Ruihan Shi

Evidence of Hepatitis E virus breaking through the blood–brain barrier and replicating in the central nervous system

Neurologic dysfunctions such as Guillain–Barre′ syndrome, encephalitis, meningitis and transverse myelitis occur frequently in patients with hepatitis E virus (HEV) infection, and this study was conducted to better characterize the role of HEV in the pathogenesis of neurologic disorders. Genotype 4 strain of swine HEV was used to inoculate Mongolian gerbils. Reverse transcription–nested polymerase chain reaction (RT‐nPCR), ELISA, histopathology, ultrastructural pathology and enzyme immunohistochemistry method were conducted to investigate the replication and localization of HEV in the central nervous system (CNS) and the consequent pathological changes. Both positive‐ and negative‐strand HEV RNA was detectable in brain and spinal cord from 7 to 28 dpi (days postinoculation) via RT‐nPCR. Various pathological changes such as perineural invasion, neuron necrosis, microglia nodule, lymphocyte infiltration, perivascular cuff and myelin degeneration were observed in HEV‐positive brains and spinal cords. Immunohistochemical (IHC) staining targeting on HEV ORF2 protein revealed positive signals concentrated mainly in the cytoplasm of neuron, ependymal epithelium and choroid plexus area. Positive area density of ZO‐1 (zonula occludens‐1) in brain of HEV‐positive gerbils decreased, while the GFAP (glial fibrillary acidic protein) expression was upregulated compared with control groups. These results provide strong evidence that HEV is able to damage the blood–brain barrier (BBB), replicate in brain and spinal cord, and hammer the causative role of HEV in the pathogenesis of neurologic disorders.

Fatal disease associated with Swine Hepatitis E virus and Porcine circovirus 2 co-infection in four weaned pigs in China

BackgroundIn recent decades, Porcine circovirus 2 (PCV2) infection has been recognized as the causative agent of postweaning multisystemic wasting syndrome, and has become a threat to the swine industry. Hepatitis E virus (HEV) is another high prevalent pathogen in swine in many regions of the world. PCV2 and HEV are both highly prevalent in pig farms in China.Case presentationIn this study, we characterized the HEV and PCV2 co-infection in 2–3 month-old piglets, based on pathogen identification and the pathological changes observed, in Hebei Province, China. The pathological changes were severe, and general hyperemia, hemorrhage, inflammatory cell infiltration, and necrosis were evident in the tissues of dead swine. PCR was used to identify the pathogen and we tested for eight viruses (HEV, Porcine reproductive and respiratory syndrome virus, PCV2, Classical swine fever virus, Porcine epidemic diarrhea virus, Transmissible gastroenteritis coronavirus, Porcine parvovirus and Pseudorabies virus) that are prevalent in Chinese pig farms. The livers, kidneys, spleens, and other organs of the necropsied swine were positive for HEV and/or PCV2. Immunohistochemical staining showed HEV- and PCV2-antigen-positive signals in the livers, kidneys, lungs, lymph nodes, and intestine.ConclusionHEV and PCV2 co-infection in piglets was detected in four out of seven dead pigs from two pig farms in Hebei, China, producing severe pathological changes. The natural co-infection of HEV and PCV2 in pigs in China has rarely been reported. We speculate that co-infection with PCV2 and HEV may bring some negative effect on pig production and recommend that more attention should be paid to this phenomenon.

Effect of swine hepatitis E virus on the livers of experimentally infected Mongolian gerbils by swine hepatitis E virus

Previous studies have shown that hepatitis E virus (HEV) can be transmitted between rats, pigs, cattle, rabbits, chicken, cats, and deer. Because wild and domestic rodents have anti-HEV antibodies, they are considered potential reservoirs of HEV. In the current study, Mongolian gerbils were experimentally infected with swine hepatitis E virus and the effects of this infection were investigated. After inoculation with HEV, the liver-to-body weight ratio increased at 7 dpi. Mongolian gerbils demonstrated significant increase (p<0.05) in Aspartate Transaminase (AST), alanine transaminase (ALT) and total bilirubin (T-BIL) concentrations in the sera, and HEV IgG was detected at 21 days post-inoculation (dpi). Real-time PCR revealed that the copies of HEV RNA in the liver were detected at 7 dpi, and peaked at 28 dpi at a concentration of 7.73 logs g(-1). Using both light and electron microscopy, hepatic lesions were observed in the HEV inoculated animals. In the experimental group, characteristic viral hepatitis lesions were prominent in the liver. HEV antigen was detected in the liver by immunohistochemistry, and HEV ORF3 antigen was detectable in liver by Western blot. These results clearly demonstrate that viral load of HEV in livers was dynamic, and ultrastructural hepatic injury in HEV infected Mongolian gerbils and anti-HEV IgG positive seroconversion were observed during infection.

