Virology
Abstract
The recent emergence of the SARS-CoV-2 Omicron variant of concern (VOC) containing a heavily mutated spike protein capable of escaping preexisting immunity identifies a continued need for interventional measures. Molnupiravir (MK-4482), an orally administered nucleoside analog, has demonstrated efficacy against earlier SARS-CoV-2 lineages and was recently approved for SARS-CoV-2 infections in high-risk adults. Here we assessed the efficacy of MK-4482 against the earlier Alpha, Beta and Delta VOCs and Omicron in the hamster COVID-19 model. Omicron replication and associated lung disease in vehicle treated hamsters was reduced compared to the earlier VOCs. MK-4482 treatment inhibited virus replication in the lungs of Alpha, Beta and Delta VOC infected hamsters. Importantly, MK-4482 profoundly inhibited virus replication in the upper and lower respiratory tract of hamsters infected with the Omicron VOC. Consistent with its mutagenic mechanism, MK-4482 treatment had a more pronounced inhibitory effect on infectious titers compared to viral RNA genome load. Histopathologic analysis showed that MK-4482 treatment caused a concomitant reduction in the level of lung disease and viral antigen load in infected hamsters across all VOCs examined. Together, our data indicate the potential of MK-4482 as an effective antiviral against known SARS-CoV-2 VOCs, especially Omicron, and likely future SARS-CoV-2 variants.
Authors
Kyle Rosenke, Atsushi Okumura, Matthew C. Lewis, Friederike Feldmann, Kimberly Meade-White, William F. Bohler, Amanda J. Griffin, Rebecca Rosenke, Carl Shaia, Michael A. Jarvis, Heinz Feldmann
Abstract
A major challenge in managing acute viral infections is ameliorating disease when treatment is delayed. Previously, we reported the success of a two-pronged monoclonal antibody (mAb) and antiviral remdesivir therapeutic approach to treat advanced illness in Marburg virus (MARV)-infected rhesus monkeys. Here, we explored the benefit of a similar combination therapy for Sudan ebolavirus (SUDV) infection. Importantly, no licensed anti-SUDV therapeutics currently exist, and infection of rhesus macaques with SUDV results in a rapid disease course similar to MARV with a mean time-to-death of 8.3 days. When initiation of therapy with either remdesivir or a pan ebolavirus mAb cocktail (MBP431) was delayed until 6 days post inoculation (dpi), only 20% of macaques survived. In contrast, when remdesivir and MBP431 treatment were combined beginning 6 dpi, significant protection (80%) was achieved. Our results suggest that combination therapy may be a viable treatment for patients with advanced filovirus disease that warrants further clinical testing in future outbreaks.
Authors
Robert W. Cross, Zachary A. Bornholdt, Abhishek N. Prasad, Courtney Woolsey, Viktoriya Borisevich, Krystle N. Agans, Daniel J. Deer, Dafna M. Abelson, Do H. Kim, William S. Shestowsky, Lioudmila A. Campbell, Elaine Bunyan, Joan B. Geisbert, Natalie S. Dobias, Karla A. Fenton, Danielle P. Porter, Larry Zeitlin, Thomas W. Geisbert
Abstract
Congenital cytomegalovirus (cCMV) infection is the leading infectious cause of neurodevelopmental disorders. However, the neuropathogenesis remains largely elusive due to a lack of informative animal models. In this study, we developed a congenital murine CMV (cMCMV) infection mouse model with high survival rate and long survival period that allowed long-term follow-up study of neurodevelopmental disorders. This model involves in utero intracranial injection and mimics many reported clinical manifestations of cCMV infection in infants, including growth restriction, hearing loss, and impaired cognitive and learning-memory abilities. We observed that abnormalities in MRI/CT neuroimaging were consistent with brain hemorrhage and loss of brain parenchyma, which was confirmed by pathological analysis. Neuropathological findings included ventriculomegaly and cortical atrophy associated with impaired proliferation and migration of neural progenitor cells in the developing brain at both embryonic and postnatal stages. Robust inflammatory responses during infection were shown by elevated inflammatory cytokine levels, leukocyte infiltration, and activation of microglia and astrocytes in the brain. Pathological analyses and CT neuroimaging revealed brain calcifications induced by cMCMV infection and cell death via pyroptosis. Furthermore, antiviral treatment with ganciclovir significantly improved neurological functions and mitigated brain damage as shown by CT neuroimaging. These results demonstrate that this model is suitable for investigation of mechanisms of infection-induced brain damage and long-term studies of neurodevelopmental disorders, including the development of interventions to limit CNS damage associated with cCMV infection.
