-
Journal of Clinical Microbiology Dec 2023Eastern equine encephalitis virus (EEEV), Madariaga virus (MADV), and Venezuelan equine encephalitis virus complex (VEEV) are New World alphaviruses transmitted by...
Real-time RT-PCR for Venezuelan equine encephalitis complex, Madariaga, and Eastern equine encephalitis viruses: application in human and mosquito public health surveillance in Panama.
Eastern equine encephalitis virus (EEEV), Madariaga virus (MADV), and Venezuelan equine encephalitis virus complex (VEEV) are New World alphaviruses transmitted by mosquitoes. They cause febrile and sometimes severe neurological diseases in human and equine hosts. Detecting them during the acute phase is hindered by non-specific symptoms and limited diagnostic tools. We designed and clinically assessed real-time reverse transcription polymerase chain reaction assays (rRT-PCRs) for VEEV complex, MADV, and EEEV using whole-genome sequences. Validation involved 15 retrospective serum samples from 2015 to 2017 outbreaks, 150 mosquito pools from 2015, and 118 prospective samples from 2021 to 2022 surveillance in Panama. The rRT-PCRs detected VEEV complex RNA in 10 samples (66.7%) from outbreaks, with one having both VEEV complex and MADV RNAs. VEEV complex RNA was found in five suspected dengue cases from disease surveillance. The rRT-PCR assays identified VEEV complex RNA in three () pools, leading to VEEV isolates in two. Phylogenetic analysis revealed the VEEV ID subtype in positive samples. Notably, 11.9% of dengue-like disease patients showed VEEV infections. Together, our rRT-PCR validation in human and mosquito samples suggests that this method can be incorporated into mosquito and human encephalitic alphavirus surveillance programs in endemic regions.
Topics: Humans; Animals; Horses; Encephalitis Virus, Eastern Equine; Encephalomyelitis, Venezuelan Equine; Culicidae; Reverse Transcriptase Polymerase Chain Reaction; Phylogeny; Encephalomyelitis, Eastern Equine; Prospective Studies; Public Health Surveillance; Retrospective Studies; Alphavirus; RNA; Dengue
PubMed: 37982611
DOI: 10.1128/jcm.00152-23 -
Vaccine Oct 2023In a phase 2 safety and immunogenicity study of a chikungunya virus virus-like particle (CHIKV VLP) vaccine in an endemic region, of 400 total participants, 78 were...
In a phase 2 safety and immunogenicity study of a chikungunya virus virus-like particle (CHIKV VLP) vaccine in an endemic region, of 400 total participants, 78 were found to be focus reduction neutralizing antibody seropositive at vaccination despite being ELISA seronegative at screening, of which 39 received vaccine. This post hoc analysis compared safety and immunogenicity of CHIKV VLP vaccine in seropositive (n = 39) versus seronegative (n = 155) vaccine recipients for 72 weeks post-vaccination. There were no differences in solicited adverse events, except injection site swelling in 10.3% of seropositive versus 0.6% of seronegative recipients (p = 0.006). Baseline seropositive vaccine recipients had stronger post-vaccination luciferase neutralizing antibody responses versus seronegative recipients (peak geometric mean titer of 3594 and 1728, respectively) persisting for 72 weeks, with geometric mean fold increases of 3.1 and 13.2, respectively. In this small study, CHIKV VLP vaccine was well-tolerated and immunogenic in individuals with pre-existing immunity. ClinicalTrials.gov Identifier: NCT02562482.
Topics: Humans; Chikungunya virus; Chikungunya Fever; Vaccines, Virus-Like Particle; Viral Vaccines; Antibodies, Viral; Antibodies, Neutralizing; Immunogenicity, Vaccine; Double-Blind Method
PubMed: 37690874
DOI: 10.1016/j.vaccine.2023.08.086 -
Cell Reports Mar 2024Alphaviruses are mosquito-transmitted pathogens that induce high levels of viremia, which facilitates dissemination and vector transmission. One prevailing paradigm is...
Alphaviruses are mosquito-transmitted pathogens that induce high levels of viremia, which facilitates dissemination and vector transmission. One prevailing paradigm is that, after skin inoculation, alphavirus-infected resident dendritic cells migrate to the draining lymph node (DLN), facilitating further rounds of infection and dissemination. Here, we assess the contribution of infiltrating myeloid cells to alphavirus spread. We observe two phases of virus transport to the DLN, one that occurs starting at 1 h post infection and precedes viral replication, and a second that requires replication in the skin, enabling transit to the bloodstream. Depletion of Ly6C monocytes reduces local chikungunya (CHIKV) or Ross River virus (RRV) infection in the skin, diminishes the second phase of virus transport to the DLN, and delays spread to distal sites. Our data suggest that infiltrating monocytes facilitate alphavirus infection at the initial infection site, which promotes more rapid spread into circulation.
