-
Journal of Virology Mar 2024Lymphocytic choriomeningitis virus (LCMV) is a bisegmented negative-sense RNA virus classified within the family of the order. LCMV is associated with fatal disease in...
UNLABELLED
Lymphocytic choriomeningitis virus (LCMV) is a bisegmented negative-sense RNA virus classified within the family of the order. LCMV is associated with fatal disease in immunocompromized populations, and as the prototypical arenavirus, acts as a model for the many serious human pathogens within this group. Here, we examined the dependence of LCMV multiplication on cellular trafficking components using a recombinant LCMV expressing enhanced green fluorescent protein in conjunction with a curated siRNA library. The screen revealed a requirement for subunits of both the coat protein 1 (COPI) coatomer and adapter protein 4 (AP-4) complexes. By rescuing a recombinant LCMV harboring a FLAG-tagged glycoprotein (GP-1) envelope spike (rLCMV-GP1-FLAG), we showed infection resulted in marked co-localization of individual COPI and AP-4 components with both LCMV nucleoprotein (NP) and GP-1, consistent with their involvement in viral processes. To further investigate the role of both COPI and AP-4 complexes during LCMV infection, we utilized the ARF-I inhibitor brefeldin A (BFA) that prevents complex formation. Within a single 12-h cycle of virus multiplication, BFA pre-treatment caused no significant change in LCMV-specific RNA synthesis, alongside no significant change in LCMV NP expression, as measured by BFA time-of-addition experiments. In contrast, BFA addition resulted in a significant drop in released virus titers, approaching 50-fold over the same 12-h period, rising to over 600-fold over 24 h. Taken together, these findings suggest COPI and AP-4 complexes are important host cell factors required for the formation and release of infectious LCMV.
IMPORTANCE
Arenaviruses are rodent-borne, segmented, negative-sense RNA viruses, with several members responsible for fatal human disease, with the prototypic member lymphocytic choriomeningitis virus (LCMV) being under-recognised as a pathogen capable of inflicting neurological infections with fatal outcome. A detailed understanding of how arenaviruses subvert host cell processes to complete their multiplication cycle is incomplete. Here, using a combination of gene ablation and pharmacological inhibition techniques, we showed that host cellular COPI and AP-4 complexes, with native roles in cellular vesicular transport, were required for efficient LCMV growth. We further showed these complexes acted on late stages of the multiplication cycle, post-gene expression, with a significant impact on infectious virus egress. Collectively, our findings improve the understanding of arenaviruses host-pathogen interactions and reveal critical cellular trafficking pathways required during infection.
Topics: Animals; Humans; Chlorocebus aethiops; Lymphocytic Choriomeningitis; Lymphocytic choriomeningitis virus; Vero Cells; Virus Replication; Adaptor Protein Complex 4; Coat Protein Complex I
PubMed: 38334330
DOI: 10.1128/jvi.02006-23 -
Nature Microbiology Mar 2024Infection with Lassa virus (LASV) can cause Lassa fever, a haemorrhagic illness with an estimated fatality rate of 29.7%, but causes no or mild symptoms in many...
Infection with Lassa virus (LASV) can cause Lassa fever, a haemorrhagic illness with an estimated fatality rate of 29.7%, but causes no or mild symptoms in many individuals. Here, to investigate whether human genetic variation underlies the heterogeneity of LASV infection, we carried out genome-wide association studies (GWAS) as well as seroprevalence surveys, human leukocyte antigen typing and high-throughput variant functional characterization assays. We analysed Lassa fever susceptibility and fatal outcomes in 533 cases of Lassa fever and 1,986 population controls recruited over a 7 year period in Nigeria and Sierra Leone. We detected genome-wide significant variant associations with Lassa fever fatal outcomes near GRM7 and LIF in the Nigerian cohort. We also show that a haplotype bearing signatures of positive selection and overlapping LARGE1, a required LASV entry factor, is associated with decreased risk of Lassa fever in the Nigerian cohort but not in the Sierra Leone cohort. Overall, we identified variants and genes that may impact the risk of severe Lassa fever, demonstrating how GWAS can provide insight into viral pathogenesis.
Topics: Humans; Lassa Fever; Genome-Wide Association Study; Seroepidemiologic Studies; Lassa virus; Fever; Human Genetics
PubMed: 38326571
DOI: 10.1038/s41564-023-01589-3 -
Cell Reports Feb 2024Viral pandemics and epidemics pose a significant global threat. While macaque models of viral disease are routinely used, it remains unclear how conserved antiviral...
