Field-deployable viral diagnostic tools for dengue virus based on Cas13a and Cas12a.

The diagnosis of dengue virus (DENV) has been challenging particularly in areas far from clinical laboratories. Early diagnosis of pathogens is a prerequisite for the timely treatment and pathogen control. An ideal diagnostic for viral infections should possess high sensitivity, specificity, and flexibility. In this study, we implemented dual amplification involving Cas13a and Cas12a, enabling sensitive and visually aided diagnostics for the dengue virus. Cas13a recognized the target RNA by crRNA and formed the assembly of the Cas13a/crRNA/RNA ternary complex, engaged in collateral cleavage of nearby crRNA of Cas12a. The Cas12a/crRNA/dsDNA activator ternary complex could not be assembled due to the absence of crRNA of Cas12a. Moreover, the probe, with 5' and 3' termini labeled with FAM and biotin, could not be separated. The probes labeled with FAM and biotin, combined the Anti-FAM and the Anti-Biotin Ab-coated gold nanoparticle, and conformed sandwich structure on the T-line. The red line on the paper strip caused by clumping of AuNPs on the T-line indicated the detection of dengue virus. This technique, utilizing an activated Cas13a system cleaving the crRNA of Cas12a, triggered a cascade that amplifies the virus signal, achieving a low detection limit of 190 fM with fluorescence. Moreover, even at 1 pM, the red color on the T-line was easily visible by naked eyes. The developed strategy, incorporating cascade enzymatic amplification, exhibited good sensitivity and may serve as a field-deployable diagnostic tool for dengue virus.

Human cytomegalovirus microRNAs: strategies for immune evasion and viral latency.

Viruses use various strategies and mechanisms to deal with cells and proteins of the immune system that form a barrier against infection. One of these mechanisms is the encoding and production of viral microRNAs (miRNAs), whose function is to regulate the gene expression of the host cell and the virus, thus creating a suitable environment for survival and spreading viral infection. miRNAs are short, single-stranded, non-coding RNA molecules that can regulate the expression of host and viral proteins, and due to their non-immunogenic nature, they are not eliminated by the cells of the immune system. More than half of the viral miRNAs are encoded and produced by Orthoherpesviridae family members. Human cytomegalovirus (HCMV) produces miRNAs that mediate various processes in infected cells to contribute to HCMV pathogenicity, including immune escape, viral latency, and cell apoptosis. Here, we discuss which cellular and viral proteins or cellular pathways and processes these mysterious molecules target to evade immunity and support viral latency in infected cells. We also discuss current evidence that their function of bypassing the host's innate and adaptive immune system is essential for the survival and multiplication of the virus and the spread of HCMV infection.

ICTV Virus Taxonomy Profile: Phasmaviridae 2024.

Phasmaviridae is a family for negative-sense RNA viruses with genomes of about 9.7-15.8 kb. These viruses are maintained in and/or transmitted by insects. Phasmavirids produce enveloped virions containing three single-stranded RNA segments that encode a nucleoprotein (N), a glycoprotein precursor (GPC), and a large (L) protein containing an RNA-directed RNA polymerase (RdRP) domain. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Phasmaviridae, which is available at ictv.global/report/phasmaviridae.

A Pilot Seroprevalence Study Suggests Silent Zika virus Transmission in Bhopal Region of Central India.

