Molecular characterisation of a rabbit Hepatitis E Virus strain detected in a chronically HEV-infected individual from Germany.

In immunocompromised individuals persisting viremia frequently leads to a chronic hepatitis E virus (HEV) infection. Zoonotic transmission of HEV from pigs and wild boar to humans is proven and sporadic infections with rabbit HEV (raHEV) have recently been reported. Here, the molecular characterisation of a raHEV strain isolated from an immunocompromised, chronically HEV-infected, heart-transplanted patient is described. After successful ribavirin (RBV) treatment of a HEV infection in 2019, the patient was again tested HEV positive in 2021 and received a second RBV therapy cycle. Full-length HEV genome amplification and next generation sequencing was performed on a plasma sample taken between first and second cycle of RBV therapy and a stool sample taken two months after starting the second cycle. The sequence of plasma (raHEV-83) and stool (raHEV-99) derived virus showed the highest nucleotide sequence identity to a Chinese raHEV and a phylogenetic relationship to a raHEV strain isolated from a French patient. Furthermore, sequence analysis revealed the presence of RBV-associated substitutions V1479I and G1634K in the HEV sequences from plasma and additionally K1398R from stool. The results underline the role of rabbits as putative sources of HEV infection and emphasize the need of a one health concept for a better understanding of HEV epidemiology and to develop tools for prevention and control of HEV infection.

Insertions and deletions in the hypervariable region of the hepatitis E virus genome in individuals with acute and chronic infection.

BACKGROUND AND AIMS: Hepatitis E virus is a major cause of acute hepatitis worldwide and can progress to chronicity in immunocompromised individuals. Various virus-host recombination events have been reported in the hypervariable region of the hepatitis E virus genome, but the patterns of assembly and selection remain unclear. METHODS: To gain further insight into viral evolution, we assessed the presence of low abundance variants in 16 samples from individuals with acute or chronic infection using a targeted next-generation sequencing approach. RESULTS: In seven samples, different variants with insertions and/or deletions were identified. Among them, eight insertions originating either from human genes or from the hepatitis E virus genome. Five different deletions could be identified. The amino acid composition of sequences with insertions showed a higher frequency of lysine and a lower abundance of proline, and additionally acetylation and ubiquitination sites were more frequent than in hepatitis E virus wild-type sequences. CONCLUSIONS: These findings suggest that the nucleotide composition of insertions and sites for post-translational modification may contribute to recombination events. Although the impact of low-level hepatitis E virus variants is uncertain, our results highlight the importance of a highly sensitive next-generation sequencing approach to capture the full diversity of hypervariable region.

Advanced sequencing approaches detected insertions of viral and human origin in the viral genome of chronic hepatitis E virus patients.

The awareness of hepatitis E virus (HEV) increased significantly in the last decade due to its unexpectedly high prevalence in high-income countries. There, infections with HEV-genotype 3 (HEV-3) are predominant which can progress to chronicity in immunocompromised individuals. Persistent infection and antiviral therapy can select HEV-3 variants; however, the spectrum and occurrence of HEV-3 variants is underreported. To gain in-depth insights into the viral population and to perform detailed characterization of viral genomes, we used a new approach combining long-range PCR with next-generation and third-generation sequencing which allowed near full-length sequencing of HEV-3 genomes. Furthermore, we developed a targeted ultra-deep sequencing approach to assess the dynamics of clinically relevant mutations in the RdRp-region and to detect insertions in the HVR-domain in the HEV genomes. Using this new approach, we not only identified several insertions of human (AHNAK, RPL18) and viral origin (RdRp-derived) in the HVR-region isolated from an exemplary sample but detected a variant containing two different insertions simultaneously (AHNAK- and RdRp-derived). This finding is the first HEV-variant recognized as such showing various insertions in the HVR-domain. Thus, this molecular approach will add incrementally to our current knowledge of the HEV-genome organization and pathogenesis in chronic hepatitis E.

