCRV-HN14) was recently isolated from local diseased fish in our laboratory. The S11 segment of GCRV-HN14 was speculated to encode the virus capsid protein VP35. In our study, the S11 segment was cloned into the eukaryotic expression vector pcDNA3.1(+) to construct the recombinant plasmid pcDNA3.1-s11, which was then transfected into CIK cells, and the VP35 protein was successfully expressed. Grass carp was immunized with pcDNA3.1-s11, and the in vivo distribution and expression of the pcDNA3.1-s11 plasmids were analyzed by PCR and Western blot. Recombinant plasmids were detected in the blood, liver, spleen, kidney, and muscle. However, protein expression could only be detected in the muscle. The immune protection of the pcDNA3.1-s11 plasmid in grass carp was evaluated using a series of experiments. Results showed that the population of white blood cells significantly increased at 1, 7, and 14 days post-immunization (dpi) and reached a peak with (9.58 ±â€¯0.72) × 107/ml at 7 dpi (P < 0.01 or P < 0.05). The percentage of neutrophils reached a peak with (24.13 ±â€¯2.38)% at 7 dpi (P < 0.01), whereas the lymphocytes peaked with (93.30 ±â€¯4.71)% at 14 dpi (P < 0.05). Serum antibody levels were significantly enhanced in immunized fish at 14, 21, and 28 dpi (P < 0.01). The mRNA expression levels of type I interferon, immunoglobulin M, Toll-like receptor 22, and major histocompatibility complex class I were significantly up-regulated in the head kidney and spleen of immunized fish (P < 0.05). Grass carp immunized with pcDNA3.1-s11 exhibited a higher survival percentage (70.4%-73.3%) than the controls (5%-13%). Overall, as a DNA vaccine, the pcDNA3.1-s11 plasmid could induce immune protection against GCRV.

High load of hepatitis E viral RNA in pork livers but absence in pork muscle at French slaughterhouses.

Pork ham muscle can be contaminated with HEV via blood vessels during viremia and represents a possible source of human contamination via the consumption of dried ham. This study evaluated the prevalence of HEV RNA in pork ham muscles and pork livers at slaughterhouses. Serology was determined on the corresponding serum samples. The apparent individual seroprevalence rate in the 49 pig farms studied was 59% [55.5%-61.4%]. None of the 1134 ham muscles tested was positive for the presence of HEV. HEV prevalence in paired liver samples was 2.8% with a level of contamination of up to 1.46 108copies/g. Sequences of viral strains isolated from positive livers belonged to genotype 3 and subtypes 3c, 3e, 3f and 3j. Our results confirmed that raw pork liver food products are a source of risk for humans but they also showed that there is a limited risk of human infection by HEV through the consumption of ham muscle.