Authors
Yue-Peng Zhou, Meng-Jie Mei, Xian-Zhang Wang, Sheng-Nan Huang, Lin Chen, Ming Zhang, Xin-Yan Li, Hai-Bin Qin, Xiao Dong, Shuang Cheng, Le Wen, Bo Yang, Xue-Fang An, Ao-Di He, Bing Zhang, Wen-Bo Zeng, Xiao-Jun Li, Youming Lu, Hong-Chuang Li, Haidong Li, Wei-Guo Zou, Alec J. Redwood, Simon Rayner, Han Cheng, Michael A. McVoy, Qiyi Tang, William J. Britt, Xin Zhou, Xuan Jiang, Min-Hua Luo
Abstract
Invariant natural killer T (iNKT) cells are implicated in viral clearance, however their role in HCV infection remains controversial. Here, iNKT cells were studied during different stages of HCV infection. iNKT cells from patients with acute HCV infection and people who inject drugs (PWID) with chronic or spontaneously resolved HCV infection were characterized by flow cytometry. In a longitudinal analysis during acute HCV infection, frequencies of activated CD38+ iNKT cells reproducibly declined in spontaneously resolving patients whereas they were persistently elevated in patients progressing to chronic infection. During the first year of infection, the frequency of activated CD38+ or CD69+ iNKT cells strongly correlated with alanine transaminase levels with particularly pronounced correlations in spontaneously resolving patients. Increased frequencies of activated iNKT cells in chronic HCV infection were confirmed in cross-sectional analyses of PWID with chronic or spontaneously resolved HCV infection, however, no apparent functional differences were observed with various stimulation protocols. Our data suggest that iNKT cells are activated during acute hepatitis C and that activation is sustained in chronic infection. The correlation between the frequency of activated iNKT cells and ALT may point towards a role of iNKT cells in liver damage.
Authors
Tina Senff, Christopher Menne, Christine Cosmovici, Lia Laura Lewis-Ximenez, Jasneet Aneja, Ruth Broering, Arthur Y. Kim, Astrid M. Westendorf, Ulf Dittmer, Norbert Scherbaum, Georg M. Lauer, Jörg Timm
Abstract
Understanding viral rebound in pediatric HIV-1 infection may inform the development of alternatives to lifelong antiretroviral therapy (ART) to achieve viral remission. We thus investigated viral rebound after analytical treatment interruption (ATI) in 10 infant macaques orally infected with SHIV.C.CH505 and treated with long-term ART. Rebound viremia was detected within 7-35 days of ATI in 9/10 animals, with post-treatment control of viremia seen in 5/5 Mamu-A*01+ macaques. Single-genome sequencing revealed initial rebound virus was similar to viral DNA present in CD4+ T cells from blood, rectum, and lymph nodes before ATI. We assessed the earliest sites of viral reactivation immediately following ATI using ImmunoPET imaging. The largest increase in signal that preceded detectable viral RNA in plasma was found in the gastrointestinal (GI) tract, a site with relatively high SHIV RNA/DNA ratios in CD4+ T cells prior to ATI. Thus, the GI tract may be an initial source of rebound virus but as ATI progresses, viral reactivation in other tissues likely contributes to the composition of plasma virus. Our study provides novel insight into the features of viral rebound in pediatric infection and highlights the application of a non-invasive technique to monitor areas of HIV-1 expression in children.
