scholarly journals Disruption of Zika Virus xrRNA1-Dependent sfRNA1 Production Results in Tissue-Specific Attenuated Viral Replication

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1177
Author(s):  
Hadrian Sparks ◽  
Brendan Monogue ◽  
Benjamin Akiyama ◽  
Jeffrey Kieft ◽  
J. David Beckham
Keyword(s):  
Zika Virus ◽  
Noncoding Rna ◽  
Defense Mechanisms ◽  
Neutralizing Antibody ◽  
Mammalian Host ◽  
Genome Replication ◽  
Genomic Rnas ◽  
Tissue Specific ◽  
Targeted Mutation ◽  
Flavivirus Vaccine

The Zika virus (ZIKV), like other flaviviruses, produces several species of sub-genomic RNAs (sfRNAs) during infection, corresponding to noncoding RNA fragments of different lengths that result from the exonuclease degradation of the viral 3′ untranslated region (UTR). Over the course of infection, these sfRNAs accumulate in the cell as a result of an incomplete viral genome degradation of the 3′ UTR by the host 5′ to 3′ exoribonuclease, Xrn1. The halting of Xrn1 in the 3′ UTR is due to two RNA pseudoknot structures in the 3′ UTR, termed exoribonuclease-resistant RNA1 and 2 (xrRNA1&2). Studies with related flaviviruses have shown that sfRNAs are important for pathogenicity and inhibiting both mosquito and mammalian host defense mechanisms. However, these investigations have not included ZIKV and there is very limited data addressing how sfRNAs impact infection in a whole animal model or specific tissues. In this study, we generate a sfRNA1-deficient ZIKV (X1) by targeted mutation in the xrRNA1 3′ UTR structure. We find that the X1 virus lacks the production of the largest ZIKV sfRNA species, sfRNA1. Using the X1 virus to infect adult Ifnar1−/− mice, we find that while the lack of sfRNA1 does not alter ZIKV replication in the spleen, there is a significant reduction of ZIKV genome replication in the brain and placenta compared to wild-type ZIKV infection. Despite the attenuated phenotype of the X1 ZIKV, mice develop a robust neutralizing antibody response. We conclude that the targeted disruption of xrRNA1 results in tissue-specific attenuation while still supporting robust neutralizing antibody responses. Future studies will need to investigate the tissue-specific mechanisms by which ZIKV sfRNAs influence infection and may utilize targeted xrRNA mutations to develop novel attenuated flavivirus vaccine approaches.

scholarly journals Disruption of Zika virus xrRNA1-dependent sfRNA1 production results in tissue-specific attenuated viral replication

2020 ◽  
Author(s):  
Hadrian Sparks ◽  
Brendan Monogue ◽  
Benjamin Akiyama ◽  
Jeffrey S. Kieft ◽  
J. David Beckham
Keyword(s):  
Zika Virus ◽  
Noncoding Rna ◽  
Defense Mechanisms ◽  
Neutralizing Antibody ◽  
Mammalian Host ◽  
Genome Replication ◽  
Genomic Rnas ◽  
Tissue Specific ◽  
Targeted Mutation ◽  
Flavivirus Vaccine

AbstractZika virus (ZIKV), like other flaviviruses, produces several species of sub-genomic RNAs (sfRNAs) during infection, corresponding to noncoding RNA fragments of different lengths derived from the viral 3’ untranslated region (UTR). Over the course of infection, these sfRNAs accumulate in the cell as a result of incomplete viral genome degradation of the 3’UTR by host 5’ to 3’ exoribonuclease (Xrn1). The halting of Xrn1 in the 3’UTR is due to two RNA pseudoknot structures in the 3’UTR termed exoribonuclease-resistant RNA1 and 2 (xrRNA1&2). Studies with related flaviviruses have shown that sfRNAs are important for pathogenicity and inhibiting both mosquito and mammalian host defense mechanisms. However, these investigations have not included ZIKV and there is very limited data addressing how sfRNAs impact infection in a whole animal model or specific tissues. In this study, we rescued a sfRNA1-deficient ZIKV (X1) by targeted mutation in the xrRNA1 3’ UTR structure. We found that virus which lacks the production of the largest ZIKV sfRNA species, sfRNA1. Using the X1 virus to infect adult IFNAR1-/- mice, we found that while the lack of sfRNA1 does not alter ZIKV replication in the spleen, there is a significant reduction of ZIKV genome replication in the brain and placenta compared to WT ZIKV infection. Despite thee attenuated phenotype of the X1 ZIKV, mice develop a robust neutralizing antibody response. We conclude that targeted disruption of xrRNA1 results in tissue-specific attenuation while still supporting robust neutralizing antibody responses. Future studies will need to investigate the tissue-specific mechanisms by which ZIKV sfRNAs influence infection and may utilize targeted xrRNA mutations to develop novel attenuated flavivirus vaccine approaches.