BACKGROUND: Several sporadic cases and outbreaks of Zika virus disease have been reported from different states of India. OBJECTIVES: This paper explored the possibility of any ongoing transmission of Zika virus (ZIKV) in the Bhopal region of Central India, where the last outbreak of this disease was reported in 2018. MATERIALS AND METHODS: We screened a group of 75 febrile patients who had already tested negative for the locally endemic causes of fever like dengue, chikungunya, enteric fever, malaria, and scrub typhus and two groups of asymptomatic healthy individuals represented by blood donors (n = 75) and antenatal mothers (n = 75). We tested blood samples of febrile patients for ZIKV RNA using real-time polymerase chain reaction (PCR), and for the healthy individuals, we determined anti-zika immunoglobulin G (IgG) antibodies using enzyme-linked immunosorbent assay. RESULTS: ZIKV RNA was not detected in any of the 75 samples tested by real-time PCR assay. Among the voluntary blood donors and antenatal mothers, a total of 10 (15.38%) and 5 (6.66%) individuals were found to be seropositive for anti-ZIKV IgG antibodies, respectively. The seropositive group was found to have higher age 33.06 (±10.83) years as compared to seronegative individuals 26.60 (±5.12) years (P = 0.037). CONCLUSION: This study, which is the first survey of seroprevalence of anti-Zika antibodies from India, reports an overall seropositivity rate of 10% for anti-Zika antibodies among the healthy population, suggesting an ongoing, low level, silent transmission of ZIKV in the local community.

Comprehensive survey of conserved RNA secondary structures in full-genome alignment of Hepatitis C virus.

Hepatitis C virus (HCV) is a plus-stranded RNA virus that often chronically infects liver hepatocytes and causes liver cirrhosis and cancer. These viruses replicate their genomes employing error-prone replicases. Thereby, they routinely generate a large 'cloud' of RNA genomes (quasispecies) which-by trial and error-comprehensively explore the sequence space available for functional RNA genomes that maintain the ability for efficient replication and immune escape. In this context, it is important to identify which RNA secondary structures in the sequence space of the HCV genome are conserved, likely due to functional requirements. Here, we provide the first genome-wide multiple sequence alignment (MSA) with the prediction of RNA secondary structures throughout all representative full-length HCV genomes. We selected 57 representative genomes by clustering all complete HCV genomes from the BV-BRC database based on k-mer distributions and dimension reduction and adding RefSeq sequences. We include annotations of previously recognized features for easy comparison to other studies. Our results indicate that mainly the core coding region, the C-terminal NS5A region, and the NS5B region contain secondary structure elements that are conserved beyond coding sequence requirements, indicating functionality on the RNA level. In contrast, the genome regions in between contain less highly conserved structures. The results provide a complete description of all conserved RNA secondary structures and make clear that functionally important RNA secondary structures are present in certain HCV genome regions but are largely absent from other regions. Full-genome alignments of all branches of Hepacivirus C are provided in the supplement.

Bacillaceae serine proteases and Streptomyces epsilon-poly-L-lysine synergistically inactivate Caliciviridae by inhibiting RNA genome release.

Human norovirus (HuNoV) is an enteric infectious pathogen belonging to the Caliciviridae family that causes occasional epidemics. Circulating alcohol-tolerant viral particles that are readily transmitted via food-borne routes significantly contribute to the global burden of HuNoV-induced gastroenteritis. Moreover, contact with enzymes secreted by other microorganisms in the environment can impact the infectivity of viruses. Hence, understanding the circulation dynamics of Caliciviridae is critical to mitigating epidemics. Accordingly, in this study, we screened whether environmentally abundant secretase components, particularly proteases, affect Caliciviridae infectivity. Results showed that combining Bacillaceae serine proteases with epsilon-poly-L-lysine (EPL) produced by Streptomyces-a natural antimicrobial-elicited anti-Caliciviridae properties, including against the epidemic HuNoV GII.4_Sydney_2012 strain. In vitro and in vivo biochemical and virological analyses revealed that EPL has two unique synergistic viral inactivation functions. First, it maintains an optimal pH to promote viral surface conformational changes to the protease-sensitive structure. Subsequently, it inhibits viral RNA genome release via partial protease digestion at the P2 and S domains in the VP1 capsid. This study provides new insights regarding the high-dimensional environmental interactions between bacteria and Caliciviridae, while promoting the development of protease-based anti-viral disinfectants.

Tissue-based T cell activation and viral RNA persist for up to 2 years after SARS-CoV-2 infection.

