A volatile from the skin microbiota of flavivirus-infected hosts promotes mosquito attractiveness.

The host-seeking activity of hematophagous arthropods is essential for arboviral transmission. Here, we demonstrate that mosquito-transmitted flaviviruses can manipulate host skin microbiota to produce a scent that attracts mosquitoes. We observed that Aedes mosquitoes preferred to seek and feed on mice infected by dengue and Zika viruses. Acetophenone, a volatile compound that is predominantly produced by the skin microbiota, was enriched in the volatiles from the infected hosts to potently stimulate mosquito olfaction for attractiveness. Of note, acetophenone emission was higher in dengue patients than in healthy people. Mechanistically, flaviviruses infection suppressed the expression of RELMα, an essential antimicrobial protein on host skin, thereby leading to the expansion of acetophenone-producing commensal bacteria and, consequently, a high acetophenone level. Given that RELMα can be specifically induced by a vitamin A derivative, the dietary administration of isotretinoin to flavivirus-infected animals interrupted flavivirus life cycle by reducing mosquito host-seeking activity, thus providing a strategy of arboviral control.

A mosquito salivary protein-driven influx of myeloid cells facilitates flavivirus transmission.

Mosquitoes transmit many disease-relevant flaviviruses. Efficient viral transmission to mammalian hosts requires mosquito salivary factors. However, the specific salivary components facilitating viral transmission and their mechanisms of action remain largely unknown. Here, we show that a female mosquito salivary gland-specific protein, here named A. aegypti Neutrophil Recruitment Protein (AaNRP), facilitates the transmission of Zika and dengue viruses. AaNRP promotes a rapid influx of neutrophils, followed by virus-susceptible myeloid cells toward mosquito bite sites, which facilitates establishment of local infection and systemic dissemination. Mechanistically, AaNRP engages TLR1 and TLR4 of skin-resident macrophages and activates MyD88-dependent NF-κB signaling to induce the expression of neutrophil chemoattractants. Inhibition of MyD88-NF-κB signaling with the dietary phytochemical resveratrol reduces AaNRP-mediated enhancement of flavivirus transmission by mosquitoes. These findings exemplify how salivary components can aid viral transmission, and suggest a potential prophylactic target.

An evolutionarily conserved ubiquitin ligase drives infection and transmission of flaviviruses.

Mosquito-borne flaviviruses such as dengue (DENV) and Zika (ZIKV) cause hundreds of millions of infections annually. The single-stranded RNA genome of flaviviruses is translated into a polyprotein, which is cleaved equally into individual functional proteins. While structural proteins are packaged into progeny virions and released, most of the nonstructural proteins remain intracellular and could become cytotoxic if accumulated over time. However, the mechanism by which nonstructural proteins are maintained at the levels optimal for cellular fitness and viral replication remains unknown. Here, we identified that the ubiquitin E3 ligase HRD1 is essential for flaviviruses infections in both mammalian hosts and mosquitoes. HRD1 directly interacts with flavivirus NS4A and ubiquitylates a conserved lysine residue for ER-associated degradation. This mechanism avoids excessive accumulation of NS4A, which otherwise interrupts the expression of processed flavivirus proteins in the ER. Furthermore, a small-molecule inhibitor of HRD1 named LS-102 effectively interrupts DENV2 infection in both mice and Aedes aegypti mosquitoes, and significantly disturbs DENV transmission from the infected hosts to mosquitoes owing to reduced viremia. Taken together, this study demonstrates that flaviviruses have evolved a sophisticated mechanism to exploit the ubiquitination system to balance the homeostasis of viral proteins for their own advantage and provides a potential therapeutic target to interrupt flavivirus infection and transmission.

ELF4 is critical for induction of type I interferon and the host antiviral response.