Viral pandemics and epidemics pose a significant global threat. While macaque models of viral disease are routinely used, it remains unclear how conserved antiviral responses are between macaques and humans. Therefore, we conducted a cross-species analysis of transcriptomic data from over 6,088 blood samples from macaques and humans infected with one of 31 viruses. Our findings demonstrate that irrespective of primate or viral species, there are conserved antiviral responses that are consistent across infection phase (acute, chronic, or latent) and viral genome type (DNA or RNA viruses). Leveraging longitudinal data from experimental challenges, we identify virus-specific response kinetics such as host responses to Coronaviridae and Orthomyxoviridae infections peaking 1-3 days earlier than responses to Filoviridae and Arenaviridae viral infections. Our results underscore macaque studies as a powerful tool for understanding viral pathogenesis and immune responses that translate to humans, with implications for viral therapeutic development and pandemic preparedness.
Topics: Animals; Humans; Immunoinformatics; Orthomyxoviridae Infections; Filoviridae; Macaca; Antiviral Agents
PubMed: 38294906
DOI: 10.1016/j.celrep.2024.113706 -
Journal of Virology Feb 2024The highly pathogenic arenavirus, Junín virus (JUNV), expresses three truncated alternative isoforms of its nucleoprotein (NP), i.e., NP, NP, and NP. While both NP and...
Alternative translation contributes to the generation of a cytoplasmic subpopulation of the Junín virus nucleoprotein that inhibits caspase activation and innate immunity.
The highly pathogenic arenavirus, Junín virus (JUNV), expresses three truncated alternative isoforms of its nucleoprotein (NP), i.e., NP, NP, and NP. While both NP and NP have been previously shown to be products of caspase cleavage, here, we show that expression of the third isoform NP is due to alternative in-frame translation from M80. Based on this information, we were able to generate recombinant JUNVs lacking each of these isoforms. Infection with these mutants revealed that, while all three isoforms contribute to the efficient control of caspase activation, NP plays the predominant role. In contrast to full-length NP (i.e., NP), which is localized to inclusion bodies, where viral RNA synthesis takes place, the loss of portions of the N-terminal coiled-coil region in these isoforms leads to a diffuse cytoplasmic distribution and a loss of function in viral RNA synthesis. Nonetheless, NP, NP, and NP all retain robust interferon antagonistic and 3'-5' exonuclease activities. We suggest that the altered localization of these NP isoforms allows them to be more efficiently targeted by activated caspases for cleavage as decoy substrates, and to be better positioned to degrade viral double-stranded (ds)RNA species that accumulate in the cytoplasm during virus infection and/or interact with cytosolic RNA sensors, thereby limiting dsRNA-mediated innate immune responses. Taken together, this work provides insight into the mechanism by which JUNV leverages apoptosis during infection to generate biologically distinct pools of NP and contributes to our understanding of the expression and biological relevance of alternative protein isoforms during virus infection.IMPORTANCEA limited coding capacity means that RNA viruses need strategies to diversify their proteome. The nucleoprotein (NP) of the highly pathogenic arenavirus Junín virus (JUNV) produces three N-terminally truncated isoforms: two (NP and NP) are known to be produced by caspase cleavage, while, here, we show that NP is produced by alternative translation initiation. Recombinant JUNVs lacking individual NP isoforms revealed that all three isoforms contribute to inhibiting caspase activation during infection, but cleavage to generate NP makes the biggest contribution. Importantly, all three isoforms retain their ability to digest double-stranded (ds)RNA and inhibit interferon promoter activation but have a diffuse cytoplasmic distribution. Given the cytoplasmic localization of both aberrant viral dsRNAs, as well as dsRNA sensors and many other cellular components of innate immune activation pathways, we suggest that the generation of NP isoforms not only contributes to evasion of apoptosis but also robust control of the antiviral response.
Topics: Humans; Apoptosis; Caspase Inhibitors; Caspases; Cytoplasm; Enzyme Activation; Hemorrhagic Fever, American; Host-Pathogen Interactions; Immunity, Innate; Interferons; Junin virus; Nucleoproteins; Protein Biosynthesis; Protein Isoforms; RNA, Double-Stranded; RNA, Viral; Virus Replication
PubMed: 38294249
DOI: 10.1128/jvi.01975-23 -
Journal of Virology Feb 2024Guanarito virus (GTOV) is the causative agent of Venezuelan hemorrhagic fever. GTOV belongs to the genus , family and has been classified as a Category A bioterrorism...
Development of reverse genetics system for Guanarito virus: substitution of E1497K in the L protein of Guanarito virus S-26764 strain changes plaque phenotype and growth kinetics.
