Who is perceived to be an expert on COVID-19 vaccination on social media? We conducted two experimental studies investigating how the presence of biomedical credentials on social media profiles impacts users’ perceived expertise. Participants (Experiment 1 N = 200; Experiment 2 N = 201) viewed a series of Twitter profiles that appeared with or without biomedical credentials and judged to what extent they believed each user was an expert on the topic of COVID-19 vaccination. We found that the presence of biomedical credentials consistently increased perceptions of expertise, including among unvaccinated, vaccine-hesitant, and conservative participants. This work supports existing observations that biomedical credentials may be leveraged by both pro- and anti-vaccine communities to increase perceived credibility and message reach, and counters the narrative that those with anti-vaccination attitudes do not recognize biomedical credentials as conferring expertise.

Background: Asia remains particularly vulnerable to new and emerging disease threats. During the COVID-19 pandemic, next generation sequencing (NGS) emerged as a powerful tool for pathogen surveillance. Understanding opportunities to accelerate adoption and scale is an urgent priority for regional health security. Methods: An end-to-end framework and semi-quantitative survey were developed to assess the national status of pathogen genomic surveillance. Cross-sectional surveys were conducted with institutions supporting pathogen genomics across countries in South and Southeast Asia with a focus on low-resource settings. Findings: Between June 2022 and March 2023, 42 institutions contributed data from 13 countries. All countries had NGS capacity and shared data through publicly available platforms, with expertise residing primarily in public health institutions and academic/research partners. Seven countries had national strategic plans that integrate pathogen genomics into wider surveillance efforts. Several pathogens are prioritized for human surveillance, including coronaviruses, tuberculosis, and influenza, with limited NGS application to environmental and human-animal interface surveillance. Barriers to NGS included a reliance on external funding, supply chain challenges, laboratory and bioinformatics capacity gaps, and limited quality assurance mechanisms. Established platforms sequenced at half capacity with a median delay of 18 days between sample collection and reporting. NGS capacity varies across countries, with most (9/13) in the early stages of NGS implementation. Interpretation: Pathogen genomics capacity exists across Asia, but its use is limited and under-resourced. Coordinated regional efforts are required to support national planning and system design, respond to supply chain challenges, address capacity gaps, enhance quality assurance and facilitate timely data sharing. Funding: The Bill & Melinda Gates Foundation (INV-037608), and the Duke-NUS Signature Research Programme funded by the Ministry of Health, Singapore Declaration of Interest: The authors declare no competing interests.

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that can have devastating health consequences. The developmental and neurological effects from a ZIKV infection arise in part from the virus triggering cellular stress pathways and perturbing transcriptional programs. To date, the underlying mechanisms of transcriptional control directing viral restriction and virus-host interaction are understudied. Activating Transcription Factor 3 (ATF3) is a stress-induced transcriptional effector that modulates the expression of genes involved in a myriad of cellular processes, including inflammation and antiviral responses, to restore cellular homeostasis. While ATF3 is known to be upregulated during ZIKV infection, the mode by which ATF3 is activated and the specific role of ATF3 during ZIKV infection is unknown. In this study, we show via inhibitor and RNA interference approaches that ZIKV infection initiates the integrated stress response pathway to activate ATF4 which in turn induces ATF3 expression. Additionally, by using CRISPR-Cas9 system to delete ATF3, we found that ATF3 acts to limit ZIKV gene expression in A549 cells. We also determined that ATF3 enhances the expression of antiviral genes such as STAT1 and other components in the innate immunity pathway to induce an ATF3-dependent anti-ZIKV response. Our study reveals crosstalk between the integrated stress response and innate immune response pathways and highlights an important role for ATF3 in establishing an antiviral effect during ZIKV infection. Importance ZIKV is a re-emerging mosquito-borne flavivirus that co-opts cellular mechanisms to support viral processes which can reprogram the host transcriptional profile. Such viral-directed transcriptional changes and the pro- or anti-viral outcomes remain understudied. We previously showed that ATF3, a stress-induced transcription factor, is significantly upregulated in ZIKV infected mammalian cells, along with other cellular and immune response genes. We now define the intracellular pathway responsible for ATF3 activation and elucidate the impact of ATF3 expression on ZIKV infection. We show that during ZIKV infection the integrated stress response pathway stimulates ATF3 which enhances the innate immune response to antagonize ZIKV infection. This study establishes a link between viral-induced stress response and transcriptional regulation of host defense pathways and thus expands our knowledge on virus-mediated transcriptional mechanisms and transcriptional control of interferon stimulated genes during ZIKV infection.

