Evidences of HEV genotype 3 persistence and reactivity in liver parenchyma from experimentally infected cynomolgus monkeys (Macaca fascicularis).

Hepatitis E virus genotype 3 (HEV-3) is an emerging zoonotic pathogen, responsible for sporadic cases of acute hepatitis E worldwide. Primate models have proven to be an essential tool for the study of HEV pathogenesis. Here we describe the outcomes of HEV infection in Macaca fascicularis (cynomolgus) inoculated experimentally with genotype 3. Eight adult cynomolgus macaques were inoculated intravenously with HEV-3 viral particles isolated from swine and human samples. Liver, spleen, duodenum, gallbladder and bile were sequential assessed up to the end-point of this study, 67 days post-inoculation (dpi). Our previously published findings showed that biochemical parameters return gradually to baseline levels at 55 dpi, whereas anti-HEV IgM and HEV RNA become undetectable in the serum and feces of all animals, indicating a non-viremic phase of recovery. Nevertheless, at a later stage during convalescence (67 dpi), the presence of HEV-3 RNA and antigen persist in central organs, even after peripheral viral clearance. Our results show that two cynomolgus inoculated with swine HEV-3 (animals I3 and O1) presented persistence of HEV RNA low titers in liver, gallbladder and bile. At this same stage of infection, HEV antigen (HEV Ag) could be detected in all infected animals, predominantly in non-reactive Kupffer cells (CD68+iNOS-) and sinusoidal lining cells. Simultaneously, CD4+, CD3+CD4+, and CD3+CD8+ immune cells were identified in hepatic sinusoids and small inflammatory clusters of lobular mononuclear cells, at the end-point of this study. Inability of HEV clearance in humans can result in chronic hepatitis, liver cirrhosis, with subsequent liver failure requiring transplantation. The results of our study support the persistence of HEV-3 during convalescence at 67 dpi, with active immune response in NHP. We alert to the inherent risk of viral transmission through liver transplantation, even in the absence of clinical and biochemical signs of acute infection. Thus, besides checking conventional serological markers of HEV infection, we strongly recommend HEV-3 RNA and antigen detection in liver explants as public health measure to prevent donor-recipient transmission and spread of hepatitis E.

Análise do controle viral e caracterização de subpopulações de células imunes em infecções pelo Vírus da hepatite E genótipo 3 de origem suína e humana em macacos cynomolgus (Macaca fascicularis) na fase de convalescência / Analysis of viral control and characterization of immune cell subpopulations in infections by hepatitis e genotype 3 of swine and human origin in cynomolgus monkeys (macaca fascicularis) in convalescence phase

O vírus da Hepatite E (HEV) apresenta-se relacionado à crescente ocorrência de casos da doença em países industrializados. Os achados deste em inúmeras espécies animais e sua transmissão associada ao consumo de produtos de origem animal, o definem como um agente zoonótico de importância para a Saúde Pública. Frente à escassez de informações sobre a patogenia da hepatite E, optou-se por avaliar o controle viral e caracterizar as células imunes envolvidas na resposta intra-hepática na fase de convalescência da infecção experimental com o HEV3 recuperado de suínos e humanos em macacos cynomolgus (Macaca fascicularis). No diagnóstico molecular por qRT-PCR em tecidos coletados 67 dpi, pôde-se detectar o RNA viral indicativo de permanência do vírus no trato biliar de dois animais e no fígado de um animal, inoculados com HEV suíno. Em imunomarcações específicas para a detecção do antígeno viral, todos os animais expressaram HEV Ag na fase tardia de infecção, principalmente em células sinusoidais. A discordância na detecção do HEV RNA e do HEV Ag sugere sensibilidades diferentes dos ensaios durante a fase da convalescência. A hiperplasia das células de Kupffer não foi observada no período de estudo, entretanto, a frequência elevada de células de kupffer comarcadas com HEV no grupo suíno sugere uma maior imunoreatividade tecidual ao HEV3 suíno em fase tardia da infecção. Quanto à expressão da enzima iNOS, observou-se pouca presença em células de Kupffer e maior expressão em células circulantes sinusoidais e hepatócitos, envolvidos na resposta inflamatória convalescente da hepatite E, com maior produção de iNOS nos animais inoculados com HEV suíno...

Hepatitis E virus (HEV) is presented related to the increasing occurrence of the disease in industrialized countries. The findings of this in several animal species and their associated transmission by consumption of animal products, define it as a zoonoticagent of importance to public health. Facing the lacking of information on thepathogenesis of hepatitis E, we chose to evaluate the viral control and characterize the immune cells involved in intrahepatic response during convalescence phase ofexperimental infection with HEV3 recovered from pigs and humans in cynomolgus monkeys (Macaca fascicularis). Through molecular diagnostics by qRT - PCR intissues collected 67 dpi, we could detect viral RNA indicative of virus persistence in the biliary tract of two animals and the liver of one animal inoculated with swine HEV. In specific immunostaining for the detection of viral antigen, all animals expressed HEVAg in the late phase of infection, especially sinusoidal cells. The discrepancy in thedetection of HEV RNA and HEV Ag suggests different sensitivities of the tests during the period of convalescence. The hyperplasia of Kupffer cells was not observed duringthe study period, however, the high frequency of Kupffer cells stained with HEV in the swine group suggests a greater tissue immunoreactivity for swine HEV3 in late stage of infection. Regarding the expression of iNOS, low presence was observed on Kupffer cells and higher expression in sinusoidal circulating cells and hepatocytes, involved in the inflammatory response of convalescent hepatitis E, with greater production of iNOS in animals inoculated with swine HEV...