Guanarito virus (GTOV) is the causative agent of Venezuelan hemorrhagic fever. GTOV belongs to the genus , family and has been classified as a Category A bioterrorism agent by the United States Centers for Disease Control and Prevention. Despite being a high-priority agent, vaccines and drugs against Venezuelan hemorrhagic fever are not available. GTOV S-26764, isolated from a non-fatal human case, produces an unclear cytopathic effect (CPE) in Vero cells, posing a significant obstacle to research and countermeasure development efforts. Vero cell-adapted GTOV S-26764 generated in this study produced clear CPE and demonstrated rapid growth and high yield in Vero cells compared to the original GTOV S-26764. We developed a reverse genetics system for GTOV to study amino acid changes acquired through Vero cell adaptation and leading to virus phenotype changes. The results demonstrated that E1497K in the L protein was responsible for the production of clear plaques as well as enhanced viral RNA replication and transcription efficiency. Vero cell-adapted GTOV S-26764, capable of generating CPE, will allow researchers to easily perform neutralization assays and anti-drug screening against GTOV. Moreover, the developed reverse genetics system will accelerate vaccine and antiviral drug development.IMPORTANCEGuanarito virus (GTOV) is a rodent-borne virus. GTOV causes fever, prostration, headache, arthralgia, cough, sore throat, nausea, vomiting, diarrhea, epistaxis, bleeding gums, menorrhagia, and melena in humans. The lethality rate is 23.1% or higher. Vero cell-adapted GTOV S-26764 shows a clear cytopathic effect (CPE), whereas the parental virus shows unclear CPE in Vero cells. We generated a reverse genetics system to rescue recombinant GTOVs and found that E1497K in the L protein was responsible for the formation of clear plaques as well as enhanced viral RNA replication and transcription efficiency. This reverse genetic system will accelerate vaccine and antiviral drug developments, and the findings of this study contribute to the understanding of the function of GTOV L as an RNA polymerase.
Topics: Animals; Female; Humans; Arenaviridae; Arenaviridae Infections; Arenaviruses, New World; Chlorocebus aethiops; Hemorrhagic Fevers, Viral; Phenotype; Reverse Genetics; Vaccines; Vero Cells
PubMed: 38289100
DOI: 10.1128/jvi.01964-23 -
Cell Reports. Medicine Feb 2024Lassa fever (LF) is an acute viral illness that causes thousands of deaths annually in West Africa. There are currently no Lassa virus (LASV) vaccines or antivirals...
Lassa fever (LF) is an acute viral illness that causes thousands of deaths annually in West Africa. There are currently no Lassa virus (LASV) vaccines or antivirals approved for human use. Recently, we showed that combinations of broadly neutralizing human monoclonal antibodies (BNhuMAbs) known as Arevirumab-2 or Arevirumab-3 protected up to 100% of cynomolgus macaques against challenge with diverse lineages of LASV when treatment was initiated at advanced stages of disease. This previous work assessed efficacy against parenteral exposure. However, transmission of LASV to humans occurs primarily by mucosal exposure to virus shed from Mastomys rodents. Here, we describe the development of a lethal intranasal exposure macaque model of LF. This model is employed to show that Arevirumab cocktails rescue 100% of macaques from lethal LASV infection when treatment is initiated 8 days after LASV exposure. Our work demonstrates BNhuMAbs have utility in treating LASV infection acquired through mucosal exposure.
Topics: Animals; Humans; Lassa virus; Lassa Fever; Macaca fascicularis; Immunotherapy; Antibodies, Monoclonal
PubMed: 38280377
DOI: 10.1016/j.xcrm.2024.101392 -
Emerging Infectious Diseases Feb 2024We identified a novel lineage of lymphocytic choriomeningitis virus, tentatively named lineage V, in wood mice (Apodemus sylvaticus) from Germany. Wood mouse-derived...
We identified a novel lineage of lymphocytic choriomeningitis virus, tentatively named lineage V, in wood mice (Apodemus sylvaticus) from Germany. Wood mouse-derived lymphocytic choriomeningitis virus can be found across a substantially greater range than previously thought. Increased surveillance is needed to determine its geographic range and zoonotic potential.
Topics: Mice; Animals; Lymphocytic choriomeningitis virus; Germany
PubMed: 38270110
DOI: 10.3201/eid3002.230868 -
Viruses Jan 2024Metagenomic analysis of and mosquitoes from diverse geographical regions of India revealed the presence of several insect viruses of human interest. Most abundant...