The mechanisms of postacute medical conditions and unexplained symptoms after SARS-CoV-2 infection [Long Covid (LC)] are incompletely understood. There is growing evidence that viral persistence, immune dysregulation, and T cell dysfunction may play major roles. We performed whole-body positron emission tomography imaging in a well-characterized cohort of 24 participants at time points ranging from 27 to 910 days after acute SARS-CoV-2 infection using the radiopharmaceutical agent [18F]F-AraG, a selective tracer that allows for anatomical quantitation of activated T lymphocytes. Tracer uptake in the postacute COVID-19 group, which included those with and without continuing symptoms, was higher compared with prepandemic controls in many regions, including the brain stem, spinal cord, bone marrow, nasopharyngeal and hilar lymphoid tissue, cardiopulmonary tissues, and gut wall. T cell activation in the spinal cord and gut wall was associated with the presence of LC symptoms. In addition, tracer uptake in lung tissue was higher in those with persistent pulmonary symptoms specifically. Increased T cell activation in these tissues was also observed in many individuals without LC. Given the high [18F]F-AraG uptake detected in the gut, we obtained colorectal tissue for in situ hybridization of SARS-CoV-2 RNA and immunohistochemical studies in a subset of five participants with LC symptoms. We identified intracellular SARS-CoV-2 single-stranded spike protein-encoding RNA in rectosigmoid lamina propria tissue in all five participants and double-stranded spike protein-encoding RNA in three participants up to 676 days after initial COVID-19, suggesting that tissue viral persistence could be associated with long-term immunologic perturbations.

Genetic diversity of astroviruses detected in wild aquatic birds in Hong Kong.

Wild waterfowl serve as a reservoir of some astroviruses. Fecal samples from wild waterfowl collected at Hong Kong's Marshes were tested using pan-astrovirus reverse transcription-PCR. Positive samples underwent subsequent host identification using DNA barcoding. Based on deduced partial sequences, noteworthy samples from three astrovirus groups (mammalian, avian and unclassified astroviruses) were further analyzed by next-generation sequencing. One sample of Avastrovirus 4 clade, MP22-196, had a nearly complete genome identified. The results of ORF2 phylogenetic analysis and genetic distance analysis indicate that Avastrovirus 4 is classified as a distinct subclade within Avastrovirus. MP22-196 has typical astrovirus genome characteristics. The unique characteristics and potential differences of this genome, compared to other avian astrovirus sequences, involve the identification of a modified sgRNA sequence situated near the ORF2 start codon, which precedes the ORF1b stop codon. Additionally, the 3' UTR of MP22-196 is shorter than other avian astroviruses. This study expands our understanding of the Avastrovirus 4 clade.

Circulating immune and plasma biomarkers of time to HIV rebound in HIV controllers treated with vesatolimod.

Background: Antiretroviral therapy (ART) for HIV-1 treatment has improved lifespan but requires lifelong adherence for people living with HIV (PLWH), highlighting the need for a cure. Evaluation of potential cure strategies requires analytic treatment interruption (ATI) with close monitoring of viral rebound. Predictive biomarkers for HIV-1 rebound and/or duration of control during ATI will facilitate these HIV cure trials while minimizing risks. Available evidence suggests that host immune, glycomic, lipid, and metabolic markers of inflammation may be associated with HIV-1 persistence in PLWH who are treated during chronic HIV-1 infection. Methods: We conducted post-hoc analysis of HIV controllers who could maintain low levels of plasma HIV-1 without ART in a phase 1b vesatolimod trial. Baseline and pre-ATI levels of immune, glycomic, lipidomic, and metabolomic markers were tested for association with ATI outcomes (time of HIV-1 rebound to 200 copies/mL and 1,000 copies/mL, duration of HIV-1 RNA ≤400 copies/mL and change in intact proviral HIV-1 DNA during ATI) using Spearman's correlation and Cox proportional hazards model. Results: Higher levels of CD69+CD8+ T-cells were consistently associated with shorter time to HIV-1 rebound at baseline and pre-ATI. With few exceptions, baseline fucosylated, non-galactosylated, non-sialylated, bisecting IgG N-glycans were associated with shorter time to HIV rebound and duration of control as with previous studies. Baseline plasma MPA and HPA binding glycans and non-galactosylated/non-sialylated glycans were associated with longer time to HIV rebound, while baseline multiply-galactosylated glycans and sialylated glycans, GNA-binding glycans, NPA-binding glycans, WGA-binding glycans, and bisecting GlcNAc glycans were associated with shorter time to HIV rebound and duration of control. Fourteen bioactive lipids had significant baseline associations with longer time to rebound and duration of control, and larger intact proviral HIV-1 DNA changes; additionally, three baseline bioactive lipids were associated with shorter time to first rebound and duration of control. Conclusion: Consistent with studies in HIV non-controllers, proinflammatory glycans, lipids, and metabolites were generally associated with shorter duration of HIV-1 control. Notable differences were observed between HIV controllers vs. non-controllers in some specific markers. For the first time, exploratory biomarkers of ATI viral outcomes in HIV-controllers were investigated but require further validation.

