Hepatitis E virus infections among patients with acute febrile jaundice in two regions of Cameroon: First molecular characterization of hepatitis E virus genotype 4.

BACKGROUND: Febrile jaundice is a common indicator of certain infectious diseases, including hepatitis E. In Cameroon, the yellow fever virus is the only pathogen that is monitored in patients who present with this symptom. However, more than 90% of the samples received as part of this surveillance are negative for yellow fever. This study aimed to describe the prevalence and hepatitis E virus (HEV) genotype among yellow fever-negative patients in the Far North and West regions of Cameroon. METHODS: In a cross-sectional study, yellow fever surveillance-negative samples collected between January 2021 and January 2023 were retrospectively analyzed. Anti-HEV IgM and IgG antibodies were tested using commercially available ELISA kits. Anti-HEV IgM and/or IgG positive samples were tested for HEV RNA by real-time RT-PCR, followed by nested RT-PCR, sequencing and phylogenetic analysis. RESULTS: Overall, 121 of the 543 samples (22.3%, 95% CI: 19.0% - 26.0%) were positive for at least one anti-HEV marker. Amongst these, 8.1% (44/543) were positive for anti-HEV IgM, 5.9% (32/543) for anti-HEV IgG, and 8.3% (45/544) for both markers. A total of 15.2% (12/79) samples were positive for HEV RNA real-time RT-PCR and 8 samples were positive for HEV RNA by nested RT-PCR. Phylogenetic analysis showed that the retrieved sequences clustered within HEV genotypes/subtypes 1/1e, 3/3f and 4/4b. CONCLUSION: Our results showed that HEV is one of the causes of acute febrile jaundice in patients enrolled in the yellow fever surveillance program in two regions of Cameroon. We described the circulation of three HEV genotypes, including two zoonotic genotypes. Further studies will be important to elucidate the transmission routes of these zoonotic HEV genotypes to humans in Cameroon.

Detection of Hepatitis E Virus in Rabbits and Rabbit Meat from Slaughterhouses in Hebei Province of China.

Background: Hepatitis E virus (HEV) is a zoonotic pathogen. HEV has been found to be widely prevalent in rabbits. Its isolates are classified into HEV-3, rabbit subgenotype (HEV-3ra). The routes of human infection with HEV-3ra remain unclear; however, foodborne transmission is possible when asymptomatically infected animals enter the food chain. The prevalence of HEV infection in slaughtered rabbits and the presence of HEV in rabbit meat were evaluated in this study. Materials and Methods: In three slaughterhouses in Hebei province, China, samples of rabbit blood were collected during the slaughter process, and muscle, liver, and cavity juice were collected from the rabbit carcasses. Anti-HEV antibody in serum samples was detected using enzyme-linked immunosorbent assay. HEV RNA was tested in all samples by reverse transcription nested PCR (RT-nested PCR). The final amplicons of RT-nested PCR were sequenced and phylogenetically analyzed. Results: Of the 459 serum samples, 50 [10.9%, 95% confidence interval (CI): 8.1-13.7] were positive for anti-HEV antibody, and 17 (3.7%, 95% CI: 2.0-5.4) were positive for HEV RNA. HEV RNA was detected in 7 of 60 liver samples (11.7%, 95% CI: 3.3-20) and 2 cavity juice samples from semi-eviscerated carcasses, but was not detected in any muscle sample from either the eviscerated or semi-eviscerated carcasses. All the detected HEV strains belonged to HEV-3ra and related most closely with the rabbit HEV sequence previously reported in China. Conclusion: A portion of rabbits were in the viremia period of HEV infection at the slaughter age, resulting in the possibility of HEV carriage by rabbit carcass, particularly semi-eviscerated carcass containing liver. These findings suggest a potential risk of HEV transmission from raw rabbit products entering the food chain, whereas the presence of HEV appeared to be lower in the eviscerated carcass than in the semi-eviscerated carcass.

Genotype 4 HEV infection triggers the initiation and development of acute pancreatitis.