Topics: Animals; Monocytes; Chikungunya virus; Mosquito Vectors; Chikungunya Fever; Myeloid Cells; Virus Replication
PubMed: 38446669
DOI: 10.1016/j.celrep.2024.113876 -
Frontiers in Immunology 2023Interferon (IFN) responses are critical in the resolution of viral infections and are actively targeted by many viruses. They also play a role in inducing protective...
BACKGROUND
Interferon (IFN) responses are critical in the resolution of viral infections and are actively targeted by many viruses. They also play a role in inducing protective responses after vaccination and have been successfully tested as vaccine adjuvants. IFN responses are well conserved and function very similar in teleosts and mammals. Like in mammals, IFN responses in piscine cells are initiated by intracellular detection of the viral infection by different pattern recognition receptors. Upon the recognition of viral components, IFN responses are rapidly induced to combat the infection. However, many viruses may still replicate and be able to inhibit or circumvent the IFN response by different means.
METHODS
By employing CRISPR Cas9 technology, we have disrupted proteins that are central for IFN signaling in the salmonid cell line CHSE-214. We successfully generated KO clones for the mitochondrial antiviral signaling protein MAVS, the transcription factors IRF3 and IRF7-1, as well as a double KO for IRF7-1/3 using an optimized protocol for delivery of CRISPR-Cas ribonucleoproteins through nucleofection.
RESULTS
We found that MAVS and IRF3 KOs inhibited IFN and IFN-stimulated gene induction after intracellular poly I:C stimulation as determined through gene expression and promoter activation assays. In contrast, the IRF7-1 KO had no clear effect. This shows that MAVS and IRF3 are essential for initiation of intracellular RNA-induced IFN responses in CHSE-214 cells. To elucidate viral interference with IFN induction pathways, the KOs were infected with Salmon alphavirus 3 (SAV3) and infectious pancreatic necrosis virus (IPNV). SAV3 infection in control and IRF7-1 KO cells yielded similar titers and no cytopathic effect, while IRF3 and MAVS KOs presented with severe cytopathic effect and increased titers 6 days after SAV 3 infection. In contrast, IPNV yields were reduced in IRF3 and MAVS KOs, suggesting a dependency on interactions between viral proteins and pattern recognition receptor signaling components during viral replication.
CONCLUSION
Aside from more insight in this signaling in salmonids, our results indicate a possible method to increase viral titers in salmonid cells.
Topics: Animals; Salmonidae; CRISPR-Cas Systems; Signal Transduction; Cell Line; Salmon; Infectious pancreatic necrosis virus; Mammals
PubMed: 37588594
DOI: 10.3389/fimmu.2023.1214912 -
Trends in Genetics : TIG May 2024Positive-strand RNA [(+)RNA] viruses include pandemic SARS-CoV-2, tumor-inducing hepatitis C virus, debilitating chikungunya virus (CHIKV), lethal encephalitis viruses,... (Review)
Review
Positive-strand RNA [(+)RNA] viruses include pandemic SARS-CoV-2, tumor-inducing hepatitis C virus, debilitating chikungunya virus (CHIKV), lethal encephalitis viruses, and many other major pathogens. (+)RNA viruses replicate their RNA genomes in virus-induced replication organelles (ROs) that also evolve new viral species and variants by recombination and mutation and are crucial virus control targets. Recent cryo-electron microscopy (cryo-EM) reveals that viral RNA replication proteins form striking ringed 'crowns' at RO vesicle junctions with the cytosol. These crowns direct RO vesicle formation, viral (-)RNA and (+)RNA synthesis and capping, innate immune escape, and transfer of progeny (+)RNA genomes into translation and encapsidation. Ongoing studies are illuminating crown assembly, sequential functions, host factor interactions, etc., with significant implications for control and beneficial uses of viruses.
PubMed: 38724328
DOI: 10.1016/j.tig.2024.04.003 -
Cell Reports Aug 2023Venezuelan equine encephalitis virus (VEEV) is an encephalitic alphavirus responsible for epidemics of neurological disease across the Americas. Low-density lipoprotein...