scholarly journals Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes

2007 ◽  
Vol 88 (2) ◽  
pp. 621-630 ◽  
Author(s):  
S. Maan ◽  
N. S. Maan ◽  
A. R. Samuel ◽  
S. Rao ◽  
H. Attoui ◽  
...  
Keyword(s):  
Bluetongue Virus ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Full Length ◽  
Molecular Diagnostic ◽  
Mammalian Host ◽  
Reading Frame ◽  
Diagnostic Assays ◽  
Primary Determinant ◽  
Serotype Identification

The outer capsid protein VP2 of Bluetongue virus (BTV) is a target for the protective immune response generated by the mammalian host. VP2 contains the majority of epitopes that are recognized by neutralizing antibodies and is therefore also the primary determinant of BTV serotype. Full-length cDNA copies of genome segment 2 (Seg-2, which encodes VP2) from the reference strains of each of the 24 BTV serotypes were synthesized, cloned and sequenced. This represents the first complete set of full-length BTV VP2 genes (from the 24 serotypes) that has been analysed. Each Seg-2 has a single open reading frame, with short inverted repeats adjacent to conserved terminal hexanucleotide sequences. These data demonstrated overall inter-serotype variations in Seg-2 of 29 % (BTV-8 and BTV-18) to 59 % (BTV-16 and BTV-22), while the deduced amino acid sequence of VP2 varied from 22.4 % (BTV-4 and BTV-20) to 73 % (BTV-6 and BTV-22). Ten distinct Seg-2 lineages (nucleotypes) were detected, with greatest sequence similarities between those serotypes that had previously been reported as serologically ‘related’. Fewer similarities were observed between different serotypes in regions of VP2 that have been reported as antigenically important, suggesting that they may play a role in the neutralizing antibody response. The data presented form an initial basis for BTV serotype identification by sequence analyses and comparison of Seg-2, and for development of molecular diagnostic assays for individual BTV serotypes (by RT-PCR).

scholarly journals A direct RNA-protein interaction atlas of the SARS-CoV-2 RNA in infected human cells

2020 ◽  
Author(s):  
Nora Schmidt ◽  
Caleb A. Lareau ◽  
Hasmik Keshishian ◽  
Randy Melanson ◽  
Matthias Zimmer ◽  
...  
Keyword(s):  
Viral Replication ◽  
Host Defense ◽  
Drug Targets ◽  
Defense Mechanisms ◽  
Human Cells ◽  
Genome Replication ◽  
Defense Strategies ◽  
Novel Therapeutic Strategies ◽  
Global Threat ◽  
Research Challenge

ABSTRACTSARS-CoV-2 infections pose a global threat to human health and an unprecedented research challenge. Among the most urgent tasks is obtaining a detailed understanding of the molecular interactions that facilitate viral replication or contribute to host defense mechanisms in infected cells. While SARS-CoV-2 co-opts cellular factors for viral translation and genome replication, a comprehensive map of the host cell proteome in direct contact with viral RNA has not been elucidated. Here, we use RNA antisense purification and mass spectrometry (RAP-MS) to obtain an unbiased and quantitative picture of the human proteome that directly binds the SARS-CoV-2 RNA in infected human cells. We discover known host factors required for coronavirus replication, regulators of RNA metabolism and host defense pathways, along with dozens of potential drug targets among direct SARS-CoV-2 binders. We further integrate the SARS-CoV-2 RNA interactome with proteome dynamics induced by viral infection, linking interactome proteins to the emerging biology of SARS-CoV-2 infections. Validating RAP-MS, we show that CNBP, a regulator of proinflammatory cytokines, directly engages the SARS-CoV-2 RNA. Supporting the functional relevance of identified interactors, we show that the interferon-induced protein RYDEN suppresses SARS-CoV-2 ribosomal frameshifting and demonstrate that inhibition of SARS-CoV-2-bound proteins is sufficient to manipulate viral replication. The SARS-CoV-2 RNA interactome provides an unprecedented molecular perspective on SARS-CoV-2 infections and enables the systematic dissection of host dependency factors and host defense strategies, a crucial prerequisite for designing novel therapeutic strategies.

scholarly journals Distinct cellular immune signatures in acute Zika virus infection are associated with high or low persisting neutralizing antibody titers