Phylogenetic and mutational analysis of H10N3 avian influenza A virus in China: potential threats to human health.

In recent years, the avian influenza virus has emerged as a significant threat to both human and public health. This study focuses on a patient infected with the H10N3 subtype of avian influenza virus, admitted to the Third People's Hospital of Kunming City on March 6, 2024. Metagenomic RNA sequencing and polymerase chain reaction (PCR) analysis were conducted on the patient's sputum, confirming the H10N3 infection. The patient presented severe pneumonia symptoms such as fever, expectoration, chest tightness, shortness of breath, and cough. Phylogenetic analysis of the Haemagglutinin (HA) and neuraminidase (NA) genes of the virus showed that the virus was most closely related to a case of human infection with the H10N3 subtype of avian influenza virus found in Zhejiang Province, China. Analysis of amino acid mutation sites identified four mutations potentially hazardous to human health. Consequently, this underscores the importance of continuous and vigilant monitoring of the dynamics surrounding the H10N3 subtype of avian influenza virus, utilizing advanced genomic surveillance techniques.

Caenorhabditis elegans RIG-I-like receptor DRH-1 signals via CARDs to activate antiviral immunity in intestinal cells.

Upon sensing viral RNA, mammalian RIG-I-like receptors (RLRs) activate downstream signals using caspase activation and recruitment domains (CARDs), which ultimately promote transcriptional immune responses that have been well studied. In contrast, the downstream signaling mechanisms for invertebrate RLRs are much less clear. For example, the Caenorhabditis elegans RLR DRH-1 lacks annotated CARDs and up-regulates the distinct output of RNA interference. Here, we found that similar to mammal RLRs, DRH-1 signals through two tandem CARDs (2CARD) to induce a transcriptional immune response. Expression of DRH-1(2CARD) alone in the intestine was sufficient to induce immune gene expression, increase viral resistance, and promote thermotolerance, a phenotype previously associated with immune activation in C. elegans. We also found that DRH-1 is required in the intestine to induce immune gene expression, and we demonstrate subcellular colocalization of DRH-1 puncta with double-stranded RNA inside the cytoplasm of intestinal cells upon viral infection. Altogether, our results reveal mechanistic and spatial insights into antiviral signaling in C. elegans, highlighting unexpected parallels in RLR signaling between C. elegans and mammals.

Complete genome sequence of a novel amalgavirus in sponge gourd, Luffa cylindrica.

A novel monopartite dsRNA virus, tentatively named "sponge gourd amalgavirus 1" (SGAV1), was discovered by high-throughput sequencing in sponge gourd (Luffa cylindrica) displaying mosaic symptoms in Jiashan County, Zhejiang Province, China. The genome of SGAV1 is 3,447 nucleotides in length and contains partially overlapping open reading frames (ORFs) encoding a putative replication factory matrix-like protein and a fusion protein, respectively. The fusion protein of SGAV1 shares 57.07% identity with the homologous protein of salvia miltiorrhiza amalgavirus 1 (accession no. DAZ91057.1). Phylogenetic analysis based on the RNA-dependent RNA polymerase (RdRp) protein suggests that SGAV1 belongs to the genus Amalgavirus of the family Amalgaviridae. Moreover, analysis of SGAV1-derived small interfering RNAs indicated that SGAV1 was actively replicating in the host plant. Semi-quantitative RT-PCR showed higher levels of SGAV1 expression in leaves than in flowers and fruits. This is the first report of a novel amalgavirus found in sponge gourd in China.