Zika virus (ZIKV) infection of central nervous system (CNS) tissue is associated with CNS inflammation, which contributes to ZIKV pathology. Similarly, ZIKV infection has been associated with increased vaginal and rectal mucosal inflammation. As mucosal dysfunction may contribute to elevated systemic inflammation, ZIKV-induced mucosal alterations could potentiate CNS disruptions, leading to ZIKV pathogenesis. However, the potential link between mucosal dysfunction, CNS inflammation and the underlying mechanisms causing these disruptions in ZIKV infection has not been well described. Here, we assessed plasma and CSF indicators of inflammation, including neopterin, tryptophan, kynurenine and serotonin by liquid chromatography tandem mass spectrometry. We observed significant increases in neopterin formation, tryptophan catabolism and serotonin levels in the plasma and CSF of ZIKV-infected pigtail macaques (PTM), rhesus macaques (RM) and in the plasma of ZIKV-infected humans. We next examined whether ZIKV infection resulted in microbial translocation across mucosal surfaces by evaluating plasma and cerebrospinal fluid (CSF) levels of soluble CD14 (sCD14) and lipopolysaccharide-binding protein (LBP) by enzyme-linked immunosorbent assay (ELISA). Increased sCD14 was observed in the CSF of PTM and rhesus macaque (RM), while increased LBP was observed in pigtail macaque (PTM) plasma. Finally, to examine whether ZIKV-induced microbial dysbiosis could underlie increased microbial translocation and inflammation, we characterized intestinal microbial communities by 16s rRNA gene sequencing and microbial functional changes by quantifying short-chain fatty acid (SCFA) concentrations by gas chromatography mass spectrometry. We observed that although ZIKV infection of PTM did not result in significant taxonomic shifts in microbial communities, there were significant reductions in SCFA levels. Loss of microbial function in ZIKV infection could cause decreased intestinal integrity, thereby contributing to elevated microbial translocation and systemic and CNS inflammation, providing a possible mechanism underlying ZIKV pathogenesis. Further, this may represent a mechanism underlying inflammation and pathogenesis in other diseases. Author Summary Zika virus (ZIKV) can be transmitted to humans via the bite of an infected mosquito or between humans during sexual intercourse, typically resulting in mild symptoms, which has been linked to elevated inflammation in the CNS and the development of more serious conditions, including severe neurological syndromes. Previous studies have observed that ZIKV infection is associated with increased mucosal dysfunction, including elevated inflammation in rectal and vaginal mucosal tissue. However, the mechanism of ZIKV-induced mucosal dysfunction may contribute to systemic and CNS inflammation has not been previously investigated. Here, we used the non-human primate (NHP) model and clinical specimens from ZIKV-infected humans to examine markers of systemic and CNS inflammation and microbial translocation. We observed elevated markers indicative of microbial translocation and inflammation in the CNS of ZIKV-infected macaques and humans. A potential association with mucosal dysfunction in ZIKV infection is shifts in microbial dysbiosis. We also observed that there were no significant overall taxonomic shifts in microbial communities, but a reduction of bacterial-derived short-chain fatty acid (SCFA) levels. Finally, we observed that the decrease in SCFA levels significantly negatively correlated with the elevated peripheral and CNS inflammatory markers, suggesting a link between ZIKV-driven disease pathology and microbial function. Taken together, our study provides new insight into a previously unconsidered mechanism underlying ZIKV pathogenesis.

Fragment screening data from 37 experiments, and 1,309 protein structures binding to 1,601 ligands were analysed. A new method to group ligands by binding sites was developed and sites clustered according to profiles of relative solvent accessibility. This identified 293 unique ligand binding sites, which are grouped into four clusters (C1-4). C1 presents larger, more buried, conserved, and missense-depleted sites, and is enriched in known functional sites. C4 comprises smaller highly accessible, more divergent, missense-enriched sites, and is depleted in functional sites. A site in C1 is 28 times more likely to be functional than a site in C4. 17 novel sites in 13 proteins are identified as likely to be functionally important with examples from human tenascin and 5-aminolevulinate synthase discussed in more detail. An artificial neural network, and a K-nearest neighbours model are presented to predict cluster labels for new ligand binding sites with an accuracy of 96% and 100%, respectively so allowing functional classification of sites for proteins not in this set. Our findings will be of interest to those studying protein-ligand interactions and developing new drugs or function modulators.

