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Combinatorial RNA Interference Therapy Prevents Selection of Pre-existing HBV Variants in Human Liver Chimeric Mice.

ABSTRACT: Selection of escape mutants with mutations within the target sequence could abolish the antiviral RNA interference activity. Here, we investigated the impact of a pre-existing shRNA-resistant HBV variant on the efficacy of shRNA therapy. We previously identified a highly potent shRNA, S1, which, when delivered by an adeno-associated viral vector, effectively inhibits HBV replication in HBV transgenic mice. We applied the "PICKY" software to systemically screen the HBV genome, then used hydrodynamic transfection and HBV transgenic mice to identify additional six highly potent shRNAs. Human liver chimeric mice were infected with a mixture of wild-type and T472C HBV, a S1-resistant HBV variant, and then treated with a single or combined shRNAs. The presence of T472C mutant compromised the therapeutic efficacy of S1 and resulted in replacement of serum wild-type HBV by T472C HBV. In contrast, combinatorial therapy using S1 and P28, one of six potent shRNAs, markedly reduced titers for both wild-type and T472C HBV. Interestingly, treatment with P28 alone led to the emergence of escape mutants with mutations in the P28 target region. Our results demonstrate that combinatorial RNAi therapy can minimize the escape of resistant viral mutants in chronic HBV patients.

SUBMITTER: Shih YM

PROVIDER: S-EPMC4612501 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Combinatorial RNA Interference Therapy Prevents Selection of Pre-existing HBV Variants in Human Liver Chimeric Mice.

Shih Yao-Ming YM Sun Cheng-Pu CP Chou Hui-Hsien HH Wu Tzu-Hui TH Chen Chun-Chi CC Wu Ping-Yi PY Enya Chen Yu-Chen YC Bissig Karl-Dimiter KD Tao Mi-Hua MH

Scientific reports 20151020

Selection of escape mutants with mutations within the target sequence could abolish the antiviral RNA interference activity. Here, we investigated the impact of a pre-existing shRNA-resistant HBV variant on the efficacy of shRNA therapy. We previously identified a highly potent shRNA, S1, which, when delivered by an adeno-associated viral vector, effectively inhibits HBV replication in HBV transgenic mice. We applied the "PICKY" software to systemically screen the HBV genome, then used hydrodyna ...[more]

PMID: 26482836

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Project description:BackgroundNovel coronavirus SARS-CoV2 is evolving continuously with emergence of several variants of increasing transmission capabilities and pandemic potential. Generation of variants occurs through accumulation of mutations due to the RNA nature of viral genome, which is further enhanced by variable selection pressures of this ongoing pandemic. COVID-19 presentations of SARS-CoV2 are mainly pulmonary manifestations with or without mild gastrointestinal (GI) and hepatic symptoms. However, the virus has evolved beyond pulmonary manifestations to multisystem disorder due to systemic inflammation and cytokine storm. Definitive cause of acute or late onset of inflammation, infection in various organs, and host response to emerging variants lacks clarity and needs elucidation. Several studies have reported underlying diseases including diabetes, hypertension, obesity, cardio- and cerebrovascular disorders, and immunocompromised conditions as significant risk factors for severe form of COVID-19. Pre-existing liver and GI diseases are also highly predominant in the population, which can alter COVID-19 outcome due to altered immune status and host response. We aim to review the emerging variants of SARS-CoV2 and host response in patients with pre-existing liver and GI diseases.MethodsIn this review, we have elucidated the emergence and characteristic features of new SARS-CoV2 variants, mechanisms of infection and host immune response, GI and hepatic manifestation with radiologic features of COVID-19, and outcomes in pre-existing liver and GI diseases.Key findingsEmerging variants of concern (VOC) have shown increased transmissibility and virulence with severe COVID-19 presentation and mortality. There is a drastic swift of variants from the first wave to the next wave of infections with predominated major VOC including alpha (B.1.1.7, UK), beta (B.1.351, South Africa), gamma (B.1.1.28.1, Brazil), and delta (B1.1.617, India) variants. The mutations in the spike protein of VOC are implicated for increased receptor binding (N501Y, P681R) and immune escape (L452R, E484K/Q, T478K/R) to host response. Pre-existing liver and GI diseases not only have altered tissue expression and distribution of viral entry ACE2 receptor but also host protease TMPRSS2, which is required for both spike protein binding and cleavage to initiate infection. Altered immune status due to pre-existing conditions results in delayed virus clearance or prolonged viremia. Even though GI and hepatic manifestations of SARS-CoV2 are less severe, the detection of virus in patient's stool indicates GI tropism, replication, and shedding from the GI tract. COVID-19-induced liver injury, acute hepatic decompensation, and incidences of acute-on-chronic liver failure may change the disease outcomes.ConclusionsThe changes in the spike protein of emerging variants, immunomodulation by viral proteins, and altered expression of host viral entry receptor in pre-existing diseases are the key determinants of host response to SARS-CoV2 and its disease outcome.