Complete genome sequence of a novel botourmiavirus infecting the fungus Phomopsis asparagi.

Here, we report a novel ourmia-like mycovirus, named "Phomopsis asparagi magoulivirus 1" (PaMV1), derived from the phytopathogenic fungus Phomopsis asparagi. The genome of PaMV1 consists of a positive-sense single-stranded RNA (+ ssRNA) that is 2,639 nucleotides in length, with a GC content of 57.13%. It contains a single open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) consisting of 686 amino acids with a molecular mass of 78.57 kDa. Phylogenetic analysis based on RdRp sequences revealed that PaMV1 grouped together with Diaporthe gulyae magoulivirus 1 (DgMV1) in a distinct clade. Sequence comparisons and phylogenetic analysis suggest that PaMV1 is a novel member of the genus Magoulivirus, family Botourmiaviridae.

An improved alphaviruses-specific RT-qPCR facilitates monitoring and prevention of alphaviruses.

Molecular surveillance is vital for monitoring arboviruses, often employing genus-specific quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Despite this, an overlooked chikungunya fever outbreak occurred in Yunnan province, China, in 2019 and false negatives are commonly encountered during alphaviruses screening practice, highlighting the need for improved detection methods. In this study, we developed an improved alphaviruses-specific RT-qPCR capable of detecting chikungunya virus, eastern equine encephalitis virus, western equine encephalitis virus, Venezuelan equine encephalitis virus, Sindbis virus, Mayaro virus, and Ross River virus with high sensitivity and specificity. The assay identified three chikungunya virus-positive cases out of 188 sera retrospectively. Later genetic characterization suggested that imported cases from neighboring countries may be responsible for the neglected chikungunya fever outbreak of 2019 in Yunnan. Our findings underscore the value of improved alphaviruses-specific RT-qPCR in bolstering alphaviruses surveillance and informing preventive strategies.

A comparison study of temporal trends of SARS-CoV2 RNAemia and biomarkers to predict success and failure of high flow oxygen therapy among patients with moderate to severe COVID-19.

Optimal timing for intubating patients with coronavirus disease 2019 (COVID-19) has been debated throughout the pandemic. Early use of high-flow nasal cannula (HFNC) can help reduce the need for intubation, but delay can result in poorer outcomes. This study examines trends in laboratory parameters and serum severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA levels of patients with COVID-19 in relation to HFNC failure. Patients requiring HFNC within three days of hospitalization between July 1 and September 30, 2021 were enrolled. The primary outcome was HFNC failure (early failure ≤Day 3; late failure ≥Day 4), defined as transfer to intensive care just before/after intubation or in-hospital death. We examined changes in laboratory markers and SARS-CoV2-RNAemia on Days 1, 4, and 7, together with demographic data, oxygenation status, and therapeutic agents. We conducted a univariate logistic regression with the explanatory variables defined as 10% change rate in each laboratory marker from Day 1 to 4. We utilized the log-rank test to assess the differences in HFNC failure rates, stratified based on the presence of SARS-CoV2 RNAemia. Among 122 patients, 17 (13.9%) experienced HFNC failure (early: n = 6, late: n = 11). Seventy-five patients (61.5%) showed an initial SpO2/FiO2 ratio ≤243, equivalent to PaO2/FiO2 ratio ≤200, and the initial SpO2/FiO2 ratio was significantly lower in the failure group (184 vs. 218, p = 0.018). Among the laboratory markers, a 10% increase from Day 1 to 4 of lactate dehydrogenase (LDH) and interleukin (IL)-6 was associated with late failure (Odds ratio [OR]: 1.42, 95% confidence interval [CI]: 1.09-1.89 and OR: 1.04, 95%CI: 1.00-1.19, respectively). Furthermore, in patients with persistent RNAemia on Day 4 or 7, the risk of late HFNC failure was significantly higher (Log-rank test, p<0.01). In conclusion, upward trends in LDH and IL-6 levels and the persistent RNAemia even after treatment were associated with HFNC failure.

