Hepatitis E virus in Norway rats (Rattus norvegicus) captured around pig farm

BACKGROUND: Hepatitis E virus (HEV) transmitted via the oral route through the consumption of contaminated water or uncooked or undercooked contaminated meat has been implicated in major outbreaks. Rats may play a critical role in HEV outbreaks, considering their negative effects on environmental hygiene and food sanitation. Although the serological evidence of HEV infection in wild rodents has been reported worldwide, the infectivity and propagation of HEV in wild rats remain unknown. To investigate if rats are a possible carrier of HEV, we studied wild Norway rats (Rattus norvegicus) that were caught near a pig farm, where HEV was prevalent among the pigs. METHODS: We examined 56 Norway rats for HEV. RNA from internal organs was examined for RT-PCR and positive samples were sequenced. Positive tissue samples were incubated with A549 cell line to isolate HEV. Anti-HEV antibodies were detected by ELISA. RESULTS: Sixteen rats were seropositive, and the HEV RNA was detected in 10 of the 56 rats. Sequencing of the partial ORF1 gene from 7 samples resulted in partially sequenced HEV, belonging to genotype 3, which was genetically identical to the HEV prevalent in the swine from the source farm. The infectious HEVs were isolated from the Norway rats by using the human A549 cell line. CONCLUSIONS: There was a relatively high prevalence (17.9%) of the HEV genome in wild Norway rats. The virus was mainly detected in the liver and spleen. The results indicate that these animals might be possible carrier of swine HEV in endemic regions. The HEV contamination risk due to rats needs to be examined in human habitats

Long-term shedding of hepatitis E virus in the feces of pigs infected naturally, born to sows with and without maternal antibodies.

Pigs are presumed reservoirs for hepatitis E virus (HEV) transmission to humans. To examine infection kinetics, two litters of domestic pigs (A and B, each containing 10 piglets) infected naturally with HEV were studied until pigs were 6 months old. Maternal IgG and IgA antibodies were detected in litter A piglets, but not in litter B ones. All pigs shed HEV in feces when they were 30-110 days old, and 17 developed viremia at 40-100 days of age. Phylogenetic analysis revealed a highly close sequence of HEV genotype 3 in all pigs. The serum levels of specific IgG and IgA were similar in all pigs, although IgA was not detected in the feces. Interestingly, the onset of both viremia and seroconversion was delayed significantly in litter A pigs. The kinetics of fecal virus shedding was similar in both litters; shedding was not detected after the pigs were 120 days old. The differences in the infection kinetics between litters A and B suggested that maternal antibodies delayed the onset of viremia and seroconversion. Quantitative real-time reverse transcriptase-polymerase chain reaction revealed that HEV RNA in feces peaked 10 days after initial shedding of approximately 10(6.0) copies/g. The viral load was much lower in the serum than in the feces. At 200 days of age, HEV RNA was found in the internal organs of 3 out of 13 pigs. These study findings improve the understanding of the dynamics of natural HEV transmission in pigs, which could help in controlling virus transmission from pigs to humans

Distribution and Propagation of Hepatitis E Virus in Experimentally Infected Swine

HEV infections in human and pigs have been reported in many countries; however, the precise distribution and multiplication of this virus in the host remains poorly understood. In this study, we examined the distribution and multiplication of HEV genotype 3 in two piglets at the early phase of intravenous infection, and also examined the virus distribution in a naturally infected pig. We developed real-time RT-PCR to determine copy numbers of the HEV genome in the sera, feces and organs. HEV-RNA was detected in serum from a pig transiently for 7 days post-inoculation (dpi). The HEV copy numbers in the feces appearing 7 dpi were increased to 3.4x106 copies/g at 14 dpi. In contrast, the higher copy numbers of HEV were widely distributed in the tissue organs from naturally HEV-infected pig. The study showed that intravenously inoculated HEV was distributed in several restricted tissues, such as in the liver and intestine

