A ribavirin-induced ORF2 single-nucleotide variant produces defective hepatitis E virus particles with immune decoy function

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and is the leading cause of enterically transmitted viral hepatitis worldwide. Ribavirin (RBV) is currently the only treatment option for many patients; however, cases of treatment failures or posttreatment relapses have been frequently reported. RBV therapy was shown to be associated with an increase in HEV genome heterogeneity and the emergence of distinct HEV variants. In this study, we analyzed the impact of eight patient-derived open reading frame 2 (ORF2) single-nucleotide variants (SNVs), which occurred under RBV treatment, on the replication cycle and pathogenesis of HEV. The parental HEV strain and seven ORF2 variants showed comparable levels of RNA replication in human hepatoma cells and primary human hepatocytes. However, a P79S ORF2 variant demonstrated reduced RNA copy numbers released in the supernatant and an impairment in the production of infectious particles. Biophysical and biochemical characterization revealed that this SNV caused defective, smaller HEV particles with a loss of infectiousness. Furthermore, the P79S variant displayed an altered subcellular distribution of the ORF2 protein and was able to interfere with antibody-mediated neutralization of HEV in a competition assay. In conclusion, an SNV in the HEV ORF2 could be identified that resulted in altered virus particles that were noninfectious in vitro and in vivo, but could potentially serve as immune decoys. These findings provide insights in understanding the biology of circulating HEV variants and may guide development of personalized antiviral strategies in the future.

Despite its rising global prevalence, hepatitis E is a disease that is mostly overlooked. Every year, more than 44,000 people die as a result of ∼20 million infections worldwide (1). Healthy individuals usually display no or only mild symptoms of viral hepatitis, such as fever, nausea, vomiting, and abdominal pain (2), while patients with preexisting liver disease, pregnant women, and immunocompromised individuals suffer from liver cirrhosis and liver failure (3). Pregnant women additionally present with increased mortality rates of >25% (4). Despite those liver-associated problems, there are also extrahepatic manifestations, such as hematopoietic disease, neurological disorders, and renal injury (5–9). The underlying agent, hepatitis E virus (HEV), is classed within the species of Paslahevepirus balayani (10), formerly known as Orthohepevirus A, which includes isolates from human, swine, wild boar, rat, and other mammals. HEV is a quasienveloped virus existing as both enveloped and non-enveloped particles (11, 12). To date, eight distinct genotypes (GT) of this species of the single-stranded RNA virus have been described (13), which display similar genomic structures. The positive orientated HEV genome is organized in three main open reading frames (ORF1 to ORF3) with a total length of 7.2 kb. Nonstructural proteins forming the HEV replicase complex, such as the RNA-dependent RNA polymerase (RdRp), RNA helicase, or methyltransferase, are encoded by ORF1, while the viral capsid protein is encoded by ORF2. During the HEV replication cycle, HEV produces at least three forms of ORF2 protein: infectious ORF2 (ORF2i), glycosylated ORF2 (ORF2g), and cleaved ORF2 (ORF2c) protein (14). The ORF2i protein is the structural component of infectious particles that is likely derived from the assembly of the intracellular ORF2 (ORF2intra) protein form. In contrast, ORF2g and ORF2c protein are not associated with infectious virions, but secreted in large amounts and are the most abundant antigens detected in patient sera (14). ORF3 encodes for a functional ion channel required for assembly and release of infectious particles by interacting with a variety of host factors (15).In immunocompetent patients, acute hepatitis E usually does not involve antiviral therapy; however, chronically infected and immunocompromised patients often require clinical intervention to clear the infection. Antiviral therapies include pegylated interferon (16–18), successfully implemented for many virus infections, and sofosbuvir (19, 20), a direct acting antiviral against hepatitis C virus, both of which have not yet been systematically evaluated in the context of HEV therapy. Recent studies have investigated the antiviral potential of silvestrol (21), zinc salts (22), and other possible drug candidates in vitro [reviewed in detail by Kinast et al. (23)], but the findings remain to be clinically validated. Lacking specific treatment options, the broad antiviral ribavirin (RBV) (24) is frequently used off-label. However, RBV therapy is often discontinued due to adverse side effects and is only effective in ∼80% of patients, implying that 20% of treated patients remain viremic (25). RBV treatment is specifically contraindicated in pregnant women and can give rise to variants such as G1634R, as well as other amino acid substitutions within the ORF1-encoded polyprotein, potentially contributing to treatment failure and poor clinical long-term outcomes (26–28). In this context, we recently identified viral populations of HEV harboring variations in the capsid-encoding ORF2 region during RBV therapy. With the use of an efficient HEV cell-culture model system, we characterized the impact of these ORF2 variants in the HEV replication cycle.     

Long Abductor Digiti Minimi Muscle: Variation of the Hypothenar Muscles and Clinical Consequences

Variations in the hypothenar muscles influence the susceptibility of Guyon's canal and surgical procedures in this region. The hypothenar regions of both hands of 38 human donors were dissected and the single muscles identified. A semiquantitative evaluation included 19 donors. The opponens digiti minimi and the abductor digiti minimi were constantly present. The flexor digiti minimi showed various appearances: in 58% it formed one belly, in 21% two bellies, and in 21% it was missing. Rare unilateral supernumerary muscles (2.5% in this study) were associated to the abductor digiti minimi. The variation of the flexor digiti minimi and the possible appearance of additional muscles should be recognized for hypothenar region pathologies. Clin. Anat., 33:643–645, 2020. © 2019 Wiley Periodicals, Inc.     

Distribution of p53 protein expression in gliosarcomas: an immunohistochemical study

Summary The wild-type p53 gene product is a nuclear phosphoprotein that suppresses cell and tumor growth. Mutations of the p53 gene are by now the most frequently recognized genetic alterations in human malignancies and occur in many types of carcinomas as well as in astrocytomas and sarcomas. Wild-type p53 protein has a short half-life, is present in very low quantities in normal cells and cannot be detected immunohistochemically. Mutant p53 proteins have longer half-lives and are usually present in immunohistologically detectable amounts. It is generally agreed that the presence of p53 immunostaining indicates the presence of an abnormal p53 protein and is strongly suggestive of a mutation in the p53 gene. In this study, we stained paraffin sections from eight samples of gliosarcomas from seven patients with an antibody to p53. All tumors contained p53-immunoreactive nuclei in both the glial and the sarcomatous component. In five tumors, a majority of nuclei was positive in the sarcomatous component while only a minority of nuclei was positive in the glial areas. In one tumor, the reverse was seen. In another tumor, approximately half the nuclei were positive in both components and in one tumor, only a minority of nuclei were positive in either component (this lesion was the recurrence of a tumor in which the majority of the sarcoma's nuclei had been positive). These data indicate that p53 mutations may play a role in the pathogenesis of gliosarcomas and suggest an origin of both the glial and sarcomatous components from a common progenitor.Supported by a grant from the Physicians Referral Service of the University of Texas M.D. Anderson Cancer Center. This work was presented in abstract form at the meeting of the American Association of Neuropathologists in St. Louis, June 18–21, 1992