The role of HEV infection in AP remains unclear. 1000 patients with AP and 1000 HCs were enrolled, and pancreatitis was evaluated in HEV-infected rhesus macaques. The positive rates of anti-HEV IgG, IgM, and HEV RNA in the AP patients were significantly higher than HCs. With the increase in the severity of AP, the percentage of HEV infection increased. AP patients were divided into AP- and AP + AHE groups. The percentage of severe AP in the AP + AHE group was significantly higher than in the AP- group. HEV infection was one of the main independent risk factors and had high predictive power for AP outcomes. A high level of HEV titer would prolong the recovery time and increase the risk of recurrent AP. Moreover, AP + AHE patients receiving conservative treatment showed a better prognosis. Furthermore, HEV can replicate in the pancreas of rhesus macaques. The pancreatic islet structure was damaged, the tissue was loose after 272 dpi, and a large amount of hyperemia appeared after 770 dpi. HEV infection also caused a large number of inflammatory cells in the pancreas. The pancreas and liver had a comparable viral load. HEV infection affects AP's occurrence, development, and prognosis.

A surveillance study of hepatitis E virus infection in household cats.

Hepatitis E virus (HEV) typically causes self-limiting acute viral hepatitis, however chronic infection and extrahepatic manifestations have increasingly become a significant health problem. Domestic pigs and wild boars are the main reservoirs of HEV genotype 3 and genotype 4 for human infections in industrialized countries, although molecular and serological evidence suggest that several additional animal species may act as HEV hosts. In this study, by assessing serologically and molecularly the sera of 324 household cats from Apulia region (Italy), HEV antibodies were detected with an overall prevalence of 3.1%. Viral RNA was not detected in the sera of the animals using both HEV-specific assays and a pan-hepevirus broadly reactive set of primers for Hepeviridae. These findings document a low seroprevalence to HEV in cats in the investigated geographical setting. The exact nature of the HEV-like strains circulating in feline population remains to be established.

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.

Hepatitis E seroprevalence and associated risk factors in Croatian liver transplant recipients

Abstract INTRODUCTION Solid-organ transplant recipients are at risk of hepatitis E virus (HEV) infection. We analyzed the seroprevalence/risk factors of HEV in Croatian liver transplant recipients. METHODS Two hundred forty-two serum samples were tested for HEV immunoglobuline IgG/IgM and HEV RNA. Sociodemographic data and risk factors were collected using a questionnaire. RESULTS HEV IgG seroprevalence rate was 24.4%. Positive/equivocal HEV IgM were found in two patients. HEV RNA was not detected. Logistic regression showed that older age, female gender, rural area/farm, water well, and septic tank were associated with HEV seropositivity. CONCLUSIONS This study revealed a high exposure rate to HEV in Croatian liver recipients.

Backyard pigs are a reservoir of zoonotic hepatitis E virus in southern Brazil.

Background: Hepatitis E virus (HEV) is the causative agent of acute hepatitis worldwide. There is no seroprevalence study in backyard farms, which are characterized by suboptimal hygienic conditions in Brazil. We aimed to determine the seroprevalence and genetic diversity of HEV in backyard pigs in Brazil. Methods: Swine serum samples collected in 2012 (n=731) and 2014 (n=713) were analysed. The presence of anti-HEV immunoglobulin G in pig serum was evaluated by indirect enzyme-linked immunosorbent assay. Reverse transcription polymerase chain reaction was performed and phylogenetic analyses were carried out based on the partial ORF1 and ORF2 coding regions. Results: Anti-HEV antibodies were detected in 77.6% (567/731; 95% confidence interval [CI] 74.5 to 90.6%) of serum samples in 2012 and 65.5% (467/713; 95% CI 62.0 to 69.0%) in 2014. The herd seroprevalence was 91.7% (187/204; 95% CI 91% to 99%) in 2012 and 83.7% (164/196; 95% CI 78% to 89%) in 2014. Further, HEV RNA was detected in 0.8% (6/713) of samples from 2014. Phylogenetic analysis showed three different genotype 3 subtypes with high similarity to human HEV strains. Conclusions: This study showed that backyard pigs are a reservoir of HEV and alerts us to the need to control infection and spillover from backyard farms. GenBank accession numbers: MF438128-MF438135.

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.

Silent hepatitis E virus infection in Dutch blood donors, 2011 to 2012.