Detection of Q129H Immune Escape Mutation in Apparently Healthy Hepatitis B Virus Carriers in Southwestern Nigeria.

As the global effort to eradicate hepatitis B continues, immune escape mutations (IEMs) and drug resistance mutations (DRMs) affecting its diagnosis, treatment, and prevention are compromising this goal. However, knowledge about the prevalence and circulation of these mutations in Nigeria is scarce. Serum samples (n = 199) from apparently healthy prospective blood donors, pregnant women, and individuals presenting with fever in southwestern Nigeria were analyzed for the presence of IEMs and DRMs by means of nested PCR in the HBV S (HBs) and HBV polymerase (Pol) genes, followed by phylogenetic and mutational analyses. In total, 25.1% (n = 50/199) of samples were positive for HBV, as measured by PCR. In 41 samples (20.6%), both fragments could be amplified, whereas the HBs gene and the Pol gene fragment alone were detected in 0.5% (n = 1/199) and 4% (n = 8/199) of samples, respectively. Sequences were successfully obtained for all 42 HBs gene fragments but for only 31/49 Pol gene fragments (totaling 73 sequences from 44 individuals). All sequences were identified as HBV genotype E. IEMs were present in 18.2% (n = 8/44) of the sequences of HBV-positive individuals with available sequences. IEM Q129H was detected in eight out of the 44 (18.2%) HBV isolates sequenced in this study; however, no DRMs were observed. This study confirms the circulation of HBV IEMs and reports the presence of Q129H IEM for the first time in Nigeria. Intensified research on the dynamics of IEM is necessary in order to enhance the elimination of HBV.

Identification of a Putative Novel Genotype of Avian Hepatitis E Virus from Apparently Healthy Chickens in Southwestern Nigeria.

Avian hepatitis E virus (aHEV) is the major etiological agent of hepatitis-splenomegaly syndrome (HSS), big liver and spleen disease (BLSD), and hepatic rupture hemorrhage syndrome (HRHS) in chickens. Infections with aHEV cause a significant decrease in egg production and increased mortality in chickens worldwide. However, studies on the prevalence of aHEV in Nigeria are scarce. In this study, serum (n = 88) and fecal samples (n = 110) obtained from apparently healthy layer chickens from three states in southwestern Nigeria were analyzed by nested reverse transcription-polymerase chain reaction (nRT-PCR) targeting the helicase and capsid gene for the presence of aHEV. Avian HEV was detected in 12.5% (n = 11/88) of serum samples and 9.1% (n = 10/110) of fecal samples tested. Phylogenetic analysis showed that five of the twelve identified aHEV sequences belonged to genotype 2. The remaining seven sequences were only distantly related to other known aHEV isolates. After amplification of the near-complete ORF2 fragment (1618 bp) and part of the ORF1 (582 bp) of isolate YF40_aHEV_NG phylogenetic analysis revealed a nucleotide sequence identity between 79.0 and 82.6% and 80.1 and 83.5%, respectively, to other known aHEV strains, indicating that the Nigerian isolate YF40_aHEV_NG belongs to a novel aHEV genotype. This is the first report of co-circulation of aHEV genotypes in chickens in Nigeria.

Hepatitis E virus infection in high-risk populations in Osun State, Nigeria.

