A complete understanding of human immune responses to Ebola virus infection is limited by the availability of specimens and the requirement for biosafety level 4 (BSL-4) containment. In an effort to bridge this gap, we evaluated cryopreserved PBMCs from 4 patients who survived Ebola virus disease (EVD) using an established mass cytometry antibody panel to characterize various cell populations during both the acute and convalescent phases. Acute loss of nonclassical monocytes and myeloid DCs, especially CD1c+ DCs, was noted. Classical monocyte proliferation and CD38 upregulation on plasmacytoid DCs coincided with declining viral load. Unsupervised analysis of cell abundance demonstrated acute declines in monocytic, NK, and T cell populations, but some populations, many of myeloid origin, increased in abundance during the acute phase, suggesting emergency hematopoiesis. Despite cell losses during the acute phase, upregulation of Ki-67 correlated with recovery of cell populations over time. These data provide insights into the human immune response during EVD.
Anita K. McElroy, Rama S. Akondy, David R. Mcllwain, Han Chen, Zach Bjornson-Hooper, Nilanjan Mukherjee, Aneesh K. Mehta, Garry Nolan, Stuart T. Nichol, Christina F. Spiropoulou
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of human coronavirus disease 2019 (COVID-19), emerged in Wuhan, China in December 2019. The virus rapidly spread globally, resulting in a public-health crisis including more than 3.1 million cases and 224,000 deaths as of May 1, 2020. Here, we describe the identification and evaluation of commercially available reagents and assays for the molecular detection of SARS-CoV-2 in infected formalin fixed paraffin embedded (FFPE) cell pellets. We identified a suitable rabbit polyclonal anti-SARS-CoV spike protein antibody and a mouse monoclonal anti-SARS-CoV nucleocapsid protein (NP) antibody for cross detection of the respective SARS-CoV-2 proteins by immunohistochemistry (IHC) and immunofluorescence assay (IFA). Next, we established RNAscope in situ hybridization (ISH) to detect SARS-CoV-2 RNA. Furthermore, we established a multiplex fluorescence ISH (mFISH) to detect positive-sense SARS-CoV-2 RNA and negative-sense SARS-CoV-2 RNA (a replicative intermediate indicating viral replication). Finally, we developed a dual staining assay using IHC and ISH to detect SARS-CoV-2 antigen and RNA in the same FFPE section. These reagents and assays will accelerate COVID-19 pathogenesis studies in humans and in COVID-19 animal models.
Jun Liu, April M. Babka, Brian J. Kearney, Sheli R. Radoshitzky, Jens H. Kuhn, Xiankun Zeng
Plasma viral load (VL) and CD4+ T-cell count are widely used as biomarkers of HIV-1 replication, pathogenesis, and response to antiretroviral therapy (ART). However, the clinical potential of cell-associated (CA) HIV-1 molecular markers is much less understood. Here, we measured CA HIV-1 RNA and DNA in HIV-infected individuals treated with temporary ART initiated during primary HIV-1 infection. We demonstrate significant predictive value of CA RNA for: (a) the virological and immunological response to early ART, (b) the magnitude and time to viral rebound after discontinuation of early ART, and (c) the disease progression in the absence of treatment. Remarkably, when adjusted for CA RNA, plasma VL no longer appeared as an independent predictor of any clinical endpoint in this cohort. The potential of CA RNA as an HIV-1 clinical marker, in particular as a predictive biomarker of virological control after stopping ART, should be explored in the context of HIV-1 curative interventions.
Alexander O. Pasternak, Marlous L. Grijsen, Ferdinand W. Wit, Margreet Bakker, Suzanne Jurriaans, Jan M. Prins, Ben Berkhout
Recovery from measles results in life-long protective immunity. To understand induction of long-term immunity, rhesus macaques were studied for six months after infection with WT measles virus (MeV). Infection caused viremia and rash with clearance of infectious virus by 14 days. MeV RNA persisted in PBMCs for 30-90 days and in lymphoid tissue for 6 months most often in B cells but was rarely detected in BM. Antibody with neutralizing activity and binding specificity for MeV nucleocapsid (N), hemagglutinin (H) and fusion proteins appeared with the rash and avidity matured over 3-4 months. Lymph nodes had increasing numbers of MeV-specific antibody-secreting cells (ASCs) and germinal centers with late hyalinization. ASCs appeared in circulation with the rash and continued to appear along with peripheral Tfh cells for the study duration. ASCs in lymph nodes and PBMCs produced antibody to both H and N, with more H-specific ASCs in BM. From 14-21 days 20-100-fold more total ASCs than MeV-specific ASCs appeared in circulation suggesting mobilization of pre-existing ASCs. Therefore, persistence of MeV RNA in lymphoid tissue was accompanied by continued germinal center formation, ASC production, avidity maturation and accumulation of H-specific ASCs in BM to sustain neutralizing antibody and protective immunity.