Assessment of performance and comparison of three commercial HDV RNA detection assays: implications for diagnosis and treatment monitoring.

Objectives: Precise HDV-RNA detection and quantification are pivotal for diagnosis and monitoring of response to newly approved treatment. We evaluate the performance of three HDV RNA detection and quantification assays. Methods: Hepatitis Delta RT-PCR system kit, EurobioPlex HDV assay, and RoboGene HDV RNA Quantification kit 2.0 were used for testing 151 HBsAg-positive samples, 90 HDV-RNA negative and 61 HDV-RNA positive. We also evaluated serial dilutions of the WHO international standard for HDV, PEI 7657/12. All HDV-RNA positive samples were genotyped using a next-generation sequencing strategy. Results: Qualitative results indicated a 100% concordance between tests. Quantitative results correlated well, r2 = 0.703 (Vircell-vs-Eurobio), r2 = 0.833 (Vircell-vs-RoboGene), r2 = 0.835 (Robogene-vs-Eurobio). Bias index was 2.083 (Vircell-vs-Eurobio), -1.283 (Vircell-vs-RoboGene), and -3.36 (Robogene-vs-Eurobio). Using the WHO IS, Vircell overestimated the viral load by 0.98 log IU/mL, Eurobio by 1.46 log IU/mL, and RoboGene underestimated it by 0.98 log IU/mL. Fifty-nine samples were successfully genotyped (Genotype 1, n=52; Genotype 5, n=7; Genotype 6, n=1), with similar results for correlation and bias. Conclusion: This study underscores the necessity of using reliable HDV-RNA detection and quantification assays, as evidenced by the high concordance rates in qualitative detection and the observed variability in quantitative results. These findings highlight the importance of consistent assay use in clinical practice to ensure accurate diagnosis and effective treatment monitoring of HDV infection.

Point mutations at specific sites of the nsp12-nsp8 interface dramatically affect the RNA polymerization activity of SARS-CoV-2.

In a recent characterization of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variability present in 30 diagnostic samples from patients of the first COVID-19 pandemic wave, 41 amino acid substitutions were documented in the RNA-dependent RNA polymerase (RdRp) nsp12. Eight substitutions were selected in this work to determine whether they had an impact on the RdRp activity of the SARS-CoV-2 nsp12-nsp8-nsp7 replication complex. Three of these substitutions were found around the polymerase central cavity, in the template entry channel (D499G and M668V), and within the motif B (V560A), and they showed polymerization rates similar to the wild type RdRp. The remaining five mutations (P323L, L372F, L372P, V373A, and L527H) were placed near the nsp12-nsp8F contact surface; residues L372, V373, and L527 participated in a large hydrophobic cluster involving contacts between two helices in the nsp12 fingers and the long α-helix of nsp8F. The presence of any of these five amino acid substitutions resulted in important alterations in the RNA polymerization activity. Comparative primer elongation assays showed different behavior depending on the hydrophobicity of their side chains. The substitution of L by the bulkier F side chain at position 372 slightly promoted RdRp activity. However, this activity was dramatically reduced with the L372P, and L527H mutations, and to a lesser extent with V373A, all of which weaken the hydrophobic interactions within the cluster. Additional mutations, specifically designed to disrupt the nsp12-nsp8F interactions (nsp12-V330S, nsp12-V341S, and nsp8-R111A/D112A), also resulted in an impaired RdRp activity, further illustrating the importance of this contact interface in the regulation of RNA synthesis.

The West Nile virus genome harbors essential riboregulatory elements with conserved and host-specific functional roles.