Project description:BackgroundHepatitis D Virus (HDV) is classified into eight genotypes with distinct clinical outcomes. Despite the maintenance of highly conserved functional motifs, it is unknown whether sequence divergence between genotypes, such as HDV-1 and HDV-3, or viral interference mechanisms may affect co-infection in the same host and cell, thus hindering the development of HDV inter-genotypic recombinants. We aimed to investigate virological differences of HDV-1 and HDV-3 and assessed their capacity to infect and replicate within the same liver and human hepatocyte in vivo.MethodsHuman liver chimeric mice were infected with hepatitis B virus (HBV) and with one of the two HDV genotypes or with HDV-1 and HDV-3 simultaneously. In a second set of experiments, HBV-infected mice were first infected with HDV-1 and after 9 weeks with HDV-3, or vice versa. Also two distinct HDV-1 strains were used to infect mice simultaneously and sequentially. Virological parameters were determined by strain-specific qRT-PCR, RNA in situ hybridization and immunofluorescence staining.ResultsHBV/HDV co-infection studies indicated faster spreading kinetics and higher intrahepatic levels of HDV-3 compared to HDV-1. In mice that simultaneously received both HDV strains, HDV-3 became the dominant genotype. Interestingly, antigenomic HDV-1 and HDV-3 RNA were detected within the same liver but hardly within the same cell. Surprisingly, sequential super-infection experiments revealed a clear dominance of the HDV strain that was inoculated first, indicating that HDV-infected cells may acquire resistance to super-infection.ConclusionInfection with two largely divergent HDV genotypes could be established in the same liver, but rarely within the same hepatocyte. Sequential super-infection with distinct HDV genotypes and even with two HDV-1 isolates was strongly impaired, suggesting that virus interference mechanisms hamper productive replication in the same cell and hence recombination events even in a system lacking adaptive immune responses.

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Project description:Infection with the hepatitis E virus (HEV) is one of the main causes of acute hepatitis worldwide. Given that, the histopathology of hepatitis E is relatively poorly characterized, and it is unclear what exactly determines its remarkable variability. The aim of our study was a systematic analysis of hepatitis E histology, especially with regard to the clinical setting. Fifty-two liver samples (48 biopsies, 1 liver explant, 3 autopsy livers) from 41 patients with molecularly proven hepatitis E (28 HEV genotype (gt) 3, three gt 1, one gt 4 and 9 undetermined gt) were systematically evaluated for 33 histopathologic features. Following one approach, the biopsies were assigned to one of five generic histologic patterns. In another approach, they were subjected to hierarchical clustering. We found that 23/41 (56%) patients were immunocompromised, whereas 18 (44%) had no known immunosuppression. Five patients (12%) had pre-existing liver disease (LD). The histopathologic spectrum ranged from almost normal to acute, chronic, and steato-hepatitis to subtotal necrosis, and was thus distributed across all five generic patterns. Hierarchical clustering, however, identified three histopathologic clusters (C1-C3), which segregated along the immune status and pre-existing LD: C1 comprised mostly patients with pre-existing LD; histology mainly reflected the respective LD without pointing to the additional hepatitis E. C2 comprised mostly immunocompetent patients; histology mainly displayed florid hepatitis. C3 comprised mostly immunocompromised patients; histology mainly displayed smoldering hepatitis. Accordingly, C1-C3 differed markedly with respect to their clinical and histopathologic differential diagnoses. Hierarchical clustering suggests three groups with distinct histopathologies, indicating biologically different manifestations of hepatitis E. The association of histopathologic changes with the patient's immune status and pre-existing LD plausibly explains the diversity of hepatitis E histopathology, and suggests that these factors are the crucial underlying determinants. We expect our results to improve patient management by guiding the clinico-pathologic diagnosis of hepatitis E.

Dataset Information

Combinatorial RNA Interference Therapy Prevents Selection of Pre-existing HBV Variants in Human Liver Chimeric Mice.

Publications

Combinatorial RNA Interference Therapy Prevents Selection of Pre-existing HBV Variants in Human Liver Chimeric Mice.

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