Inactivated and Immunogenic SARS-CoV-2 for Safe Use in Immunoassays and as an Immunization Control for Non-Clinical Trials.

Successful SARS-CoV-2 inactivation allows its safe use in Biosafety Level 2 facilities, and the use of the whole viral particle helps in the development of analytical methods and a more reliable immune response, contributing to the development and improvement of in vitro and in vivo assays. In order to obtain a functional product, we evaluated several inactivation protocols and observed that 0.03% beta-propiolactone for 24 h was the best condition tested, as it promoted SARS-CoV-2 inactivation above 99.99% and no cytopathic effect was visualized after five serial passages. Moreover, RT-qPCR and transmission electron microscopy revealed that RNA quantification and viral structure integrity were preserved. The antigenicity of inactivated SARS-CoV-2 was confirmed by ELISA using different Spike-neutralizing monoclonal antibodies. K18-hACE2 mice immunized with inactivated SARS-CoV-2, formulated in AddaS03TM, presented high neutralizing antibody titers, no significant weight loss, and longer survival than controls from a lethal challenge, despite RNA detection in the oropharyngeal swab, lung, and brain. This work emphasizes the importance of using different techniques to confirm viral inactivation and avoid potentially disastrous contamination. We believe that an efficiently inactivated product can be used in several applications, including the development and improvement of molecular diagnostic kits, as an antigen for antibody production as well as a control for non-clinical trials.

Uncovering neglected subtypes and zoonotic transmission of Hepatitis E virus (HEV) in Brazil.

Hepatitis E virus (HEV) circulation in humans and swine has been extensively studied in South America over the last two decades. Nevertheless, only 2.1% of reported HEV strains are available as complete genome sequences. Therefore, many clinical, epidemiological, and evolutionary aspects of circulating HEV in the continent still need to be clarified. Here, we conducted a retrospective evolutionary analysis of one human case and six swine HEV strains previously reported in northeastern, southern, and southeastern Brazil. We obtained two complete and four nearly complete genomic sequences. Evolutionary analysis comparing the whole genomic and capsid gene sequences revealed high genetic variability. This included the circulation of at least one unrecognized unique South American subtype. Our results corroborate that sequencing the whole capsid gene could be used as an alternative for HEV subtype assignment in the absence of complete genomic sequences. Moreover, our results substantiate the evidence for zoonotic transmission by comparing a larger genomic fragment recovered from the sample of the autochthonous human hepatitis E case. Further studies should continuously investigate HEV genetic diversity and zoonotic transmission of HEV in South America.

Detection and characterization of hepatitis E virus genotype 3 in HIV-infected patients and blood donors from southern Brazil.

BACKGROUND: Hepatitis E virus genotype 3 (HEV-3) infection usually causes self-limited acute hepatitis. In immunosuppressed patients, HEV-3 infection can rapidly progress to chronic hepatitis and cirrhosis. In southern Brazil, data on HEV seroprevalence are scarce. METHODS: Testing for HEV RNA and antibodies (anti-HEV) was performed for 320 HIV-infected patients followed at the HIV/AIDS Service of the Federal University of Rio Grande between 2012 and 2013, as well as 281 blood donor samples obtained in 2015. Variables associated with anti-HEV positivity were assessed by multivariable logistic regression analysis. RESULTS: HIV and blood donor groups showed similar HEV seroprevalence (6.7% and 7.1%, respectively). Risk factors associated with anti-HEV detection were older age, marital status, a higher number of sexual partners, poor sanitation, and alcohol use (HIV group), and living in a rural area (blood donors). HEV RNA was detected in eight serum samples from HIV-infected patients and in one blood donor, who was also positive for anti-HEV IgM and IgG. CONCLUSIONS: The prevalence rates of HEV infection were comparable between HIV-seropositive patients who were not severely immunocompromised and blood donors. The blood donor's HEV isolate showed high similarity with swine HEV strains from Brazilian herds in the same region, thus indicating a potential risk of foodborne and parenteral transmission via blood transfusion.

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.

Cynomolgus monkeys (Macaca fascicularis) experimentally and naturally infected with hepatitis E virus: The bone marrow as a possible new viral target.