Induction of type I interferon is a central event of innate immunity, essential for host defense. Here we report that the transcription factor ELF4 is induced by type I interferon and upregulates interferon expression in a feed-forward loop. ELF4 deficiency leads to reduced interferon production, resulting in enhanced susceptibility to West Nile virus encephalitis in mice. After viral infection, ELF4 is recruited by STING, interacts with and is activated by the MAVS-TBK1 complex, and translocates into the nucleus to bind interferon promoters. Cooperative binding with ELF4 increases the binding affinity of interferon regulatory factors IRF3 and IRF7, which is mediated by EICE elements. Thus, in addition to identifying a regulator of innate immune signaling, we uncovered a role for EICE elements in interferon transactivation.

A C-type lectin collaborates with a CD45 phosphatase homolog to facilitate West Nile virus infection of mosquitoes.

West Nile virus (WNV) is the most common arthropod-borne flavivirus in the United States; however, the vector ligand(s) that participate in infection are not known. We now show that an Aedes aegypti C-type lectin, mosGCTL-1, is induced by WNV, interacts with WNV in a calcium-dependent manner, and facilitates infection in vivo and in vitro. A mosquito homolog of human CD45 in A. aegypti, designated mosPTP-1, recruits mosGCTL-1 to enable viral attachment to cells and to enhance viral entry. In vivo experiments show that mosGCTL-1 and mosPTP-1 function as part of the same pathway and are critical for WNV infection of mosquitoes. A similar phenomenon was also observed in Culex quinquefasciatus, a natural vector of WNV, further demonstrating that these genes participate in WNV infection. During the mosquito blood-feeding process, WNV infection was blocked in vivo with mosGCTL-1 antibodies. A molecular understanding of flaviviral-arthropod interactions may lead to strategies to control viral dissemination in nature.

Lipases secreted by a gut bacterium inhibit arbovirus transmission in mosquitoes.

Arboviruses are etiological agents of various severe human diseases that place a tremendous burden on global public health and the economy; compounding this issue is the fact that effective prophylactics and therapeutics are lacking for most arboviruses. Herein, we identified 2 bacterial lipases secreted by a Chromobacterium bacterium isolated from Aedes aegypti midgut, Chromobacterium antiviral effector-1 (CbAE-1) and CbAE-2, with broad-spectrum virucidal activity against mosquito-borne viruses, such as dengue virus (DENV), Zika virus (ZIKV), Japanese encephalitis virus (JEV), yellow fever virus (YFV) and Sindbis virus (SINV). The CbAEs potently blocked viral infection in the extracellular milieu through their lipase activity. Mechanistic studies showed that this lipase activity directly disrupted the viral envelope structure, thus inactivating infectivity. A mutation in the lipase motif of CbAE-1 fully abrogated the virucidal ability. Furthermore, CbAEs also exert lipase-dependent entomopathogenic activity in mosquitoes. The anti-arboviral and entomopathogenic properties of CbAEs render them potential candidates for the development of novel transmission control strategies against vector-borne diseases.

Neighboring mutation-mediated enhancement of dengue virus infectivity and spread.

Frequent turnover of dengue virus (DENV) clades is one of the major forces driving DENV persistence and prevalence. In this study, we assess the fitness advantage of nine stable substitutions within the envelope (E) protein of DENV serotypes. Two tandem neighboring substitutions, threonine to lysine at the 226th (T226K) and glycine to glutamic acid at the 228th (G228E) residues in the DENV2 Asian I genotype, enhance virus infectivity in either mosquitoes or mammalian hosts, thereby promoting clades turnover and dengue epidemics. Mechanistic studies indicate that the substitution-mediated polarity changes in these two residues increase the binding affinity of E for host C-type lectins. Accordingly, we predict that a G228E substitution could potentially result in a forthcoming epidemic of the DENV2 Cosmopolitan genotype. Investigations into the substitutions associated with DENV fitness in hosts may offer mechanistic insights into dengue prevalence, thus providing a warning of potential epidemics in the future.

A mutation-mediated evolutionary adaptation of Zika virus in mosquito and mammalian host.