West Nile virus (WNV) is an arthropod-borne, positive-sense RNA virus that poses an increasing global threat due to warming climates and lack of effective therapeutics. Like other enzootic viruses, little is known about how host context affects the structure of the full-length RNA genome. Here, we report a complete secondary structure of the entire WNV genome within infected mammalian and arthropod cell lines. Our analysis affords structural insights into multiple, conserved aspects of flaviviral biology. We show that the WNV genome folds with minimal host dependence, and we prioritize well-folded regions for functional validation using structural homology between hosts as a guide. Using structure-disrupting, antisense locked nucleic acids, we then demonstrate that the WNV genome contains riboregulatory structures with conserved and host-specific functional roles. These results reveal promising RNA drug targets within flaviviral genomes, and they highlight the therapeutic potential of ASO-LNAs as both WNV-specific and pan-flaviviral therapeutic agents.

IFIH1 (MDA5) is required for innate immune detection of intron-containing RNA expressed from the HIV-1 provirus.

Intron-containing RNA expressed from the HIV-1 provirus activates type 1 interferon in primary human blood cells, including CD4+ T cells, macrophages, and dendritic cells. To identify the innate immune receptor required for detection of intron-containing RNA expressed from the HIV-1 provirus, a loss-of-function screen was performed with short hairpin RNA-expressing lentivectors targeting twenty-one candidate genes in human monocyte-derived dendritic cells. Among the candidate genes tested, only knockdown of XPO1 (CRM1), IFIH1 (MDA5), or MAVS prevented activation of the interferon-stimulated gene ISG15. The importance of IFIH1 protein was demonstrated by rescue of the knockdown with nontargetable IFIH1 coding sequence. Inhibition of HIV-1-induced ISG15 by the IFIH1-specific Nipah virus V protein, and by IFIH1-transdominant 2-CARD domain-deletion or phosphomimetic point mutations, indicates that IFIH1 (MDA5) filament formation, dephosphorylation, and association with MAVS are all required for innate immune activation in response to HIV-1 transduction. Since both IFIH1 (MDA5) and DDX58 (RIG-I) signal via MAVS, the specificity of HIV-1 RNA detection by IFIH1 was demonstrated by the fact that DDX58 knockdown had no effect on activation. RNA-Seq showed that IFIH1 knockdown in dendritic cells globally disrupted the induction of IFN-stimulated genes by HIV-1. Finally, specific enrichment of unspliced HIV-1 RNA by IFIH1 (MDA5), over two orders of magnitude, was revealed by formaldehyde cross-linking immunoprecipitation (f-CLIP). These results demonstrate that IFIH1 is the innate immune receptor for intron-containing RNA from the HIV-1 provirus and that IFIH1 potentially contributes to chronic inflammation in people living with HIV-1, even in the presence of effective antiretroviral therapy.

Comparative pathogenesis of three Mayaro virus genotypes in the cynomolgus macaque.

Mayaro virus (MAYV), a mosquito-borne alphavirus, is considered an emerging threat to public health with epidemic potential. Phylogenetic studies show the existence of three MAYV genotypes. In this study, we provide a preliminary analysis of the pathogenesis of all three MAYV genotypes in cynomolgus macaques (Macaca facicularis, Mauritian origin). Significant MAYV-specific RNAemia and viremia were detected during acute infection in animals challenged intravenously with the three MAYV genotypes, and strong neutralizing antibody responses were observed. MAYV RNA was detected at high levels in lymphoid tissues, joint muscle and synovia over 1 month after infection, suggesting that this model could serve as a promising tool in studying MAYV-induced chronic arthralgia, which can persist for years. Significant leucopenia was observed across all MAYV genotypes, peaking with RNAemia. Notable differences in the severity of acute RNAemia and composition of cytokine responses were observed among the three MAYV genotypes. Our model showed no outward signs of clinical disease, but several major endpoints for future MAYV pathology and intervention studies are described. Disruptions to normal blood cell counts and cytokine responses were markedly distinct from those observed in macaque models of CHIKV infection, underlining the importance of developing non-human primate models specific to MAYV infection.