2021 ◽  
Author(s):  
Elizabeth E. McCarthy ◽  
Pamela M. Odorizzi ◽  
Emma Lutz ◽  
Carolyn P. Smullin ◽  
Iliana Tenvooren ◽  
...  
Keyword(s):  
Zika Virus ◽  
Neutralizing Antibodies ◽  
Viral Infections ◽  
Immune Cell ◽  
Acute Infection ◽  
Neutralizing Antibody ◽  
Natural Immunity ◽  
Zikv Infection ◽  
Antibody Titers ◽  
Cellular Immune

Although the formation of a durable neutralizing antibody response after an acute viral infection is a key component of protective immunity, little is known about why some individuals generate high versus low neutralizing antibody titers to infection or vaccination. Infection with Zika virus (ZIKV) during pregnancy can cause devastating fetal outcomes, and efforts to understand natural immunity to this infection are essential for optimizing vaccine design. In this study, we leveraged the high-dimensional single-cell profiling capacity of mass cytometry (CyTOF) to deeply characterize the cellular immune response to acute and convalescent ZIKV infection in a cohort of blood donors in Puerto Rico incidentally found to be viremic during the 2015-2016 epidemic in the Americas. During acute ZIKV infection, we identified widely coordinated responses across innate and adaptive immune cell lineages. High frequencies of multiple activated innate immune subsets, as well as activated follicular helper CD4+ T cells and proliferating CD27-IgD- B cells, during acute infection were associated with high titers of ZIKV neutralizing antibodies at 6 months post-infection. On the other hand, low titers of ZIKV neutralizing antibodies were associated with immune features that suggested a cytotoxic-skewed immune "set-point." Our study offers insight into the cellular coordination of immune responses and identifies candidate cellular biomarkers that may offer predictive value in vaccine efficacy trials for ZIKV and other acute viral infections aimed at inducing high titers of neutralizing antibodies.

scholarly journals Zika virus is transmitted in neural progenitor cells via cell-to-cell spread and infection is inhibited by the autophagy inducer trehalose

2020 ◽  
pp. JVI.02024-20
Author(s):  
Alex E Clark ◽  
Zhe Zhu ◽  
Florian Krach ◽  
Jeremy N Rich ◽  
Gene W. Yeo ◽  
...  
Keyword(s):  
Viral Replication ◽  
Progenitor Cells ◽  
Zika Virus ◽  
Neural Progenitor Cells ◽  
Neutralizing Antibody ◽  
Neural Progenitor ◽  
Health Concern ◽  
Free Virus ◽  
Zikv Infection ◽  
Infected Cells

Zika virus (ZIKV) is a mosquito-borne human pathogen that causes congenital Zika syndrome and neurological symptoms in some adults. There are currently no approved treatments or vaccines for ZIKV, and exploration of therapies targeting host processes could avoid viral development of drug resistance. The purpose of our study was to determine if the non-toxic and widely used disaccharide trehalose, which showed antiviral activity against Human Cytomegalovirus (HCMV) in our previous work, could restrict ZIKV infection in clinically relevant neural progenitor cells (NPCs). Trehalose is known to induce autophagy, the degradation and recycling of cellular components. Whether autophagy is proviral or antiviral for ZIKV is controversial and depends on cell type and specific conditions used to activate or inhibit autophagy. We show here that trehalose treatment of NPCs infected with recent ZIKV isolates from Panama and Puerto Rico significantly reduces viral replication and spread. In addition, we demonstrate that ZIKV infection in NPCs spreads primarily cell-to-cell as an expanding infectious center, and NPCs are infected via contact with infected cells far more efficiently than by cell-free virus. Importantly, ZIKV was able to spread in NPCs in the presence of neutralizing antibody.Importance Zika virus causes birth defects and can lead to neurological disease in adults. While infection rates are currently low, ZIKV remains a public health concern with no treatment or vaccine available. Targeting a cellular pathway to inhibit viral replication is a potential treatment strategy that avoids development of antiviral resistance. We demonstrate in this study that the non-toxic autophagy-inducing disaccharide trehalose reduces spread and output of ZIKV in infected neural progenitor cells (NPCs), the major cells infected in the fetus. We show that ZIKV spreads cell-to-cell in NPCs as an infectious center and that NPCs are more permissive to infection by contact with infected cells than by cell-free virus. We find that neutralizing antibody does not prevent the spread of the infection in NPCs. These results are significant in demonstrating anti-ZIKV activity of trehalose and in clarifying the primary means of Zika virus spread in clinically relevant target cells.