Antigen detection and apoptosis in Mongolian gerbil’s kidney experimentally intraperitoneally infected by swine hepatitis E virus

We examined the effect of hepatitis E virus (HEV) on the renal tissue pathogenesis, morphological damages and related molecular mechanisms following swine HEV suspension intraperitoneally inoculation in Mongolian gerbils. The microscopic and ultramicroscopic analyses of kidney tissue structure were carried out at different points after inoculation of HEV. The immunohistochemistry, real-time PCR and Western blot were performed to explore the molecular mechanisms associated with HEV presence in the renal tissues. Real-time PCR revealed that the copies of HEV RNA in the kidney were detected at 7 dpi, and peaked at 14 dpi at a concentration was 7.18 logs g(-1), with detection of HEV ORF2 antigen by immunohistochemistry. Hematoxylin and eosin (HE) staining showed pathological lesions including glomerular atrophy, degeneration, edema and necrosis of renal tubular epithelial cells and Mallory and Sirius red staining indicated the presence of collagen fibers and fibrosis in kidney tissues of inoculated gerbils. Ultrastructural studies of basal membrane of renal tubules demonstrated the rough and uneven with mitochondria swelling and vacuolation in the tissues of HEV inoculated animals. Similarly, significantly higher number of (TUNEL)-positive cells were seen in renal tubule tissues compared to control group. Moreover, immuno histochemical results indicated that significant increase expression of the B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), FAS and Caspase-3 in HEV inoculated Mongolian gerbils at each time points. Relative mRNA expression by real-time PCR revealed a significantly higher (P<0.05) mRNA level of BAX, Bcl-2 and caspase-3 transcription in HEV inoculated Mongolian gerbils. Our results demonstrates that activation of mitochondria and Caspase-3 protease might be induced the apoptosis which subsequently cause the necrosis and cell death of renal epithelial cells during acute phase of HEV infection in HEV inoculated Mongolian gerbils.

Case Report Associated with Aspergillosis and Hepatitis E Virus Coinfection in Himalayan Griffons

This study involved a death which occurred in four Himalayan griffons housed in Beijing zoo, China. Based on pathogen identification and the pathological changes observed, we did characterize the fungi and Hepatitis E virus (HEV) in four dead Himalayan griffons. Pathological changes were severe. Membranous-like material was observed on the surface of the internal organs. Spleen was necrotic. Focal lymphocyte infiltration in the liver and many sunflower-like fungi nodules were evident in the tissues, especially in the kidney. PCR was used to identify the pathogen. Based on the 18SrRNA genomic sequence of known fungi, the results confirmed that all four dead Himalayan griffons were infected with Aspergillus. At the same time the detection of HEV also showed positive results. To the best of our knowledge, this work appears to be the first report of concurrent presence of Aspergillosis and Hepatitis E virus in rare avian species.

Antimicrobial activity and safety evaluation of peptides isolated from the hemoglobin of chickens

BackgroundHemoglobin is a rich source of biological peptides. As a byproduct and even wastewater of poultry-slaughtering facilities, chicken blood is one of the most abundant source of hemoglobin.ResultsIn this study, the chicken hemoglobin antimicrobial peptides (CHAP) were isolated and the antimicrobial and bactericidal activities were tested by the agarose diffusion assay, minimum inhibitory concentration (MIC) analysis, minimal bactericidal concentration (MBC) analysis, and time-dependent inhibitory and bactericidal assays. The results demonstrated that CHAP had potent and rapid antimicrobial activity against 19 bacterial strains, including 9 multidrug-resistant bacterial strains. Bacterial biofilm and NaCl permeability assays, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were further performed to detect the mechanism of its antimicrobial effect. Additionally, CHAP showed low hemolytic activity, embryo toxicity, and high stability in different temperatures and animal plasma.ConclusionCHAP may have great potential for expanding production and development value in animal medication, the breeding industry and environment protection.

Detection of Genotype 4 Swine Hepatitis E Virus in Systemic Tissues in Cross-Species Infected Rabbits