Hepatitis E virus (HEV) infection is an emerging infection that is of major public health concern, especially in some vulnerable groups like immunosuppressed individuals, pregnant women and HBV-coinfected individuals. HEV is transmitted faecal/oral or zoonotically depending on the HEV-genotype. This study aimed at investigating HEV infections among different at-risk populations in Osun State, Southwestern Nigeria. A total of 720 serum samples were collected from animal handlers, pregnant women, people living with HIV/AIDS, and Hepatitis B virus (HBV) infected individuals. Commercially available Enzyme-Linked Immunosorbent Assays (ELISA) were used for the detection of anti-HEV total and IgM antibodies. Polymerase chain reaction (PCR) was carried out in the HEV seropositive samples and all the samples from individuals infected with HBV. Descriptive analysis and chi-square test of association were performed. The anti-HEV total antibody seroprevalence in HIV-positive individuals, animal handlers and pregnant women was 11.4% (n = 47/411), 7.9% (n = 7/89), and 6.3% (n = 10/160), respectively. Markers of acute HEV infection (anti-HEV IgM) were detected in 2.2% of HIV-positive individuals (n = 9/411) and 1.8% of animal handlers (n = 2/89), respectively, and in 0.6% of pregnant women (n = 1/160). However, all samples were HEV RNA negative. This study analysed the presence of markers of HEV infection among different at-risk populations without clinical symptoms of HEV infection. Our results showed that HEV is an underestimated threat to public health in Nigeria and underlines the need of an HEV surveillance system to understand the distribution and transmission of HEV infection in animals and/to humans.

Serological evidence of avian HEV antibodies in apparently healthy chickens in southwest Nigeria.

Avian hepatitis E virus (aHEV) is associated with hepatitis-splenomegaly syndrome, big liver and spleen disease and hepatic rupture haemorrhage syndrome. However, the knowledge about aHEV in commercial layer chickens in Nigeria is scarce. In this study, 460 serum samples obtained from 36 apparently healthy commercial layer chicken flocks in three states (Ogun, Osun and Oyo States) of southwestern Nigeria were analysed by enzyme linked immunosorbent assay for the presence of anti-aHEV immunoglobulin Y (IgY) antibodies. In total, the overall seroprevalence of anti-aHEV antibodies was 14.6%. The serological analysis revealed that 75% of the flocks examined were positive for anti-aHEV IgY antibodies from chickens of various ages in all three states. The percentage of the seropositive chickens in the three states varied from flock to flock ranging from 60% to 88.8% and seropositive chickens were detected at any age (24-52 weeks of age) without significant differences between the age groups. This is the first report assessing the presence of aHEV antibodies in chickens from Nigeria. The detection of anti-aHEV antibodies in commercial layer chickens in this study emphasizes the importance of serosurveillance in disease monitoring due to the economic threat posed by aHEV as a result of decreased egg production and increased mortality in affected commercial layer chicken farms. However, further studies are essential to reveal the clinical implications and to assess the real burden of aHEV in Nigeria.

Prevalence and characteristics of hepatitis B and D virus infections among HIV-positive individuals in Southwestern Nigeria.

BACKGROUND: Coinfections of HIV-positive individuals with Hepatitis B and D virus (HBV and HDV) are common and can be associated with rapid liver damage. Several antiretroviral drugs for HIV exhibit anti-HBV effect; however, the selection of HBV drug resistance mutations (DRMs) in individuals under HIV antiretroviral therapy (ART) has been reported but rarely in Nigeria. In this study the HBV/HDV prevalence and HBV DRMs in HIV-positive individuals in Southwestern Nigeria were assessed. METHODS: Plasma samples collected from 310 HIV-positive individuals including 295 ART-experienced and 15 ART-naïve persons attending the HIV clinic in three south-western states of Nigeria between June 2017 and August 2017 were analysed by ELISA for HBsAg and anti-HDV. The presence of HDV RNA and HBV DNA was analysed by (RT)-PCR followed by sequencing and phylogenetic analyses for genotyping. The HBV reverse transcription (RT) region was amplified and sequenced for the analysis of drug resistance mutations. RESULTS: Overall, 16.1% (n = 50/310) of the HIV-positive individuals were positive for HBsAg, most of which were ART-experienced (94.0%; n = 47/50). From the 50 HBsAg-positive samples, 72.0% (n = 36/50) were positive for HBV DNA and 16.0% (n = 8/50) had detectable HDV RNA while 5.6% (n = 2/36) of the HBV-DNA positive samples had anti-HDV total antibodies. Sequences were available for 31/36 of the HBV DNA-positive and 3/8 HDV RNA-positive samples. HBV DNA-positive samples were characterised as HBV genotype E infections exclusively, while HDV genotype 1 was detected in the HDV RNA-positive samples. HBV DRMs V173L, L180M, S202I and M204V/I, which are associated with lamivudine resistance, were detected in 32.2% (n = 10/31) of the HBV DNA-positive samples. Most of these mutations (90.0%; n = 9/10) were present in the ART-experienced cohort. CONCLUSIONS: This study indicates that HBV/HDV coinfections are common in HIV-positive individuals under ART in Nigeria. Furthermore, a high proportion of HBV DRMs which potentially compromise future treatment options were detected, underscoring the need for HBV screening prior to starting ART. Further studies should be performed to monitor a possible increase in the spread of HDV among populations at risk of HIV and HBV infections.