In Europe, the dynamics of endemic hepatitis E virus (HEV) infection remain enigmatic. We studied the presence of silent HEV infection among Dutch blood donors. Using donations collected throughout the Netherlands in 2011 and 2012, 40,176 donations were tested for HEV RNA in 459 pools of 48 or 480 donations. Deconstruction of the reactive pools identified 13 viraemic donors. In addition, 5,239 donors were tested for presence of anti-HEV IgG and IgM and for HEV RNA when IgM-positive. Of the 5,239 donations, 1,401 (27%) tested repeat-positive for HEV IgG, of which 49 (3.5%) also tested positive for anti-HEV IgM. Four of the HEV IgM-positive donors tested positive for HEV RNA. HEV IgG seroprevalence ranged from 13% among donors younger than 30 years to 43% in donors older than 60 years. The finding of 17 HEV RNA-positive donations among 45,415 donations corresponds to one HEV-positive blood donation per day in the Netherlands. For 16 of the 17 HEV RNA-positive donors, genotyping succeeded, revealing HEV genotype 3, which is circulating among Dutch pigs. Apparently, silent HEV infection is common in the Netherlands, which possibly applies to larger parts of Europe.

Prevalence and clinical significance of hepatitis D virus co-infection in patients with chronic hepatitis B in Korea.

Hepatitis D virus (HDV) infection can cause severe acute and chronic liver disease in patients infected with hepatitis B virus (HBV). Despite the significant decline in the global HDV infection, it remains a major health concern in some countries. This study aimed to investigate the prevalence and clinical features of HDV co-infection in patients with chronic HBV infection in Korea, where HBV infection is endemic. Nine hundred forty patients [median age, 48 (18-94) years; men, 64.5%] infected chronically with HBV were enrolled consecutively. All patients who were positive for hepatitis B surface antigen (HBsAg) for at least 6 months and were tested for anti-HDV. A portion of the HDV delta antigen was amplified, sequenced, and subjected to molecular and phylogenetic analysis using sera from the patients who were anti-HDV positive. Clinical features and virologic markers were evaluated. Inactive HBsAg carriers, chronic hepatitis B, cirrhosis and hepatocellular carcinoma accounted for 29.5%, 44.7%, 17.9%, and 8.0%, respectively. Only three patients were positive for anti-HDV, corresponding to a 0.32% positive rate. All patients who were positive for anti-HDV were inactive HBsAg carriers. HDV RNA could be amplified by PCR from the sera of two patients. Phylogenetic analysis showed that both carried HDV genotype 1. In conclusion, the prevalence of HDV infection is very low (0.32%) in Korea. All HDVs were genotype 1 and detected in inactive HBsAg carriers. Therefore, HDV co-infection may not have a significant clinical impact in Korean patients with chronic HBV infection.

[Optimization of human anti-HBsAg scFv secretary expression in Escherichia coli].

OBJECTIVE: To explore the conditions for high expression of anti-HBsAg scFv A-15 in E. coli, increase the production of the scFv in the culture medium. METHODS: By changing induction occasion, concentration of inductor IPTG and induction time, influence of various conditions on expression of anti-HBsAg scFv A-15 was analyzed through ELISA. In addition, the effects of sucrose, glycine and Triton X-100 at different concentrations on the scFv excretion into culture medium was evaluation. RESULTS: The optimal expression conditions were as follows: the induction was started after culturing for 4 h, the concentration of IPTG was 0.5 mmol/L, and the induction lasted for 8 h. The scFv affinity in culture medium with 0.3 mol/L sucrose, 2% glycine, 1% Triton X-100, 16.78-fold higher, respectively than that without the three chemicals. The final yield of anti-HBsAg scFv A-15 was estimated to be 7.4 mg/L. CONCLUSION: The conditions for production of anti-HBsAg scFv A-15 were optimized, which provides a practical method for more efficient production of the scFv in E. coli for further studying structure and function.

Epidemiological study of hepatitis E virus infection in the general population of Okinawa, Kyushu, Japan.

BACKGROUND AND AIM: The aim of this study was to investigate the prevalence of hepatitis E virus (HEV) infection in the general population of Japan by determining presence of the antibody to HEV (anti-HEV). METHODS: The prevalence of HEV infection was determined by positivity of serum antibody to HEV (anti-HEV). RESULTS: On retrospective analysis, a significant decrease in anti-HEV prevalence was found in Okinawa healthy residents from 1995 (15.8%) to 2005 (5.5%) (P < 0.0001). In 2005, the anti-HEV prevalence was significantly higher in Okinawa wild boar hunters (25.3%) than in the residents (male 7.7% and female 4.1%) (P < 0.0001). A significant difference was found in the history of consumption of undercooked or raw boar meat between anti-HEV positive and negative hunters (100% vs 64.3%) (P = 0.0018). CONCLUSIONS: In conclusion, the anti-HEV prevalence has decreased in the residents of this area, but HEV infection has continued at a high rate in the hunters through the custom of eating undercooked or raw boar meat.

