Hepatitis E virus in swine and effluent samples from slaughterhouses in Brazil

Submitted by Sandra Infurna ([email protected]) on 2018-11-18T14:08:42Z No. of bitstreams: 1 vanessas_paula_marcelopinto_etal_IOC_2011.pdf: 327062 bytes, checksum: cca9f8b8fa7606b729de769e96bf7e1f (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2018-11-18T16:05:01Z (GMT) No. of bitstreams: 1 vanessas_paula_marcelopinto_etal_IOC_2011.pdf: 327062 bytes, checksum: cca9f8b8fa7606b729de769e96bf7e1f (MD5)Made available in DSpace on 2018-11-18T16:05:01Z (GMT). No. of bitstreams: 1 vanessas_paula_marcelopinto_etal_IOC_2011.pdf: 327062 bytes, checksum: cca9f8b8fa7606b729de769e96bf7e1f (MD5) Previous issue date: 2011Universidade Federal Rural do Rio de Janeiro. Instituto de Veterinária. Departamento de Microbiologia e Imunologia Veterinária. Laboratório de Imunologia e Virologia Veterinária. Seropédica, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Desenvolvimento Tecnológico em Virologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Desenvolvimento Tecnológico em Virologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Bio-Manguinhos. Laboratório de Neuro-Virulência. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Desenvolvimento Tecnológico em Virologia. Rio de Janeiro, RJ, Brasil.Hepatitis E is an infectious disease which virus (HEV) is highly disseminated in swine herd populations. Sporadic acute human hepatitis E cases have been associated to genotype 3 and 4 strains of HEV also reported in swine populations of endemic and non-endemic areas. With the aim to evaluate the incidence of animals with current infection of HEV, 115 bile samples were collected from three slaughterhouses under inspection by Animal Sanitary Protection Agency of Rio de Janeiro, Brazil. In parallel, effluent samples were collected from six sewage pipe exit sites of two slaughterhouses. HEV RNA was detected in 11 out of 115 (9.6%) bile samples collected and three waste samples from one slaughterhouse. Viral loads observed for bile samples varied from 10(1)-10(5) genome copies/mL and for effluent samples mean load was 10(2) genome copies/mL. Sequencing and phylogenetic analysis classified samples within genotype 3 subtype 3b closely related to the sample obtained from the first reported autochthonous human case and samples from swine of commercial herds in Brazil. Our data demonstrates that although most animals achieve slaughter age (around 20 weeks old) already immune to HEV, a significant number of animals are with current infection at commercial age. Further studies should be addressed to consider risk analysis and possible evaluation of inspection regulations considering food safety measures regarding hepatitis E zoonotic aspect in Brazil

Cynomolgus monkeys are successfully and persistently infected with hepatitis E virus genotype 3 (HEV-3) after long-term immunosuppressive therapy

<div><p>Epidemiological studies found that hepatitis E virus genotype 3 (HEV-3) infection was associated with chronic hepatitis and cirrhosis in immunocompromised patients. Our study aimed to investigate the relationship between the host immunosuppressive status and the occurrence of HEV-related chronic hepatitis. Here we describe a successful experimental study, using cynomolgus monkeys previously treated with tacrolimus, a potent calcineurin inhibitor immunosuppressant, and infected with a Brazilian HEV-3 strain isolated from naturally infected pigs. HEV infected monkeys were followed up during 160 days post infection (dpi) by clinical signs; virological, biochemical and haematological parameters; and liver histopathology. The tacrolimus blood levels were monitored throughout the experiment. Immunosuppression was confirmed by clinical and laboratorial findings, such as: moderate weight loss, alopecia, and herpes virus opportunistic infection. In this study, chronic HEV infection was characterized by the mild increase of liver enzymes serum levels; persistent RNA viremia and viral faecal shedding; and liver histopathology. Three out of four immunosuppressed monkeys showed recurrent HEV RNA detection in liver samples, evident hepatocellular ballooning degeneration, mild to severe macro and microvesicular steatosis (zone 1), scattered hepatocellular apoptosis, and lobular focal inflammation. At 69 dpi, liver biopsies of all infected monkeys revealed evident ballooning degeneration (zone 3), discrete hepatocellular apoptosis, and at most mild portal and intra-acinar focal inflammation. At 160 dpi, the three chronically HEV infected monkeys showed microscopic features (piecemeal necrosis) corresponding to chronic hepatitis in absence of fibrosis and cirrhosis in liver parenchyma. Within 4-months follow up, the tacrolimus-immunosuppressed cynomolgus monkeys infected with a Brazilian swine HEV-3 strain exhibited more severe hepatic lesions progressing to chronic hepatitis without liver fibrosis, similarly as shown in tacrolimus-immunosuppressed solid organ transplant (SOT) recipients. The cause-effect relationship between HEV infection and tacrolimus treatment was confirmed in this experiment.</p></div

Gender, age, sex, and body weight of the cynomolgus monkeys used in this study.