Zika virus (ZIKV) caused millions of infections during its rapid and expansive spread from Asia to the Americas from 2015 to 2017. Here, we compared the infectivity of ZIKV mutants with individual stable substitutions which emerged throughout the Asian ZIKV lineage and were responsible for the explosive outbreaks in the Americas. A threonine (T) to alanine (A) mutation at the 106th residue of the ZIKV capsid (C) protein facilitated the transmission by its mosquito vector, as well as infection in both human cells and immunodeficient mice. A mechanistic study showed that the T106A substitution rendered the C a preferred substrate for the NS2B-NS3 protease, thereby facilitating the maturation of structural proteins and the formation of infectious viral particles. Over a complete "mosquito-mouse-mosquito" cycle, the ZIKV C-T106A mutant showed a higher prevalence of mosquito infection than did the preepidemic strain, thus promoting ZIKV dissemination. Our results support the contribution of this evolutionary adaptation to the occasional widespread reemergence of ZIKV in nature.

Mechanisms of SARS-CoV-2 Transmission and Pathogenesis.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) marks the third highly pathogenic coronavirus to spill over into the human population. SARS-CoV-2 is highly transmissible with a broad tissue tropism that is likely perpetuating the pandemic. However, important questions remain regarding its transmissibility and pathogenesis. In this review, we summarize current SARS-CoV-2 research, with an emphasis on transmission, tissue tropism, viral pathogenesis, and immune antagonism. We further present advances in animal models that are important for understanding the pathogenesis of SARS-CoV-2, vaccine development, and therapeutic testing. When necessary, comparisons are made from studies with SARS to provide further perspectives on coronavirus infectious disease 2019 (COVID-19), as well as draw inferences for future investigations.

Autophagy ameliorates Pseudomonas aeruginosa-infected diabetic wounds by regulating the toll-like receptor 4/myeloid differentiation factor 88 pathway.

Diabetic foot ulcers (DFUs) are among the most common complications in patients with diabetes and a leading cause of lower extremity amputation. DFUs are exacerbated by prolonged bacterial infection; therefore, there is an urgent need for effective treatments to alleviate the burden associated with this condition. Although autophagy plays a unique role in pathogen phagocytosis and inflammation, its role in diabetic foot infections (DFIs) remains unclear. Pseudomonas aeruginosa (PA) is the most frequently isolated gram-negative bacterium from DFUs. Here, we evaluated the role of autophagy in ameliorating PA infection in wounds in a diabetic rat model and a bone marrow-derived macrophage (BMDM) hyperglycemia model. Both models were pretreated with or without rapamycin (RAPA) and then infected with or without PA. Pretreatment of rats with RAPA significantly enhanced PA phagocytosis, suppressed wound inflammation, reduced the M1:M2 macrophage ratio, and improved wound healing. In vitro investigation of the underlying mechanisms revealed that enhanced autophagy resulted in decreased macrophage secretion of inflammatory factors such as TNF-α, IL-6, and IL-1ß but increased that of IL-10 in response to PA infection. Additionally, RAPA treatment significantly enhanced autophagy in macrophages by increasing LC3 and beclin-1 levels, which led to altered macrophage function. Furthermore, RAPA blocked the PA-induced TLR4/MyD88 pathway to regulate macrophage polarisation and inflammatory cytokine production, which was validated by RNA interference and use of the autophagy inhibitor 3-methyladenine (3-MA). These findings suggest enhancing autophagy as a novel therapeutic strategy against PA infection to ultimately improve diabetic wound healing.

UBX Domain Protein 6 Positively Regulates JAK-STAT1/2 Signaling.