A Recent Human Immunodeficiency Virus Outbreak Among People Who Inject Drugs in Munich, Germany, Is Associated With Consumption of Synthetic Cathinones.

BACKGROUND: Needle and syringe sharing among people who inject drugs (PWID) can result in a rapid regional spread of a human immunodeficiency virus (HIV) variant. Such outbreaks have been identified recently in several countries and have raised public health attention because of an association with new psychoactive substances (NPS). METHODS: Dried serum spots from approximately 60% of newly diagnosed HIV cases in Germany in 2013-2018 were received together with statutory notification data. Samples were sequenced in the pol-region, genotyped, and viral phylogenies were analyzed. For selected samples, the hepatitis C virus (HCV) status and the presence of NPS were determined. RESULTS: An outbreak of closely related 27 subtype C infections with a core of 11 cases with almost identical sequences was identified using phylogenetic analyses. The first case of the outbreak was diagnosed in 2015, and the last one was in 2018. With exception of 3 infections, all were reported from Munich, the capital of the federal state of Bavaria. Of 26 analyzed outbreak members, 24 (92.3%) had a resolved or viremic HCV coinfection. In 8 of 18 (44%) cases, α-pyrrolidinopentiothiophenone and/or the related substance α-pyrrolidinoheptiophenone was identified. CONCLUSIONS: Despite harm reduction services in place, HIV outbreaks of considerable size can occur in PWID. The establishment of a real-time molecular surveillance is advised to rapidly identify outbreaks and target prevention measures.

Hepatitis E Virus Seroprevalence and Associated Risk Factors in Apparently Healthy Individuals from Osun State, Nigeria.

Hepatitis E virus (HEV) infection is a major public health concern in low-income countries, yet incidence and prevalence estimates are often lacking. Serum (n = 653) and faecal (n = 150) samples were collected from apparently healthy individuals using convenience sampling technique in six communities (Ore, Oke-Osun, Osogbo, Ede, Esa-Odo, and Iperindo) from Osun State, Nigeria. Serum samples were analysed for total anti-HEV IgG/IgM and anti-HEV IgM using commercially available HEV ELISA kits. Total anti-HEV positive serum and all stool samples were analysed for HEV RNA by RT-PCR. Overall, 15.0% (n = 98/653) and 3.8% (n = 25/653) of the serum samples were positive for anti-HEV total and IgM antibodies, respectively. Total anti-HEV and IgM in Ore, Oke-Osun, Osogbo, Ede, Esa-Odo, and Iperindo was 21.0% (n = 13/62) and 3.2% (n = 2/62), 19.4% (n = 20/103) and 6.8% (n = 7/103), 11.4% (n = 12/105) and 2.9% (n = 3/105), 8.0% (n = 16/199) and 1.5% (n = 3/199), 22.0% (n = 22/100) and 10.0% (n = 10/100), and 17.9% (n = 15/84) and 0.0% (n = 0/84), respectively. All samples (stool and serum) were HEV RNA negative. Anti-HEV seroprevalence was associated with rural location, increasing age, alcohol consumption, and rearing of animals. This study demonstrated a high anti-HEV seroprevalence in Osun State, indicating the need to implement surveillance and asses the hepatitis E burden in Nigeria.