scholarly journals Characterization of two engineered dimeric Zika virus envelope proteins as immunogens for neutralizing antibody selection and vaccine design

2019 ◽  
Vol 294 (27) ◽  
pp. 10638-10648 ◽  
Author(s):  
Chunpeng Yang ◽  
Fang Zeng ◽  
Xinyu Gao ◽  
Shaojuan Zhao ◽  
Xuan Li ◽  
...  
Keyword(s):  
Zika Virus ◽  
Vaccine Design ◽  
Neutralizing Antibody ◽  
Envelope Proteins ◽  
Virus Envelope

scholarly journals Interleukin-23 (IL-23)-IL-17 Cytokine Axis in Murine Pneumocystis carinii Infection

2007 ◽  
Vol 75 (6) ◽  
pp. 3055-3061 ◽  
Author(s):  
Xiaowen L. Rudner ◽  
Kyle I. Happel ◽  
Erana A. Young ◽  
Judd E. Shellito
Keyword(s):  
T Cells ◽  
Host Defense ◽  
Defense Mechanisms ◽  
Pneumocystis Carinii ◽  
Neutralizing Antibody ◽  
Wild Type ◽  
Pneumocystis Carinii Infection ◽  
Interleukin 23

ABSTRACT Host defense mechanisms against Pneumocystis carinii are not fully understood. Previous work in the murine model has shown that host defense against infection is critically dependent upon host CD4+ T cells. The recently described Th17 immune response is predominantly a function of effector CD4+ T cells stimulated by interleukin-23 (IL-23), but whether these cells are required for defense against P. carinii infection is unknown. We tested the hypothesis that P. carinii stimulates the early release of IL-23, leading to increases in IL-17 production and lung effector CD4+ T-cell population that mediate clearance of infection. In vitro, stimulation of alveolar macrophages with P. carinii induced IL-23, and IL-23p19 mRNA was expressed in lungs of mice infected with this pathogen. To address the role of IL-23 in resistance to P. carinii, IL-23p19−/− and wild-type control C57BL/6 mice were infected and their fungal burdens and cytokine/chemokine responses were compared. IL-23p19−/− mice displayed transient but impaired clearance of infection, which was most apparent 2 weeks after inoculation. In confirmatory studies, the administration of either anti-IL-23p19 or anti-IL-17 neutralizing antibody to wild-type mice infected with P. carinii also caused increases in fungal burdens. IL-17 and the lymphocyte chemokines IP-10, MIG, MIP-1α, MIP-1β, and RANTES were decreased in the lungs of infected IL-23p19−/− mice in comparison to their levels in the lungs of wild-type mice. In IL-23p19−/− mice infected with P. carinii, there were fewer effector CD4+ T cells in the lung tissue. Collectively, these studies indicate that the IL-23-IL-17 axis participates in host defense against P. carinii.

scholarly journals Seroprevalence of Zika virus in pregnant women from central Thailand

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257205
Author(s):  
Chayawat Phatihattakorn ◽  
Artit Wongsa ◽  
Kirakorn Pongpan ◽  
Sanitra Anuwuthinawin ◽  
Sakita Mungmanthong ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Zika Virus ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
First Trimester ◽  
Effective Prevention ◽  
Denv Serotypes ◽  
Siriraj Hospital ◽  
Health Care Monitoring ◽  
Central Thailand

Zika virus (ZKV) infection in a pregnant woman, especially during the first trimester, often results in congenital anomalies. However, the pathogenic mechanism is unknown and one-third of ZKV infected pregnancies are asymptomatic. Neutralizing antibodies against ZKV has been reported in 70% of Thai adults, but the prevalence among pregnant women is unknown. Currently, vaccines and specific treatments for ZKV are under development. A better understanding of the immune status of pregnant women will increase the success of effective prevention guidelines. The prevalence of ZKV infection in pregnant women in antenatal care clinics was investigated during the rainy season from May to October 2019 at Siriraj Hospital, Bangkok, Thailand. We recruited 650 pregnant women (39.42% first, 52.26% second and 7.36% third trimester) and found that 30.77% had ZKV-specific IgG, and 39.81% had neutralizing antibodies (nAb) against ZKV (titer ≥10). Specific and neutralizing antibody levels varied by maternal age, trimester, and month. We further characterized the cross-reaction between ZKV and the four Dengue virus (DENV) serotypes by focused reduction neutralization test (FRNT) and found that cross-reactions were common. In conclusion, about 60% of pregnant women who living in central Thailand may be at risk of ZKV infection due to the absence of neutralizing antibodies against ZKV. The functions of cross-reactive antibodies between related viral genotypes require further study. These findings have implications for health care monitoring in pregnant women including determining the risk of ZKV infection, assisting the development of a flavivirus vaccine, and informing the development of preventative health policies.