Increasing evidence demonstrates that hepatitis E virus (HEV) can be transmitted across species. According to previous reports, swine HEV has two genotypes, genotype 3 and 4, and both can infect humans by the fecal-oral route. Thus, it is crucial for the control of HEV zoonotic transmission to evaluate the dynamics of viral shedding and distribution in different tissues during cross-species infection by HEV. In this study, rabbits were infected with genotype 4 swine HEV by the intraperitoneal route. The results showed that HEV RNA not only shed in the feces but also in the saliva of some rabbits during infection with swine HEV. Viremia appeared late after infection, and anti-HEV IgG was not obvious until the appearance of high viremia levels. After the rabbits were euthanized, a histopathological examination showed that the livers developed overt hepatitis accompanied by an elevation of alanine aminotransferase (ALT) and aspartate transaminase (AST). Furthermore, HEV RNA was detected in various tissues, especially in the salivary glands and tonsils. Subsequently, negative-stranded HEV RNA was practiced in tissues with positive HEV RNA, which demonstrated that HEV replicated in the tissues. Next, we harvested additional tissues from the liver, salivary gland, tonsil, spleen, thymus gland, lymph node and intestine, which are known as replication sites of swine HEV. Additionally, we also observed the HEV antigen distributed in the organs above through immunohistochemical staining. These results demonstrate that rabbits could be used as an animal model for researching cross-species infection of genotype 4 HEV. It is also noteworthy that HEV can shed in the saliva and presents the risk of droplet transmission. These new data provide valuable information for understanding cross-species infection by HEV.

Induction of Apoptosis in the Immature Mouse Testes by a Mixture of Melamine and Cyanuric Acid

Abstract The toxicity of melamine has attracted much attention since the recent outbreaks of renal injury in pets and infants. Previous studies indicated that melamine by itself had low toxicity, whereas a mixture of melamine and cyanuric acid (M+CA) could cause serious renal damage. At present, most researches on the toxicity of M+CA are focused on the kidney. However, little is known about the adverse effects of this mixture on the reproductive system. In the present study, the toxicity of M+CA to testes was investigated. Immature male mice were orally dosed with 0, 0.6, 3, and 15 mg kg−1 d−1 of a 1:1 M+CA for 28 d. Pathological changes occurred in germ cells, such as loose arrangement, reduced numbers and karyopyknosis, indicating that this mixture was toxic to spermatogenesis. Compared with the control group, the TUNEL-positive germ cells increased significantly and the ratio of Bcl-2 to Bax, total antioxidant capacity and superoxide dismutase activity decreased significantly in the 3 and 15 mg kg−1 d−1 M+CA treated group, while the activities of caspase-3, caspase-8 and caspase-9 remained unchanged. The results suggest that M+CA can induce apoptosis in the mice testes. The downregulation of Bcl-2/Bax and oxidative stress may play a pivotal role in the induction of apoptosis by M+CA in mice testes.

Molecular and structural changes related to hepatitis E virus antigen and its expression in testis inducing apoptosis in Mongolian gerbil model

Hepatitis E virus (HEV) infection has been associated with a wide range of extrahepatic manifestations, so this study was designed to examine the effect and role of HEV on structural and molecular changes in the testicular tissues of Mongolian gerbils experimentally infected with swine HEV. HEV RNA was first detected in testis at 14 days post‐inoculation and reached a peak between 28 and 42 days later with viral load between 3.12 and 6.23 logs/g by PCR assays. Changes including vacuolation, sloughing of germ cells, formation of multinuclear giant cells, degeneration, necrosis of tubules and damaged blood‐testis barrier were observed through transmission electron microscopy. HEV ORF2 antigen was detected in the sperm cell cytoplasm along with decrease in relative protein of zonula occludens‐1 through immunohistochemistry. HEV ORF3 antigen and ZO‐1 protein were detectable by Western blotting. Lower (P<.05) serum testosterone and higher (P<.05) blood urea nitrogen level was observed in inoculated Mongolian gerbils. Likewise, increased (P<.05) germ cell apoptosis rate was detected with significant increased expression of Fas‐L and Fas in HEV‐inoculated groups at each time points. Up‐regulation (P<.05 or P<.01) in mRNA level of Fas‐L, Fas, Bax, Bcl‐2 and caspase‐3 was observed in HEV RNA‐positive testes. Our study demonstrated that after experimental inoculation, HEV can be detected in testis tissues and viral proteins produce structural and molecular changes that in turn disrupt the blood‐testis barrier and induce germ cell apoptosis.

Hepatitis E Virus Genotype 4 Sequences Detected in Sewage from Treatment Plants of China

The aim of this study was to investigate the occurrence of hepatitis E virus (HEV) in sewage samples in Shen Zhen, China. Sewage samples were collected from 152 sewage plants including livestock sewage, domestic sewage and treated sewage from May to July of 2015. Two of 152 samples were HEV positive (1.32%) from the livestock sewage plants. Partial ORF2 fragments of HEV were sequenced and a phylogenetic tree was constructed using MEGA5.1. Blast and phylogenetic analyses showed that both of these two sequences belonged to HEV Genotype 4. To the best of our knowledge, this is the first study on the molecular characterization of HEV in wastewater in China and the first time to detect Genotype 4 in the sewage. Results from this study indicate that the possibilities of sporadic infections of HEV should be emphasized because virus still has the possibility to be circulating in the sewage in China.