[Construction and screening of hepatitis E virus-specific phage antibody combinatorial library].

AIM: To construct HEV-specific phage combinatorial anti-body library and screen anti-HEV antibodies with neutralizing activity from the library. METHODS: The total RNA was extracted from B-lymphocytes of 6 HE patients. Kappa chain and Fd segment of IgG gene were amplified respectively by RT-PCR using a set of Fab-specific primers. The amplified gene were inserted successively into vector pComb3 and electrotransformed E. coli XLI-Blue cells. Furthermore, the recombinant phage was rescued by being concultured with helper phage VCSM13 to construct HEV-specific phage anti-body library. RESULTS: Fab displayed on the surface a as fusion protein with the N terminal of coat protein III, and 1. 8 x 10(7) clone library was established. Specific antibodies to HEV ORF2 recombinant antigen were acquired after five rounds of panning with HEV ORF2 recombinant antigen including neutralizing epitope. CONCLUSION: Four clones exhibited specific binding to HEV ORF2 recombinant antigen including neutralizing epitope is identified by ELISA. The results show that we have got the recombinant phage antibodies.

Epidemiology of GB virus c/hepatitis g virus infection in patients on peritoneal dialysis.

BACKGROUND: A new genus in the family Flaviviridae has recently been discovered; it has provisionally been designated GBV-C/HGV. As determined by virologic techniques [reverse-transcription polymerase chain reaction (RT-PCR)], infection with GBV-C/HGV is frequent in renal transplant (RT) recipients and in patients on chronic hemodialysis (HD). The epidemiology of GBV-C/HGV infection in patients on peritoneal dialysis is scarce and mostly based on RT-PCR technology. PURPOSE: We report on the prevalence (as detected by serologic and virologic techniques) and the risk factors for GBV-C/HGV infection in a cohort of patients on continuous ambulatory peritoneal dialysis (CAPD). We also tested a control group of blood donors. METHODS: Infection by GBV-C/HGV was assessed by serologic and virologic techniques. Cases of GBV-C/HGV viremia (GBV-C/HGV RNA) were detected by RT-PCR. Antibodies to the envelope protein of GBV-C/HGV (anti-E2 GBV-C/HGV antibody) were analyzed by serologic methods. RESULTS: We found a high frequency [17/85 (20%)] of GBV-C/HGV. The rates of GBV-C/HGV viremia and anti-E2 GBV-C/HGV positivity were 10.5% (9/85) and 10.5% (9/85) respectively. In most patients [17/18 (94%)], the presence of anti-E2 GBV-C/HGV antibody was associated with clearance of GBV-C/HGV from serum. No relationship was noted between anti-E2 GBV-C/HGV antibody (or GBV-C/ HGV viremia) and age, sex, race, time on dialysis, anti-HCV antibody, HBsAg status, and anti-HIV positivity. The frequency of GBV-C/HGV infection in CAPD patients was much higher than that in blood donors, even if the difference did not approach statistical significance. No associations between GBV-C/HGV positivity and biochemical liver tests [aminotransferase and gamma glutamyl transpeptidase (GGT)] were apparent. CONCLUSIONS: Infection by GBV-C/HGV as detected by RT-PCR and anti-E2 antibody was common in patients on CAPD and in controls alike. No association was seen between GBV-C/HGV and various demographic or clinical factors. The clinical significance of GBV-C/HGV in CAPD remains unclear. Larger investigations are in progress.

Four chimpanzee monoclonal antibodies isolated by phage display neutralize hepatitis a virus.