Authors
Veronica Obregon-Perko, Katherine M. Bricker, Gloria Mensah, Ferzan Uddin, Laura Rotolo, Daryll Vanover, Yesha Desai, Philip J. Santangelo, Sherrie Jean, Jennifer S. Wood, Fawn C. Connor-Stroud, Stephanie Ehnert, Stella J. Berendam, Shan Liang, Thomas H. Vanderford, Katharine J. Bar, George M. Shaw, Guido Silvestri, Amit Kumar, Genevieve G. Fouda, Sallie R. Permar, Ann Chahroudi
Abstract
The ectocervix is part of the lower female reproductive tract (FRT), which is susceptible to sexually transmitted infections (STI). Comprehensive knowledge of the phenotypes and T cell receptor (TCR) repertoire of tissue resident memory T cells (TRM) in human FRT is lacking. We have taken single-cell RNA sequencing approaches to simultaneously define gene expression and TCR clonotypes of the human ectocervix. There are significantly more CD8 than CD4 T cells. Unsupervised clustering and trajectory analysis identify distinct populations of CD8 T cells with IFNGhiGZMBlowCD69hiCD103low or IFNGlowGZMBhiCD69medCD103hi phenotypes. Little overlap was seen between their TCR repertoires. Immunofluorescent staining shows that CD103+ CD8 TRM cells preferentially localize in epithelium while CD69+ CD8 TRM distribute evenly in epithelium and stroma. Ex vivo assays indicate up to 14% of cervical CD8 TRM clonotypes are HSV-2 reactive in HSV-2-seropositive persons, reflecting physiologically relevant localization. Our studies identify subgroups of CD8 TRM in the human ectocervix that exhibit distinct expression of antiviral defense and tissue residency markers, anatomic locations, and TCR repertoires that target anatomically relevant viral antigens. Optimization of the location, number, and function of FRT TRM is an important approach for improving host defenses to STI.
Authors
Tao Peng, Khamsone Phasouk, Emily Bossard, Alexis Klock, Lei Jin, Kerry J. Laing, Christine Johnston, Noel A. Williams, Julie L. Czartoski, Dana Varon, Annalyssa N. Long, Jason H. Bielas, Thomas M. Snyder, Harlan Robins, David M. Koelle, M Juliana McElrath, Anna Wald, Lawrence Corey, Jia Zhu
Abstract
Virus-induced respiratory tract infections are a major health burden in childhood, and available treatments are supportive rather than disease modifying. Rhinoviruses (RVs), the cause of approximately 80% of common colds, are detected in nearly half of all infants with bronchiolitis and the majority of children with an asthma exacerbation. Bronchiolitis in early life is a strong risk factor for the development of asthma. Here, we found that RV infection induced the expression of miRNA 122 (miR-122) in mouse lungs and in human airway epithelial cells. In vivo inhibition specifically in the lung reduced neutrophilic inflammation and CXCL2 expression, boosted innate IFN responses, and ameliorated airway hyperreactivity in the absence and in the presence of allergic lung inflammation. Inhibition of miR-122 in the lung increased the levels of suppressor of cytokine signaling 1 (SOCS1), which is an in vitro–validated target of miR-122. Importantly, gene silencing of SOCS1 in vivo completely reversed the protective effects of miR-122 inhibition on RV-induced lung disease. Higher miR-122 expression in nasopharyngeal aspirates was associated with a longer time on oxygen therapy and a higher rate of treatment failure in 87 infants hospitalized with moderately severe bronchiolitis. These results suggest that miR-122 promotes RV-induced lung disease via suppression of its target SOCS1 in vivo. Higher miR-122 expression was associated with worse clinical outcomes, highlighting the potential use of anti-miR-122 oligonucleotides, successfully trialed for treatment of hepatitis C, as potential therapeutics for RV-induced bronchiolitis and asthma exacerbations.
Authors
Adam M. Collison, Leon A. Sokulsky, Elizabeth Kepreotes, Ana Pereira de Siqueira, Matthew Morten, Michael R. Edwards, Ross P. Walton, Nathan W. Bartlett, Ming Yang, Thi Hiep Nguyen, Sebastian L. Johnston, Paul S. Foster, Joerg Mattes
Abstract
HIV-1 is capable of integrating its genome into that of its host cell. We examined the influence of the activation state of CD4+ T-cells, the effect of antiretroviral therapy (ART), and the clinical stage of HIV-1 infection on HIV-1 integration site features and selection. HIV-1 integration sites were sequenced from longitudinally sampled resting and activated CD4+ T-cells from 12 HIV-1 infected individuals. In total, 589 unique HIV-1 integration sites were analyzed: 147, 391, and 51 during primary, chronic, and late presentation of HIV-1 infection, respectively. As early as during primary HIV-1 infection and independent of the activation state of CD4+ T-cells collected on and off ART, HIV-1 integration sites were preferentially detected in recurrent integration genes (RIGs), genes associated with clonal expansion of latently HIV-1 infected CD4+ T-cells, cancer related genes, and highly expressed genes. The preference for cancer related genes was more pronounced at late stages of HIV-1 infection. Host genomic features of HIV-1 integration site selection remained stable during the course of HIV-1 infection in both resting and activated CD4+ T-cells. In summary, characteristic HIV-1 integration site features are pre-established as early as during primary HIV-1 infection and are found in both resting and activated CD4+ T-cells.