Venezuelan equine encephalitis virus (VEEV) is an encephalitic alphavirus responsible for epidemics of neurological disease across the Americas. Low-density lipoprotein receptor class A domain-containing 3 (LDLRAD3) is a recently reported entry receptor for VEEV. Here, using wild-type and Ldlrad3-deficient mice, we define a critical role for LDLRAD3 in controlling steps in VEEV infection, pathogenesis, and neurotropism. Our analysis shows that LDLRAD3 is required for efficient VEEV infection and pathogenesis prior to and after central nervous system invasion. Ldlrad3-deficient mice survive intranasal and intracranial VEEV inoculation and show reduced infection of neurons in different brain regions. As LDLRAD3 is a determinant of pathogenesis and an entry receptor required for VEEV infection of neurons of the brain, receptor-targeted therapies may hold promise as countermeasures.
Topics: Animals; Mice; Brain; Central Nervous System; Encephalitis Virus, Venezuelan Equine; Encephalomyelitis, Venezuelan Equine; Receptors, LDL
PubMed: 37556325
DOI: 10.1016/j.celrep.2023.112946 -
PLoS Pathogens Nov 2023Hepatitis C virus (HCV) exploits the four entry factors CD81, scavenger receptor class B type I (SR-BI, also known as SCARB1), occludin, and claudin-1 as well as the...
Hepatitis C virus (HCV) exploits the four entry factors CD81, scavenger receptor class B type I (SR-BI, also known as SCARB1), occludin, and claudin-1 as well as the co-factor epidermal growth factor receptor (EGFR) to infect human hepatocytes. Here, we report that the disintegrin and matrix metalloproteinase 10 (ADAM10) associates with CD81, SR-BI, and EGFR and acts as HCV host factor. Pharmacological inhibition, siRNA-mediated silencing and genetic ablation of ADAM10 reduced HCV infection. ADAM10 was dispensable for HCV replication but supported HCV entry and cell-to-cell spread. Substrates of the ADAM10 sheddase including epidermal growth factor (EGF) and E-cadherin, which activate EGFR family members, rescued HCV infection of ADAM10 knockout cells. ADAM10 did not influence infection with other enveloped RNA viruses such as alphaviruses and a common cold coronavirus. Collectively, our study reveals a critical role for the sheddase ADAM10 as a HCV host factor, contributing to EGFR family member transactivation and as a consequence to HCV uptake.
Topics: Humans; Hepacivirus; Scavenger Receptors, Class B; Virus Internalization; Hepatitis C; Carrier Proteins; ErbB Receptors; Tetraspanin 28; ADAM10 Protein; Membrane Proteins; Amyloid Precursor Protein Secretases
PubMed: 37967063
DOI: 10.1371/journal.ppat.1011759 -
Vaccines Mar 2024SARS-CoV-2 virus, the causative agent of COVID-19, has produced the largest pandemic in the 21st century, becoming a very serious health problem worldwide. To prevent... (Review)
Review
SARS-CoV-2 virus, the causative agent of COVID-19, has produced the largest pandemic in the 21st century, becoming a very serious health problem worldwide. To prevent COVID-19 disease and infection, a large number of vaccines have been developed and approved in record time, including new vaccines based on mRNA encapsulated in lipid nanoparticles. While mRNA-based vaccines have proven to be safe and effective, they are more expensive to produce compared to conventional vaccines. A special type of mRNA vaccine is based on self-amplifying RNA (saRNA) derived from the genome of RNA viruses, mainly alphaviruses. These saRNAs encode a viral replicase in addition to the antigen, usually the SARS-CoV-2 spike protein. The replicase can amplify the saRNA in transfected cells, potentially reducing the amount of RNA needed for vaccination and promoting interferon I responses that can enhance adaptive immunity. Preclinical studies with saRNA-based COVID-19 vaccines in diverse animal models have demonstrated the induction of robust protective immune responses, similar to conventional mRNA but at lower doses. Initial clinical trials have confirmed the safety and immunogenicity of saRNA-based vaccines in individuals that had previously received authorized COVID-19 vaccines. These findings have led to the recent approval of two of these vaccines by the national drug agencies of India and Japan, underscoring the promising potential of this technology.
PubMed: 38543952
DOI: 10.3390/vaccines12030318 -
Journal of Virology Aug 2023Arthropod-borne viruses (arboviruses) are an emerging and evolving global public health threat, with limited antiviral treatments or vaccines available. La Crosse virus...