Sequence Variation in Hepatitis E Virus Genotypes 3 and 4 from Swine Fecal Samples in Japan

Hepatitis E virus (HEV) is a causative agent for hepatitis. HEV is transmitted via the fecal-oral route through contaminated drinking water and induces zoonotic infections through eating uncooked and undercooked meat of deer, wild boar, and swine. In Japan, genotypes 3 (G3) and 4 (G4) are prevalent in domestic swine. Here, we examined the genetic variation among HEVs derived from swine fecal samples in Japan. A total of 320 samples were collected at 32 commercial farm facilities (1 fecal sample from each of 10 pig houses in individual farms). Viral RNA amplification at open reading frame (ORF) 3 was possible in 159 (49.7%) of the fecal samples. For genotyping, the same samples were subjected to amplification at ORF2 and the resulting amplicons were sequenced. The results revealed that all the HEVs in each farm belonged to the same cluster of G3 and G4: G3JP in 8 farms, G3SP in 4 farms, G3US in 6 farms, and G4JP in 2 farms, unclassified G3 in 2 farms, unable to decide due to a low rate of amplification in 5 farms, and no detection in 5 farms. Interestingly, the HEVs from one farm were more homogeneous than those of the same cluster that was derived from other farms. Thus, the efficiency of farm-to-farm transmission of HEVs is likely to be low and HEV seems to have evolved independently at each farm in Japan

Ligand-Directed Chemistry of AMPA Receptors Confers Live-Cell Fluorescent Biosensors

AMPA-type glutamate receptors (AMPARs) mediate fast excitatory synaptic transmission in the central nervous system. Dysregulation of AMPAR function is associated with many kinds of neurological, neurodegenerative, and psychiatric disorders. As a result, molecules capable of controlling AMPAR functions are potential therapeutic agents. Fluorescent semisynthetic biosensors have attracted considerable interest for the discovery of ligands selectively acting on target proteins. Given the large protein complex formation of AMPARs in live cells, biosensors using full-length AMPARs retaining original functionality are ideal for drug screening. Here, we demonstrate that fluorophore-labeled AMPARs prepared by ligand-directed acyl imidazole chemistry can act as turn-on fluorescent biosensors for AMPAR ligands in living cells. These biosensors selectively detect orthosteric ligands of AMPARs among the glutamate receptor family. Notably, the dissociation constants of agonists and antagonists for AMPARs were determined in live cells, which revealed that the ligand-binding properties of AMPARs to agonists are largely different in living cells, compared with noncellular conditions. We also show that these sensors can be applied to detecting allosteric modulators or subunit-selective ligands of AMPARs. Thus, our protein-based biosensors can be useful for discovering pharmaceutical agents to treat AMPAR-related neurological disorders

Tyrosinase-Based Proximity Labeling in Living Cells and <i>In Vivo</i>

Characterizing the protein constituents of a specific organelle and protein neighbors of a protein of interest (POI) is essential for understanding the function and state of the organelle and protein networks associated with the POI. Proximity labeling (PL) has emerged as a promising technology for specific and efficient spatial proteomics. Nevertheless, most enzymes adopted for PL still have limitations: APEX requires cytotoxic H2O2 for activation and thus is poor in biocompatibility for in vivo application, BioID shows insufficient labeling kinetics, and TurboID suffers from high background biotinylation. Here, we introduce a bacterial tyrosinase (BmTyr) as a new PL enzyme suitable for H2O2-free, fast (≤10 min in living cells), and low-background protein tagging. BmTyr is genetically encodable and enables subcellular-resolved PL and proteomics in living cells. We further designed a strategy of ligand-tethered BmTyr for in vivo PL, which unveiled the surrounding proteome of a neurotransmitter receptor (Grm1 and Drd2) in its resident synapse in a live mouse brain. Overall, BmTyr is one promising enzyme that can improve and expand PL-based applications and discoveries