Metagenomic analysis of and mosquitoes from diverse geographical regions of India revealed the presence of several insect viruses of human interest. Most abundant reads found in mosquitoes were of Phasi Charoen-like virus (PCLV), granulovirus (CfGV), Cell fusing agent virus (CFAV), and Wenzhou sobemo-like virus 4 (WSLV4), whereas WSLV4 and CfGV constituted the highest percentage of reads in viromes. Other reads that were of low percentage included Hubei mosquito virus 2 (HMV2), Porcine astrovirus 4 (PAstV4), and Wild Boar astrovirus (WBAstV). PCLV and CFAV, which were found to be abundant in . viromes were absent in viromes. Among the viromes analyzed, sampled from Pune showed the highest percentage (79.82%) of viral reads, while . mosquitoes sampled from Dibrugarh showed the lowest percentage (3.47%). Shamonda orthobunyavirus (SHAV), African swine fever virus (ASFV), Aroa virus (AROAV), and Ilheus virus (ILHV), having the potential to infect vertebrates, including humans, were also detected in both mosquito species, albeit with low read numbers. Reads of gemykibivirus, avian retrovirus, bacteriophages, herpesviruses, and viruses infecting protozoans, algae, etc., were also detected in the mosquitoes. A high percentage of reads in the mosquito samples belonged to unclassified viruses and warrant further investigation. The data generated in the present work may not only lead to studies to explain the influence of these viruses on the replication and transmission of viruses of clinical importance but also to find applications as biocontrol agents against pathogenic viruses.
Topics: Animals; Swine; Humans; Aedes; African Swine Fever Virus; Virome; India; Bacteriophages; Arenaviridae; Granulovirus
PubMed: 38257809
DOI: 10.3390/v16010109 -
Epidemiology and Infection Jan 2024Lymphocytic choriomeningitis virus (LCMV) is one of the arenaviruses infecting humans. LCMV infections have been reported worldwide in humans with varying levels of...
Lymphocytic choriomeningitis virus (LCMV) is one of the arenaviruses infecting humans. LCMV infections have been reported worldwide in humans with varying levels of severity. To detect arenavirus RNA and LCMV-reactive antibodies in different geographical regions of Finland, we screened human serum and cerebrospinal fluid (CSF) samples, taken from suspected tick-borne encephalitis (TBE) cases, using reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence assay (IFA). No arenavirus nucleic acids were detected, and the overall LCMV seroprevalence was 4.5%. No seroconversions were detected in paired serum samples. The highest seroprevalence (5.2%) was detected among individuals of age group III (40-59 years), followed by age group I (under-20-year-olds, 4.9%), while the lowest seroprevalence (3.8%) was found in age group IV (60 years or older). A lower LCMV seroprevalence in older age groups may suggest waning of immunity over time. The observation of a higher seroprevalence in the younger age group and the decreasing population size of the main reservoir host, the house mouse, may suggest exposure to another LCMV-like virus in Finland.
Topics: Animals; Mice; Humans; Aged; Adult; Middle Aged; Encephalitis, Tick-Borne; Finland; Seroepidemiologic Studies; Lymphocytic Choriomeningitis; Lymphocytic choriomeningitis virus; Antibodies, Viral
PubMed: 38250808
DOI: 10.1017/S0950268824000128 -
Journal of Immunology (Baltimore, Md. :... Mar 2024Chronic viral infections, such as HIV and hepatitis C virus, represent a major public health problem. Although it is well understood that neonates and adults respond...
Chronic viral infections, such as HIV and hepatitis C virus, represent a major public health problem. Although it is well understood that neonates and adults respond differently to chronic viral infections, the underlying mechanisms remain unknown. In this study, we transferred neonatal and adult CD8+ T cells into a mouse model of chronic infection (lymphocytic choriomeningitis virus clone 13) and dissected out the key cell-intrinsic differences that alter their ability to protect the host. Interestingly, we found that neonatal CD8+ T cells preferentially became effector cells early in chronic infection compared with adult CD8+ T cells and expressed higher levels of genes associated with cell migration and effector cell differentiation. During the chronic phase of infection, the neonatal cells retained more immune functionality and expressed lower levels of surface markers and genes related to exhaustion. Because the neonatal cells protect from viral replication early in chronic infection, the altered differentiation trajectories of neonatal and adult CD8+ T cells is functionally significant. Together, our work demonstrates how cell-intrinsic differences between neonatal and adult CD8+ T cells influence key cell fate decisions during chronic infection.
Topics: Mice; Animals; Lymphocytic Choriomeningitis; Persistent Infection; Lymphocytic choriomeningitis virus; CD8-Positive T-Lymphocytes; Cell Differentiation; Mice, Inbred C57BL; Chronic Disease
PubMed: 38231127
DOI: 10.4049/jimmunol.2300396