Arthropod-borne viruses (arboviruses) are an emerging and evolving global public health threat, with limited antiviral treatments or vaccines available. La Crosse virus (LACV) from the order is responsible for pediatric encephalitis cases in the United States, yet little is known about the infectivity of LACV. Given the structural similarities between class II fusion glycoproteins of LACV and chikungunya virus (CHIKV), an alphavirus from the family, we hypothesized that LACV would share similar entry mechanisms with CHIKV. To test this hypothesis, we performed cholesterol-depletion and repletion assays and used cholesterol-modulating compounds to study LACV entry and replication. We found that LACV entry was cholesterol dependent, while replication was less affected by cholesterol manipulation. In addition, we generated single-point mutants in the LACV Gc loop that corresponded to known CHIKV residues important for virus entry. We found that a conserved histidine and alanine residue in the Gc loop impaired virus infectivity and attenuated LACV replication and . Finally, we took an evolution-based approach to explore how the LACV glycoprotein evolves in mosquitoes and mice. We found multiple variants that cluster in the Gc glycoprotein head domain, providing evidence for the Gc glycoprotein as a contributor to LACV adaptation. Together, these results begin to characterize the mechanisms of LACV infectivity and how the LACV glycoprotein contributes to replication and pathogenesis. IMPORTANCE Vector-borne viruses are significant health threats that lead to devastating disease worldwide. The emergence of arboviruses, in addition to the lack of effective antivirals or vaccines, highlights the need to study how arboviruses replicate at the molecular level. One potential antiviral target is the class II fusion glycoprotein. Alphaviruses, flaviviruses, and bunyaviruses encode a class II fusion glycoprotein that contains strong structural similarities at the tip of domain II. Here, we show that the bunyavirus La Crosse virus uses a cholesterol-dependent entry pathway similar to the alphavirus chikungunya virus, and residues in the loop are important for virus infectivity and replication in mice. These studies show that genetically diverse viruses may use similar pathways through conserved structure domains, suggesting that these viruses may be targets for broad-spectrum antivirals in multiple arboviral families.
Topics: Animals; Mice; Antiviral Agents; Arboviruses; Encephalitis, California; Glycoproteins; La Crosse virus; Mosquito Vectors; United States; Virus Replication
PubMed: 37578236
DOI: 10.1128/jvi.00819-23 -
Vaccine Oct 2023Chikungunya virus (CHIKV) is an alphavirus transmitted by mosquitos that causes a debilitating disease characterized by fever and long-lasting polyarthralgia. To date,...
Chikungunya virus (CHIKV) is an alphavirus transmitted by mosquitos that causes a debilitating disease characterized by fever and long-lasting polyarthralgia. To date, no vaccine has been licensed, but multiple vaccine candidates are under evaluation in clinical trials. One of these vaccines is based on a measles virus vector encoding for the CHIKV structural genes C, E3, E2, 6K, and E1 (MV-CHIK), which proved safe in phase I and II clinical trials and elicited CHIKV-specific antibody responses in adult measles seropositive vaccine recipients. Here, we predicted T-cell epitopes in the CHIKV structural genes and investigated whether MV-CHIK vaccination induced CHIKV-specific CD4 and/or CD8 T-cell responses. Immune-dominant regions containing multiple epitopes in silico predicted to bind to HLA class II molecules were found for four of the five structural proteins, while no such regions were predicted for HLA class I. Experimentally, CHIKV-specific CD4 T-cells were detected in six out of twelve participants after a single MV-CHIK vaccination and more robust responses were found 4 weeks after two vaccinations (ten out of twelve participants). T-cells were mainly directed against the three large structural proteins C, E2 and E1. Next, we sorted and expanded CHIKV-specific T cell clones (TCC) and identified human CHIKV T-cell epitopes by deconvolution. Interestingly, eight out of nine CD4 TCC recognized an epitope in accordance with the in silico prediction. CHIKV-specific CD8 T-cells induced by MV-CHIK vaccination were inconsistently detected. Our data show that the MV-CHIK vector vaccine induced a functional transgene-specific CD4 T cell response which, together with the evidence of neutralizing antibodies as correlate of protection for CHIKV, makes MV-CHIK a promising vaccine candidate in the prevention of chikungunya.
Topics: Adult; Humans; Antibodies, Neutralizing; Antibodies, Viral; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Chikungunya Fever; Chikungunya virus; Epitopes, T-Lymphocyte; Measles Vaccine; Measles virus; Viral Vaccines
PubMed: 37726181
DOI: 10.1016/j.vaccine.2023.09.022