Chimpanzee immunoglobulins are virtually identical to human immunoglobulins and may have clinically useful applications. Four chimpanzee monoclonal antibodies (MAbs) to the hepatitis A virus (HAV) capsid were isolated from a combinatorial cDNA library of gamma1/kappa antibody genes using phage display. Competition assays indicated that three of the MAbs recognized the same or overlapping epitopes, whereas the fourth recognized a different, nonoverlapping epitope on the HAV capsid. All four MAbs neutralized the homologous HAV strain, HM-175, in a radioimmunofocus assay and two of the four MAbs neutralized a heterologous simian HAV strain, AGM-27. From these data, we conclude that the MAbs must recognize at least three epitopes on the HAV capsid. Furthermore, competition assays performed with neutralizing murine MAbs suggested that three of the chimpanzee MAbs recognized epitopes on the HAV capsid which have not been defined previously.

[Selection of human phage antibodies against HAV and DNA sequence analysis].

OBJECTIVE: To obtain the anti-HAV antibodies. METHODS: The antibody-capturing antigen strategy was used in panning antibody library with impure antigen. The HAV-binding activity and specificity of phage antibodies were assayed by sandwich ELISA and competitive inhibition ELISA. RESULTS: Phage antibodies(Fab) against HAV were screened from a human immunoglobulin combinatorial library. Fd and light chain genes were sequenced. Comparison of the Fd gene and light chain gene with KABAT database showed that the heavy chain belonged to IgG1 subclass, and its variable region was derived from rearranged germ-line gene of DP88, D3-3 and JH5; while the light chain was a member of V kappa III family, the germ-line gene was DP kappa 22 and J kappa 4. CONCLUSION: Phage antibodies against HAV were selected from the antibody library by using the antibody-capturing antigen strategy. The result also suggested that the antibody library was constructed successfully.

[Dynamics of anti-HEV ORF2, ORF3, IgM and IgG in serial sera of patients with hepatitis E and their clinical significance].

OBJECTIVE: To study the dynamics of anti-HEV ORF2, ORF3, IgM and IgG in serial sera of patients with hepatitis E. METHODS: Anti-HEV was detected in serial sera of 52 patients, by an enzyme-linked immunosorbent assay (EIA) based on two synthetic peptides separated or combined from open reading frame (ORF) 2 and 3 of HEV genome. RESULTS: Both anti-ORF2 and anti-ORF3 in serum were at the highest levels in early acute phase of the disease, and declined gradually with the course, especially anti-ORF3. The sensitivity of the EIA using both ORF2 and ORF3 peptides was higher than that of either alone. The positive rates of anti-HEV IgM and anti-HEV IgG were separately 71.1% (32/45) and 97.8% (44/45) within 15 days after onset, then decreased more dramatically in IgM than in IgG with the course. CONCLUSION: Both ORF2 and ORF3 proteins should be used for the development of anti-HEV EIA kit. It seems to be more specific in anti-HEV IgM and more sensitive in anti-HEV IgG as a diagnostic marker of hepatitis E.

Selection of human anti-HAV McAb from a phage antibody library.

The phage displaying antibody fragments were subjected to three rounds of panning with hepatitis A virus (HAV) antigen in solid phase. The eluted phage was enriched nearly 100 fold, and the percentage of recombinant clones increased from 25% to 100% after three rounds of panning. The HAV antigen-specific monoclonal antibody (McAb) was screened by sandwich ELISA, and the specificity of these antibodies was further confirmed by competitive inhibition ELISA.

Genotype-dependent serologic reactivities in patients infected with hepatitis C virus in the United States.

OBJECTIVE: To evaluate the serologic reactivities in patients infected with different hepatitis C virus (HCV) genotypes to four HCV proteins that are components of the second-generation recombinant immunoblot assay. MATERIAL AND METHODS: Serum samples from 36 patients with chronic HCV infection were obtained. RNA was extracted by using chaotropic lysis and isopropanol precipitation. Reverse-transcriptase polymerase chain reaction of the NS-5 region was performed, followed by automated single-pass dideoxy sequencing of desalted amplification products. Classification of isolated HCV subtypes was based on Simmonds' system. All samples were tested for antibodies to proteins 5-1-1, C100-3, C33c, and C22-3 with the second-generation recombinant immunoblot assay. RESULTS: Reactivity to protein 5-1-1 was significantly lower for patients with genotypes 2b and 3a than for those infected with HCV types 1a or 1b (P < 0.05). Antibody reactivity to the C100-3 protein was also reduced in patients infected with HCV types 2b and 3a. CONCLUSION: These data indicate that the genotype-dependent differences in serologic reactivities are substantial among patients with chronic HCV infection.