Ashley N. Nelson, Wen-Hsuan W. Lin, Rupak Shivakoti, Nicole E. Putnam, Lisa M. Mangus, Robert J. Adams, Debra Hauer, Victoria K. Baxter, Diane E. Griffin
To investigate the nationwide severe fever with thrombocytopenia syndrome virus (SFTSV) infection status, we isolated SFTSVs from severe fever with thrombocytopenia syndrome (SFTS)-suspected patients in 207 hospitals throughout South Korea between 2013 and April of 2017. A total of 116 SFTSVs were isolated from 3,137 SFTS-suspected patients with an overall 21.6% case fatality rate. Genetic characterization revealed that at least six genotypes of SFTSVs are co-circulating in South Korea with multiple reassortments among them. Of these, the genotype B-2 strains were the most prevalent (n = 48, 36.1%) followed by the A and F genotypes. Clinical and epidemiologic investigations revealed that genotype B strains were associated with the highest case-fatality rate (34.8%, 32/92), while genotype A caused only one fatality out of ten patients. Further, ferret infection studies demonstrated varied clinical manifestations and case mortality rates of different strains of SFTSV, which suggests this virus could exhibit genotype-dependent pathogenicity.Keywords: severe fever with thrombocytopenia syndrome virus (SFTSV), clinical manifestations, genotypes, pathogenesis
Seok-Min Yun, Su-Jin Park, Young-Il Kim, Sun-Whan Park, Min-Ah Yu, Hyeok-Il Kwon, Eun-Ha Kim, Kwang-Min Yu, Hye Won Jeong, Jungsang Ryou, Won-Ja Lee, Youngmee Jee, Joo-Yeon Lee, Young Ki Choi
Influenza is a highly contagious viral pathogen with more than 200,000 cases reported in the U.S. during the 2017-2018 season. Annual vaccination is recommended by the World Health Organization with the goal to reduce influenza severity and transmission. Currently available vaccines are ~60% effective and vaccine effectiveness varies from season to season, as well as between different influenza subtypes within a single season. Immunological imprinting from early life influenza infection can prominently shape the immune response to subsequent infections. Here, the impact of pre-existing B cell memory in the response to quadrivalent influenza vaccine was assessed using blood samples collected from healthy subjects (18 to 85 years old) prior to and 21-28 days following influenza vaccination. Influenza vaccination increased both HA-specific antibodies and memory B cells frequency. Despite no apparent differences in antigenicity between vaccine components, most individuals were biased towards one of the vaccine strains. Specifically, responses to H3N2 were reduced in magnitude relative to the other vaccine components. Overall, this study unveils a new mechanism underlying differential vaccine effectiveness against distinct influenza subtypes.
Rodrigo B. Abreu, Greg A. Kirchenbaum, Emily F. Clutter, Giuseppe A. Sautto, Ted M. Ross
Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012 in the Kingdom of Saudi Arabia and has caused over 2400 cases and more than 800 deaths. Epidemiological studies identified diabetes as the primary comorbidity associated with severe and/or lethal MERS-CoV infection. Understanding how diabetes affects MERS is important due to the global burden of diabetes and pandemic potential of MERS-CoV. We used a model in which mice were made susceptible to MERS-CoV by expressing human DPP4 and type 2 diabetes was induced by administering a high fat diet. Upon infection with MERS-CoV, diabetic mice had a prolonged phase of severe disease and delayed recovery which was independent of virus titers. Histological analysis revealed that diabetic mice had delayed inflammation which was then prolonged through 21 dpi. Diabetic mice had fewer inflammatory monocyte/macrophages and CD4+ T cells which correlated with lower levels of Ccl2 and Cxcl10 expression. Diabetic mice also had lower levels of Tnfa, Il6, Il12b, and Arg1 expression and higher levels of Il17a expression. These data suggest that the increased disease severity observed in individuals with MERS and comorbid type 2 diabetes is likely due to a dysregulated immune response which results in more severe and prolonged lung pathology.