Type I/III IFNs induce expression of hundreds of IFN-stimulated genes through the JAK/STAT pathway to combat viral infections. Although JAK/STAT signaling is seemingly straightforward, it is nevertheless subjected to complex cellular regulation. In this study, we show that an ubiquitination regulatory X (UBX) domain-containing protein, UBXN6, positively regulates JAK-STAT1/2 signaling. Overexpression of UBXN6 enhanced type I/III IFNs-induced expression of IFN-stimulated genes, whereas deletion of UBXN6 inhibited their expression. RNA viral replication was increased in human UBXN6-deficient cells, accompanied by a reduction in both type I/III IFN expression, when compared with UBXN6-sufficient cells. Mechanistically, UBXN6 interacted with tyrosine kinase 2 (TYK2) and inhibited IFN-ß-induced degradation of both TYK2 and type I IFNR. These results suggest that UBXN6 maintains normal JAK-STAT1/2 signaling by stabilizing key signaling components during viral infection.

Evolutionary enhancement of Zika virus infectivity in Aedes aegypti mosquitoes.

Zika virus (ZIKV) remained obscure until the recent explosive outbreaks in French Polynesia (2013-2014) and South America (2015-2016). Phylogenetic studies have shown that ZIKV has evolved into African and Asian lineages. The Asian lineage of ZIKV was responsible for the recent epidemics in the Americas. However, the underlying mechanisms through which ZIKV rapidly and explosively spread from Asia to the Americas are unclear. Non-structural protein 1 (NS1) facilitates flavivirus acquisition by mosquitoes from an infected mammalian host and subsequently enhances viral prevalence in mosquitoes. Here we show that NS1 antigenaemia determines ZIKV infectivity in its mosquito vector Aedes aegypti, which acquires ZIKV via a blood meal. Clinical isolates from the most recent outbreak in the Americas were much more infectious in mosquitoes than the FSS13025 strain, which was isolated in Cambodia in 2010. Further analyses showed that these epidemic strains have higher NS1 antigenaemia than the FSS13025 strain because of an alanine-to-valine amino acid substitution at residue 188 in NS1. ZIKV infectivity was enhanced by this amino acid substitution in the ZIKV FSS13025 strain in mosquitoes that acquired ZIKV from a viraemic C57BL/6 mouse deficient in type I and II interferon (IFN) receptors (AG6 mouse). Our results reveal that ZIKV evolved to acquire a spontaneous mutation in its NS1 protein, resulting in increased NS1 antigenaemia. Enhancement of NS1 antigenaemia in infected hosts promotes ZIKV infectivity and prevalence in mosquitoes, which could have facilitated transmission during recent ZIKV epidemics.

Nlrp9b inflammasome restricts rotavirus infection in intestinal epithelial cells.

Rotavirus, a leading cause of severe gastroenteritis and diarrhoea in young children, accounts for around 215,000 deaths annually worldwide. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling, raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo, especially by NOD-like receptor (NLR) inflammasomes, is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that, via RNA helicase Dhx9, Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens.

Baseline Gut Microbiome Signatures Correlate with Immunogenicity of SARS-CoV-2 mRNA Vaccines.

The powerful immune responses elicited by the mRNA vaccines targeting the SARS-CoV-2 Spike protein contribute to their high efficacy. Yet, their efficacy can vary greatly between individuals. For vaccines not based on mRNA, cumulative evidence suggests that differences in the composition of the gut microbiome, which impact vaccine immunogenicity, are some of the factors that contribute to variations in efficacy. However, it is unclear if the microbiome impacts the novel mode of immunogenicity of the SARS-CoV-2 mRNA vaccines. We conducted a prospective longitudinal cohort study of individuals receiving SARS-CoV-2 mRNA vaccines where we measured levels of anti-Spike IgG and characterized microbiome composition, at pre-vaccination (baseline), and one week following the first and second immunizations. While we found that microbial diversity at all timepoints correlated with final IgG levels, only at baseline did microbial composition and predicted function correlate with vaccine immunogenicity. Specifically, the phylum Desulfobacterota and genus Bilophila, producers of immunostimulatory LPS, positively correlated with IgG, while Bacteroides was negatively correlated. KEGG predicted pathways relating to SCFA metabolism and sulfur metabolism, as well as structural components such as flagellin and capsular polysaccharides, also positively correlated with IgG levels. Consistent with these findings, depleting the microbiome with antibiotics reduced the immunogenicity of the BNT162b2 vaccine in mice. These findings suggest that gut microbiome composition impacts the immunogenicity of the SARS-CoV-2 mRNA vaccines.