Authors
Yik Lim Kok, Valentina Vongrad, Sandra E. Chaudron, Mohaned Shilaih, Christine Leemann, Kathrin Neumann, Katharina Kusejko, Francesca Di Giallonardo, Herbert Kuster, Dominique L. Braun, Roger D. Kouyos, Huldrych F. Günthard, Karin J. Metzner
Abstract
In search of new prognostic markers, many mutation analyses of the HBV genome were performed. However, the Kozak sequence preceding precore was covered only infrequently in these analyses. In this study, HBV core promoter/precore region was sequenced in serum samples of European inactive HBV carriers (n=560). Quadruple mutation GCAC1809-1812TTCT was found with a high prevalence of 42% in the Kozak sequence preceding precore among all HBV genotypes. GCAC1809-1812TTCT was strongly associated with coexistence of basal core promoter (BCP) double mutation A1762T/G1764A and lower HBV DNA levels (p<0.0001). In vitro GCAC1809-1812TTCT leads to drastically diminished synthesis of pregenomic(pg)RNA, precore mRNA, core, HBsAg and HBeAg. Calculation of the pgRNA secondary structure suggests a destabilization of the pgRNA structure by A1762T/G1764A that is compensated by GCAC1809-1812TTCT. In 125 patients with HBV-related cirrhosis, GCAC1809-1812TTCT was not detected. While a strong association of GCAC1809-1812TTCT with inactive carrier status (p<0.0001) was observed, BCP double mutation was strongly correlated with cirrhosis (p<0.0001), but this was only observed in absence of GCAC1809-1812TTCT. In conclusion, our data reveal that GCAC1809-1812TTCT is highly prevalent in inactive carriers, and acts as a compensatory mutation for BCP double mutation. GCAC1809-1812TTCT seems to be a biomarker of good prognosis in HBV infection.
Authors
Kai-Henrik Peiffer, Catrina Spengler, Michael Basic, Bingfu Jiang, Lisa Kuhnhenn, Wiebke Obermann, Tobias Zahn, Mirco Glitscher, Alessandro Loglio, Floriana Facchetti, Gert Carra, Alica Kubesch, Johannes Vermehren, Viola Knop, Christiana Graf, Julia Dietz, Fabian Finkelmeier, Eva Herrmann, Jonel Trebicka, Arnold Grünweller, Stefan Zeuzem, Christoph Sarrazin, Pietro Lampertico, Eberhard Hildt
Abstract
The challenge of discovering a completely new human tumor virus of unknown phylogeny or sequence depends on detecting viral molecules and differentiating them from host molecules in the virus-associated neoplasm. We developed differential peptide subtraction (DPS) using differential mass-spectrometry (dMS) followed by targeted analysis to facilitate this discovery. We validated this approach by analyzing Merkel cell carcinoma (MCC), an aggressive human neoplasm, in which ~80% of cases are caused by the human Merkel cell polyomavirus (MCV). Approximately 20% of MCC have a high mutational burden and are negative for MCV, but are microscopically indistinguishable from virus positive cases. Using 23 (12 MCV positive, 11 MCV negative) formalin-fixed MCC, DPS identified both viral and human biomarkers (MCV Large T antigen, CDKN2AIP, SERPINB5 and TRIM29) that discriminates MCV positive and negative MCC. Statistical analysis of 498,131 dMS features not matching the human proteome by DPS revealed 562 (0.11%) to be up-regulated in virus-infected samples. Remarkably, four (20%) of the top 20 candidate MS spectra originated from MCV T oncoprotein peptides and confirmed by reverse translation degenerate oligonucleotide sequencing. DPS is a robust proteomic approach to identify novel viral sequences in infectious tumors when nucleic acid-based methods are not feasible.
Authors
Tuna Toptan, Pamela S. Cantrell, Xuemei Zeng, Yang Liu, Mai Sun, Nathan A. Yates, Yuan Chang, Patrick S. Moore
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