scholarly journals Dysregulation of Long Non-coding RNA (lncRNA) Genes and Predicted lncRNA-Protein Interactions during Zika Virus Infection

2016 ◽  
Author(s):  
Arunachalam Ramaiah ◽  
Deisy Contreras ◽  
Vineela Gangalapudi ◽  
Masumi Sameer Padhye ◽  
Jie Tang ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Zika Virus ◽  
Mammalian Cells ◽  
Noncoding Rna ◽  
Target Genes ◽  
Vero Cells ◽  
Viral Pathogens ◽  
Developmental Abnormalities ◽  
Non Coding Rna ◽  
Cellular Factors

ABSTRACTZika Virus (ZIKV) is a causative agent for poor pregnancy outcome and fetal developmental abnormalities, including microcephaly and eye defects. As a result, ZIKV is now a confirmed teratogen. Understanding host-pathogen interactions, specifically cellular perturbations caused by ZIKV, can provide novel therapeutic targets. In order to complete viral replication, viral pathogens control the host cellular machineries and regulate various factors, including long noncoding RNA (lncRNA) genes, at transcriptional levels. The role of lncRNA genes in the pathogenesis of ZIKV-mediated microcephaly and eye defects is currently unknown. To gain additional insights, we focused on profiling the differentially expressed lncRNA genes during ZIKV infection in mammalian cells. For this study, we employed a contemporary clinical Zika viral isolate, PRVABC59, of Asian genotype. We utilized an unbiased RNA sequencing approach to profile the lncRNA transcriptome in ZIKV infected Vero cells. We identified a total of 121 lncRNA genes that are differentially regulated at 48 hours post-infection. The majority of these genes are independently validated by reverse-transcription qPCR. A notable observation was that the lncRNAs, MALAT1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) and NEAT1 (Nuclear Paraspeckle Assembly Transcript 1), are down-regulated upon Zika viral infection. MALAT1 and NEAT1 are known as nuclear localized RNAs that regulate gene expression and cell proliferation. Protein-lncRNA interaction maps revealed that MALAT1 and NEAT1 share common interacting partners and form a larger network comprising of 71 cellular factors. ZIKV-mediated dysregulation of these two regulatory lncRNAs can alter the expression of respective target genes and associated biological functions, an important one being cell division. In conclusion, this investigation is the first to provide insight into the biological connection of lncRNAs and ZIKV which can be further explored for developing antiviral therapy and understanding fetal developmental processes.

scholarly journals Protective Efficacy of a Chimeric Insect-Specific Flavivirus Vaccine against West Nile Virus

Vaccines ◽  
2020 ◽  
Vol 8 (2) ◽  
pp. 258 ◽  
Author(s):  
Laura J. Vet ◽  
Yin Xiang Setoh ◽  
Alberto A. Amarilla ◽  
Gervais Habarugira ◽  
Willy W. Suen ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Protective Efficacy ◽  
Neutralizing Antibody ◽  
Human Infection ◽  
Chimeric Virus ◽  
E Proteins ◽  
West Nile ◽  
Lethal Challenge ◽  
Virulent Strains ◽  
Flavivirus Vaccine

Virulent strains of West Nile virus (WNV) are highly neuro-invasive and human infection is potentially lethal. However, no vaccine is currently available for human use. Here, we report the immunogenicity and protective efficacy of a vaccine derived from a chimeric virus, which was constructed using the structural proteins (prM and E) of the Kunjin strain of WNV (WNVKUN) and the genome backbone of the insect-specific flavivirus Binjari virus (BinJV). This chimeric virus (BinJ/WNVKUN-prME) exhibits an insect-specific phenotype and does not replicate in vertebrate cells. Importantly, it authentically presents the prM-E proteins of WNVKUN, which is antigenically very similar to other WNV strains and lineages. Therefore BinJ/WNVKUN-prME represents an excellent candidate to assess as a vaccine against virulent WNV strains, including the highly pathogenic WNVNY99. When CD1 mice were immunized with purified BinJ/WNVKUN-prME, they developed robust neutralizing antibody responses after a single unadjuvanted dose of 1 to 5 μg. We further demonstrated complete protection against viremia and mortality after lethal challenge with WNVNY99, with no clinical or subclinical pathology observed in vaccinated animals. These data suggest that BinJ/WNVKUN-prME represents a safe and effective WNV vaccine candidate that warrants further investigation for use in humans or in veterinary applications.

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