Caspase-12 controls West Nile virus infection via the viral RNA receptor RIG-I.

Caspase-12 has been shown to negatively modulate inflammasome signaling during bacterial infection. Its function in viral immunity, however, has not been characterized. We now report an important role for caspase-12 in controlling viral infection via the pattern-recognition receptor RIG-I. After challenge with West Nile virus (WNV), caspase-12-deficient mice had greater mortality, higher viral burden and defective type I interferon response compared with those of challenged wild-type mice. In vitro studies of primary neurons and mouse embryonic fibroblasts showed that caspase-12 positively modulated the production of type I interferon by regulating E3 ubiquitin ligase TRIM25-mediated ubiquitination of RIG-I, a critical signaling event for the type I interferon response to WNV and other important viral pathogens.

National Benchmark Study of Employment Equity Among Multiply Marginalized Persons of Color with Disabilities During the COVID-19 Pandemic: A Bootstrap Approach.

BACKGROUND: Research examining state vocational rehabilitation agency (SVRA) sponsored service patterns during the COVID-19 pandemic is needed to improve employment outcomes among multiply marginalized persons of color with disabilities (i.e., African Americans, Asian Americans, Native Americans or Alaska Natives, Latinx, and Native Hawaiians/Pacific Islanders). Scarce attention has been paid to examining outcome inequities in the crisis. OBJECTIVE: This study applied a stratified bootstrap data expansion approach to assess the relationship between race/ethnicity, gender, level of educational attainment at closure and employment outcomes among target group members. METHODS: National fiscal year (FY) 2019 Rehabilitation Services Administration (RSA)-911 case records (N =114,229) closed between January 20, 2020 (date of first reported COVID-19 infection in the U.S.) to June 30 2020 were extracted and re-sampled across multiple trials using bootstrap procedures to increase logistic regression model accuracy. RESULTS: The findings indicated that African Americans, Asian Americans and Native American or Alaska Natives were statistically significantly less likely to achieve successful employment than non-Latinx Whites. Success probabilities in the COVID-19 pandemic were 'poorest' for Native American or Alaska Native VR consumers followed by African Americans, Asian Americans, Latinx, non-Latinx Whites, and then Native Hawaiians/Pacific Islanders. African Americans and Native Americans or Alaska Natives were more often closed unsuccessful because they could not be located when compared to non-Latinx Whites. CONCLUSIONS: These findings call for new targeted SVRA service initiatives.

A Critical Role for STING Signaling in Limiting Pathogenesis of Chikungunya Virus.

The stimulator of interferon gene (STING) pathway controls both DNA and RNA virus infection. STING is essential for induction of innate immune responses during DNA virus infection, while its mechanism against RNA virus remains largely elusive. We show that STING signaling is crucial for restricting chikungunya virus infection and arthritis pathogenesis. Sting-deficient mice (Stinggt/gt) had elevated viremia throughout the viremic stage and viral burden in feet transiently, with a normal type I IFN response. Stinggt/gt mice presented much greater foot swelling, joint damage, and immune cell infiltration than wild-type mice. Intriguingly, expression of interferon-γ and Cxcl10 was continuously upregulated by approximately 7 to 10-fold and further elevated in Stinggt/gt mice synchronously with arthritis progression. However, expression of chemoattractants for and activators of neutrophils, Cxcl5, Cxcl7, and Cxcr2 was suppressed in Stinggt/gt joints. These results demonstrate that STING deficiency leads to an aberrant chemokine response that promotes pathogenesis of CHIKV arthritis.

The GRA15 protein from Toxoplasma gondii enhances host defense responses by activating the interferon stimulator STING.