<p>Gender, age, sex, and body weight of the cynomolgus monkeys used in this study.</p

Dynamic of HEV RNA measured by quantitative reverse transcription PCR qRT-PCR.

<p>Immunocompetent monkeys (G1) were inoculated with HEV-3; tacrolimus-treated monkeys inoculated with HEV-3 (G2) and tacrolimus-treated monkeys (G3). qRT-PCR assay were performed on RNA extracted from sera, liver biopsies and faeces samples. The viral load results are show in: <b>(A)</b> sera, <b>(B)</b> liver biopsies, and <b>(C)</b> faeces.</p

Tacrolimus concentrations measured by LC-MS/MS in whole blood, HEV viral load and blood glucose level in AC7 monkey previously treated with tacrolimus and infected with HEV.

<p>Tacrolimus concentrations measured by LC-MS/MS in whole blood, HEV viral load and blood glucose level in AC7 monkey previously treated with tacrolimus and infected with HEV.</p

Distributions of anti-HEV antibodies, ALT, AST, platelets and CHOL observed in inoculated groups (G1 and G2).

<p>Anti-HEV IgM, IgG and IgA are shown in <b>A</b>, <b>B</b>, and <b>C</b>, respectively. Samples with OD/cutoff ratios above 1.0 are considered positive for anti-HEV. ALT and AST levels are show in <b>D</b> and <b>E</b>, and platelets and cholesterol levels in <b>F</b> and <b>G</b>.</p

Phylogenetic analysis of Hepatitis E virus strains from human and animal samples.

<p>The Bayesian phylogenetic tree was constructed by using concatenated partial nucleotide sequence of ORF1 and ORF2 (546 nt) of HEV. For each sequence used, the GenBank accession number, animal species from which it was isolated and the corresponding genotype are shown. The tree was rooted at midpoint. Posterior probabilities (pp) are shown at the branch label. Numerical value ≥ 0.7 indicates the pp replicates that supported the interior branch. Newly described HEV sequences in this study are indicated. Scale bar indicates evolutionary distance of 0.2 substitutions per position in the sequence.</p

Chronological analysis of histopathological features of liver biopsies from immunocompetent cynomolgus monkeys infected with HEV (G1), monkeys previously treated with tacrolimus and infected with HEV (G2) and monkeys only treated with tacrolimus (G3).

<p><b>Hematoxylin-eosin (H&E)–stained paraffin section. (G1A)</b> Lobular architecture disorganization, ballooning degeneration of the hepatocytes with lytic necrosis. Acidophilic clumps (arrow) are located in a perinuclear position (Mallory bodies). <b>(G1B)</b> The liver cytoarchitecture was modified by ballooning and lytic necrosis of hepatocytes. Lipid droplets and inflammatory cell infiltration were observed. <b>(G1C)</b> Hepatic parenchyma shows significantly fat droplets deposition, mixture of macrosteatosis and microsteatosis. <b>(G1D)</b> Focal collection of lymphocytes and macrophages localized in the pericentral area. Ballooned hepatocytes and lytic necrosis were noted. <b>(G2A)</b> Irregular distribution pattern of hepatocytes, ballooning ad cytolitic necrosis associated with fatty changes. <b>(G2B)</b> Normal liver architecture. Lymphohistiocytic infiltration of portal liver tract. Microvesicular steatosis is also present. <b>(G2C)</b> Disarray of the cytoarchitecture of the parenchyma. Hepatocytes exhibiting ballooning, lytic necrosis and steatosis. Note glycogen accumulation in hepatocyte nuclei. <b>(G2D)</b> Interface hepatitis surrounded by micro and macrosteatosis. <b>(G3A)</b> Normal hepatic venule. Hepatocytes plates shows regular distribution. <b>(G3B)</b> Lobular disarray and hepatocellular ballooning predominantly distributed in zone 3. <b>(G3C)</b> Significative and strong diffusely distributed mixture of hepatocellular macrosteatosis and microsteatosis in all zones from zone 1 to 3. <b>(G3D)</b> Hepatocytes cords converging towards portal tract. Minimal fat droplets deposition in the hepatocyte cytoplasm.</p

Predictive factors associated with chronic HEV infection.

<p><b>(A)</b> Predictive factors obtained based on faeces viral load. <b>(B)</b> Predictive factors obtained based on sera viral load.</p