Kirsten A. Kulcsar, Christopher M. Coleman, Sarah E. Beck, Matthew B. Frieman
Filoviruses of the genus Ebolavirus include five species with marked differences in their ability to cause disease in humans. From the highly virulent Ebola virus to the seemingly nonpathogenic Reston virus, case-fatality rates can range between 0-90%. In order to understand the molecular basis of these differences it is imperative to establish disease models that recapitulate human disease as faithfully as possible. Non-human primates are the gold-standard models for filovirus pathogenesis, but comparative studies are skewed by the fact that Reston virus infection can be lethal for NHP. Here we have used HLA-A2 transgenic, NOD-scid-interleukin 2γ receptor knockout (NSG-A2) mice reconstituted with human hematopoiesis to compare Ebola virus and Reston virus pathogenesis in a human-like environment. While significantly less pathogenic than Ebola virus, Reston virus killed 20% of infected mice, a finding that was linked to exacerbated inflammation and viral replication in the liver. In addition, ‘humanized’ mice recapitulated the case-fatality ratios of different Ebolavirus species in humans. Our findings point out at humanized mice as a putative model to test the pathogenicity of newly discovered filoviruses, and warrants further investigations on Reston virus pathogenesis in humans.
Beatriz Escudero-Pérez, Paula Ruibal, Monika Rottstegge, Anja Lüdtke, Julia R. Port, Kristin Hartmann, Sergio Gómez-Medina, Juergen Müller-Guhl, Emily V. Nelson, Susanne Krasemann, Estefanía Rodríguez, César Muñoz-Fontela
Nipah virus (NiV) is an emerging zoonotic paramyxovirus that causes highly lethal henipavirus encephalitis in humans. Survivors develop various neurologic sequelae, including late-onset and relapsing encephalitis, several months up to several years following initial infection. However, the underlying pathology and disease mechanisms of persistent neurologic complications remain unknown. Here, we demonstrate persistent NiV infection in the brains of grivets that survived experimental exposure to NiV. Encephalitis affected the entire brains, with the majority of NiV detected in the neurons and microglia of the brainstems, cerebral cortices, and cerebella. We identified the vascular endothelium in the brain as an initial target of NiV infection during the acute phase of disease, indicating a primary path of entry for NiV into the brain. Notably, we were unable to detect NiV anywhere else except the brains in the examined survivors. Our findings indicate that late-onset and relapsing encephalitis of NiV in human survivors may be due to viral persistence in the brain and shed light on the pathogenesis of chronic henipavirus encephalitis.
Jun Liu, Kayla M. Coffin, Sara C. Johnston, April M. Babka, Todd M. Bell, Simon Y. Long, Anna N. Honko, Jens H. Kuhn, Xiankun Zeng
The activation and recruitment of NK cells to the site of viral infection are crucial for virus control. However, it remains largely unknown what controls the recruitment of the activated NK cells to the infection site. In a model of intraperitoneal infection with vaccinia virus (VV), we showed that poly(ADP-ribose) polymerase-1 (PARP-1), a sensor of DNA damage, is critical for NK cell recruitment to the site of infection and viral control in vivo. We further demonstrated that PARP-1 promotes the production of CCL2 and that the CCL2-CCR2 axis is essential for NK cell recruitment to the infection site. In addition, we demonstrated that peritoneal macrophages are the main producer of PARP-1–dependent CCL2 secretion. Mechanistically, PARP-1 functions as a regulator of NF-κB by promoting its nuclear translocation and binding to its response sequences in macrophages upon VV infection. Taken together, our results reveal a potentially previously unknown role for PARP-1–dependent CCL2 production in NK cell migration and viral control and may provide important insights into the design of effective NK cell–based therapies for viral infections and cancer.
Qiyang Shou, Huiying Fu, Xiaopei Huang, Yiping Yang
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