Toxoplasma gondii is an important neurotropic pathogen that establishes latent infections in humans that can cause toxoplasmosis in immunocompromised individuals. It replicates inside host cells and has developed several strategies to manipulate host immune responses. However, the cytoplasmic pathogen-sensing pathway that detects T. gondii is not well-characterized. Here, we found that cyclic GMP-AMP synthase (cGAS), a sensor of foreign dsDNA, is required for activation of anti-T. gondii immune signaling in a mouse model. We also found that mice deficient in STING (Stinggt/gt mice) are much more susceptible to T. gondii infection than WT mice. Of note, the induction of inflammatory cytokines, type I IFNs, and interferon-stimulated genes in the spleen from Stinggt/gt mice was significantly impaired. Stinggt/gt mice exhibited more severe symptoms than cGAS-deficient mice after T. gondii infection. Interestingly, we found that the dense granule protein GRA15 from T. gondii is secreted into the host cell cytoplasm and then localizes to the endoplasmic reticulum, mediated by the second transmembrane motif in GRA15, which is essential for activating STING and innate immune responses. Mechanistically, GRA15 promoted STING polyubiquitination at Lys-337 and STING oligomerization in a TRAF protein-dependent manner. Accordingly, GRA15-deficient T. gondii failed to elicit robust innate immune responses compared with WT T. gondii. Consequently, GRA15-/-T. gondii was more virulent and caused higher mortality of WT mice but not Stinggt/gt mice upon infection. Together, T. gondii infection triggers cGAS/STING signaling, which is enhanced by GRA15 in a STING- and TRAF-dependent manner.

Interferon-stimulated TRIM69 interrupts dengue virus replication by ubiquitinating viral nonstructural protein 3.

In order to eliminate viral infections, hundreds of interferon-stimulated genes (ISGs) are induced via type I interferons (IFNs). However, the functions and mechanisms of most ISGs are largely unclear. A tripartite motif (TRIM) protein encoding gene TRIM69 is induced by dengue virus (DENV) infection as an ISG. TRIM69 restricts DENV replication, and its RING domain, which has the E3 ubiquitin ligase activity, is critical for its antiviral activity. An in vivo study further confirmed that TRIM69 contributes to the control of DENV infection in immunocompetent mice. Unlike many other TRIM family members, TRIM69 is not involved in modulation of IFN signaling. Instead, TRIM69 interacts with DENV Nonstructural Protein 3 (NS3) directly and mediates its polyubiquitination and degradation. Finally, Lys104 of NS3 is identified as the target of TRIM69-mediated ubiquitination. Our study demonstrates that TRIM69 restricts DENV replication by specifically ubiquitinating a viral nonstructural protein.

Combination of Azithromycin and Gentamicin for Efficient Treatment of Pseudomonas aeruginosa Infections.

BACKGROUND: Trans-translation is a ribosome rescue system that plays an important role in bacterial tolerance to environmental stresses. It is absent in animals, making it a potential treatment target. However, its role in antibiotic tolerance in Pseudomonas aeruginosa remains unknown. METHODS: The role and activity of trans-translation during antibiotic treatment were examined with a trans-translation-deficient strain and a genetically modified trans-translation component gene, respectively. In vitro assays and murine infection models were used to examine the effects of suppression of trans-translation. RESULTS: We found that the trans-translation system plays an essential role in P. aeruginosa tolerance to azithromycin and multiple aminoglycoside antibiotics. We further demonstrated that gentamicin could suppress the azithromycin-induced activation of trans-translation. Compared with each antibiotic individually, gentamicin and azithromycin combined increased the killing efficacy against planktonic and biofilm-associated P. aeruginosa cells, including a reference strain PA14 and its isogenic carbapenem-resistance oprD mutant, the mucoid strain FRD1, and multiple clinical isolates. Furthermore, the gentamicin-azithromycin resulted in improved bacterial clearance in murine acute pneumonia, biofilm implant, and cutaneous abscess infection models. CONCLUSIONS: Combination treatment with gentamicin and azithromycin is a promising strategy in combating P. aeruginosa infections.