Hepatitis E virus (HEV) transmission through infected blood and blood products has already been described. However, little is known about the bone marrow (BM) as source of HEV infection. Our study aimed to investigate the presence of HEV antigen (Ag) and histological changes in BM of cynomolgus monkeys (Macaca fascicularis) experimentally and naturally infected with HEV. Four cynomolgus monkeys with acute, and two with chronic hepatitis E ─ after immunosuppressive therapy with tacrolimus ─ were compared with one colony-bred animal naturally infected. Both, natural and experimental infections were characterized by anti-HEV IgG seroconversion detected by ELISA, and viral RNA isolation confirmed by RT-qPCR and qualitative nested RT-PCR. BM biopsies were collected from all animals, submitted to histology and indirect immunofluorescence techniques and observed, respectively, by light and confocal microscopy. The HEV Ag-fluorescent-labeled cells were detected from BM biopsies obtained from three monkeys with acute and one with chronic hepatitis E, and also from the naturally infected monkey. In the experimentally infected animals with acute hepatitis, HEV Ag detection occurred at 160 days post-infection, even after viral clearance in serum, feces, and liver. Double-stranded RNA, a replicative marker, was detected in BM cells from both acute and chronically infected animals. Major histological findings included vacuolization in mononuclear and endosteal cells, an absence of organized inflammatory infiltrates, and also some fields suggesting displasic focal BM disease. These findings support the hypothesis of BM cells as secondary target sites of HEV persistence. Further experimental studies should be carried out to confirm the assumption of HEV transmission through BM transplantation.

Cynomolgus Monkeys (Macaca fascicularis) as an Experimental Infection Model for Human Group A Rotavirus.

Group A rotaviruses (RVA) are one of the most common causes of severe acute gastroenteritis in infants worldwide. Rotaviruses spread from person to person, mainly by faecal⁻oral transmission. Almost all unvaccinated children may become infected with RVA in the first two years of life. The establishment of an experimental monkey model with RVA is important to evaluate new therapeutic approaches. In this study, we demonstrated viral shedding and viraemia in juvenile⁻adult Macaca fascicularis orally inoculated with Wa RVA prototype. Nine monkeys were inoculated orally: seven animals with human RVA and two control animals with saline solution. During the study, the monkeys were clinically monitored, and faeces and blood samples were tested for RVA infection. In general, the inoculated animals developed an oligosymptomatic infection pattern. The main clinical symptoms observed were diarrhoea in two monkeys for three days, associated with a reduction in plasmatic potassium content. Viral RNA was detected in seven faecal and five sera samples from inoculated animals, suggesting virus replication. Cynomolgus monkeys are susceptible hosts for human Wa RVA infection. When inoculated orally, they presented self-limited diarrhoea associated with presence of RVA infectious particles in faeces. Thus, cynomolgus monkeys may be useful as animal models to evaluate the efficacy of new antiviral approaches.

Hepatitis E: Update on Prevention and Control.

Hepatitis E virus (HEV) is a common etiology of acute viral hepatitis worldwide. Recombinant HEV vaccines have been developed, but only one is commercially available and licensed in China since 2011. Epidemiological studies have identified genotype 3 as the major cause of chronic infection in immunocompromised individuals. Ribavirin has been shown to be effective as a monotherapy to induce HEV clearance in chronic patients who have undergone solid organ transplant (SOT) under immunosuppressive therapy. Efforts and improvements in prevention and control have been made to reduce the instances of acute and chronic hepatitis E in endemic and nonendemic countries. However, this review shows that further studies are required to demonstrate the importance of preventive vaccination and treatment worldwide, with emphasis on hepatitis E infection in the public health system.

Hepatitis E seroprevalence and associated factors in rural settlers in Central Brazil.

INTRODUCTION: Prevalence of hepatitis E virus (HEV) infection and associated factors were investigated in rural settlements in Central Brazil. METHODS: A total of 464 settlers were interviewed, and serum samples were tested for anti-HEV IgG/IgM. Positive samples were tested for HEV RNA. RESULTS: Sixteen participants (3.4%; 95% CI 2.0-5.7) were positive for anti-HEV IgG. None was positive for anti-HEV IgM. HEV RNA was not detected. Dwelling in a rural settlement for >5 years was associated with HEV seropositivity. CONCLUSIONS: The results revealed the absence of acute infection and a low prevalence of previous exposure to HEV.

Hepatitis E seroprevalence and associated factors in rural settlers in Central Brazil

Abstract INTRODUCTION: Prevalence of hepatitis E virus (HEV) infection and associated factors were investigated in rural settlements in Central Brazil. METHODS: A total of 464 settlers were interviewed, and serum samples were tested for anti-HEV IgG/IgM. Positive samples were tested for HEV RNA. RESULTS: Sixteen participants (3.4%; 95% CI 2.0-5.7) were positive for anti-HEV IgG. None was positive for anti-HEV IgM. HEV RNA was not detected. Dwelling in a rural settlement for >5 years was associated with HEV seropositivity. CONCLUSIONS: The results revealed the absence of acute infection and a low prevalence of previous exposure to HEV.

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

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.

Hepatitis E virus infection in patients with acute non-A, non-B, non-C hepatitis in Central Brazil

Hepatitis E virus (HEV) infection has a worldwide distribution and represents an important cause of acute hepatitis. This study aims to investigate the occurrence of HEV infection and factors associated with this infection in patients with acute non-A, non-B, non-C hepatitis in Central Brazil. From April 2012 to October 2014, a cross-sectional study was conducted among 379 patients with acute non-A, non-B, non-C hepatitis in the City of Goiania, Central Brazil. Serum samples of all patients were tested for serological markers of HEV infection (anti-HEV IgM and IgG) by ELISA. Positive samples were confirmed using immunoblot test. Anti-HEV IgM and IgG positive samples were tested for HEV RNA. Of the 379 serum samples, one (0.3%) and 20 (5.3%) were positive for anti-HEV IgM and IgG, respectively. HEV RNA was not found in any sample positive for IgM and/or IgG anti-HEV. After multivariate analysis, low education level was independently associated with HEV seropositivity (p = 0.005), as well as living in rural area, with a borderline p-value (p = 0.056). In conclusion, HEV may be responsible for sporadic self-limited cases of acute hepatitis in Central Brazil.

Hepatitis E virus infection in patients with acute non-A, non-B, non-C hepatitis in Central Brazil.

Hepatitis E virus (HEV) infection has a worldwide distribution and represents an important cause of acute hepatitis. This study aims to investigate the occurrence of HEV infection and factors associated with this infection in patients with acute non-A, non-B, non-C hepatitis in Central Brazil. From April 2012 to October 2014, a cross-sectional study was conducted among 379 patients with acute non-A, non-B, non-C hepatitis in the City of Goiania, Central Brazil. Serum samples of all patients were tested for serological markers of HEV infection (anti-HEV IgM and IgG) by ELISA. Positive samples were confirmed using immunoblot test. Anti-HEV IgM and IgG positive samples were tested for HEV RNA. Of the 379 serum samples, one (0.3%) and 20 (5.3%) were positive for anti-HEV IgM and IgG, respectively. HEV RNA was not found in any sample positive for IgM and/or IgG anti-HEV. After multivariate analysis, low education level was independently associated with HEV seropositivity (p = 0.005), as well as living in rural area, with a borderline p-value (p = 0.056). In conclusion, HEV may be responsible for sporadic self-limited cases of acute hepatitis in Central Brazil.

Evaluation of hepatitis E virus infection between different production systems of pigs in Brazil.

The objectives of this study were to (1) investigate the occurrence of hepatitis E virus (HEV) in pigs from large-scale and family-scale farms, (2) genetically characterize the strains isolated, and (3) study the pathogenesis of swine HEV infection via immunohistochemistry. A total of 50 pigs from 10 farms in Mato Grosso State, Brazil were divided according to type of production system into either large-scale farms (n = 5) or family-scale farms (n = 5). Samples of liver, gallbladder, small and large intestines, bile, and feces from the pigs were analyzed by nested PCR with primers targeting the ORF2 region of HEV and by immunohistochemistry. Of the eight HEV-positive samples from pigs of family-scale farms, phylogenetic analysis revealed that seven of the swine HEV isolates clustered with subtype 3b of genotype 3 and one isolate was categorized with subtype 3 f. The HEV antigen was detected mainly in the small intestine samples from family-scale farms, suggesting an early stage HEV infection. HEV was not detected in the samples of pigs from large-scale farms, reinforcing the need for additional studies to evaluate the risk of transmission of HEV to humans from pigs from family-scale farms in Mato Grosso State.

Hepatitis E virus in liver and bile samples from slaughtered pigs of Brazil.

The objective of this study was to detect and identify hepatitis E virus (HEV) strains in liver and bile samples from slaughtered pigs in the state of Paraná, Brazil. Liver and bile samples were collected from 118 asymptomatic adult pigs at a slaughterhouse in a major Brazilian pork production area. The samples were assayed using a nested reverse transcription-polymerase chain reaction protocol with primer sets targeting open reading frames (ORF)1 and 2 of the HEV genome. HEV RNA was detected in two (1.7%) liver samples and one (0.84%) bile sample using both primers sets. The HEV strains were classified as genotype 3b on the basis of their nucleotide sequences. These data suggest that healthy pigs may be a source of HEV infection for consumers of pig liver and slaughterhouse workers in Brazil.

Hepatitis E virus in liver and bile samples from slaughtered pigs of Brazil

The objective of this study was to detect and identify hepatitis E virus (HEV) strains in liver and bile samples from slaughtered pigs in the state of Paraná, Brazil. Liver and bile samples were collected from 118 asymptomatic adult pigs at a slaughterhouse in a major Brazilian pork production area. The samples were assayed using a nested reverse transcription-polymerase chain reaction protocol with primer sets targeting open reading frames (ORF)1 and 2 of the HEV genome. HEV RNA was detected in two (1.7%) liver samples and one (0.84%) bile sample using both primers sets. The HEV strains were classified as genotype 3b on the basis of their nucleotide sequences. These data suggest that healthy pigs may be a source of HEV infection for consumers of pig liver and slaughterhouse workers in Brazil.