Abstract Background: The corona virus SARS-CoV-2 is the causative agent of recent most global pandemic. Its genome encodes various proteins categorized as non-structural, accessory, and structural proteins. The non-structural proteins, NSP1-16, are located within the ORF1ab. The NSP3, 4, and 6 together are involved in formation of double membrane vesicle (DMV) in host Golgi apparatus. These vesicles provide anchorage to viral replicative complexes, thus assist replication inside the host cell. While the accessory genes coded by ORFs 3a, 3b, 6, 7a, 7b, 8a, 8b, 9b, 9c, and 10 contribute in cell entry, immunoevasion, and pathological progression. Methods: This in silico study is focused on designing sequence specific siRNA molecules as a tool for silencing the non-structural and accessory genes of the virus. The gene sequences of NSP3, 4, and 6 along with ORF3a, 6, 7a, 8, and 10 were retrieved for conservation, phylogenetic, and sequence logo analyses. siRNA candidates were predicted using siDirect 2.0 targeting these genes. The GC content, melting temperatures, and various validation scores were calculated. Secondary structures of the guide strands and siRNA-target duplexes were predicted. Finally, tertiary structures were predicted and subjected to structural validations. Results: This study revealed that NSP3, 4, and 6 and accessory genes ORF3a, 6, 7a, 8, and 10 have high levels of conservation across globally circulating SARS-CoV-2 strains. A total of 71 siRNA molecules were predicted against the selected genes. Following rigorous screening including binary validations and minimum free energies, final siRNAs with high therapeutic potential were identified, including 7, 2, and 1 against NSP3, NSP4, and NSP6, as well as 3, 1, 2, and 1 targeting ORF3a, ORF7a, ORF8, and ORF10, respectively. Conclusion: Our novel in silico pipeline integrates effective methods from previous studies to predict and validate siRNA molecules, having the potential to inhibit viral replication pathway in vitro. In total, this study identified 17 highly specific siRNA molecules targeting NSP3, 4, and 6 and accessory genes ORF3a, 7a, 8, and 10 of SARS-CoV-2, which might be used as an additional antiviral treatment option especially in the cases of life-threatening urgencies.

Background Persistent infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), reactivation of dormant viruses, and immune-oxidative responses are involved in Long COVID. Objectives To investigate whether Long COVID and depressive, anxiety and chronic fatigue syndrome (CFS) symptoms, are associated with IgA/IgM/IgG to SARS-CoV-2, human Herpesvirus type 6 (HHV-6), Epstein-Barr Virus (EBV), and immune-oxidative biomarkers. Methods We examined 90 Long COVID patients and 90 healthy controls. We measured serum IgA/IgM/IgG against HHV-6 and EBV and their deoxyuridine 5′-triphosphate nucleotidohydrolase (duTPase), SARS-CoV-2, and activin-A, C-reactive protein (CRP), advanced oxidation protein products (AOPP), and insulin resistance (HOMA2-IR). Results Long COVID patients showed significant elevations in IgG/IgM-SARS-CoV-2, IgG/IgM-HHV-6 and HHV-6-duTPase, IgA/IgM-activin-A, CRP, AOPP, and HOMA2-IR. Neural network analysis yielded a highly significant predictive accuracy of 80.6% for the Long COVID diagnosis (sensitivity: 78.9%, specificity: 81.8%, area under the ROC curve=0.876); the topmost predictors were: IGA-activin-A, IgG-HHV-6, IgM-HHV-6-duTPase, IgG-SARS-CoV-2, and IgM-HHV-6 (all positively) and a factor extracted from all IgA levels to all viral antigens (inversely). The top-5 predictors of affective symptoms due to Long COVID were: IgM-HHV-6-duTPase, IgG-HHV-6, CRP, education, IgA-activin-A (predictive accuracy of r=0.636). The top-5 predictors of CFS due to Long COVID were in descending order: CRP, IgG-HHV-6-duTPase, IgM-activin-A, IgM-SARS-CoV-2, and IgA-activin-A (predictive accuracy: r=0.709). Conclusion Reactivation of HHV-6, SARS-CoV-2 persistence, and autoimmune reactions to activin-A combined with activated immune-oxidative pathways play a major role in the pathophysiology of Long COVID as well as the severity of affective symptoms and CFS due to Long COVID.

Background: The SARS-CoV-2 virus is continually evolving, and the worldwide epidemic is still ongoing. There is conflicting evidence regarding how SAS-CoV-2 infection affects the outcomes of assisted reproductive technology (ART). The aim of the current study was to investigate whether the outcomes of in vitro fertilization (IVF) treatment were affected during the acute period of SARS-CoV-2 infection or immediately after recovery from COVID-19. Methods: : In this retrospective cohort study, SARS-CoV-2-infected couples who underwent IVF treatment at Wuhan Union Hospital within the first three months following the lifting of the pandemic policy in mainland China were propensity-score matched (PSM) to uninfected couples who received IVF during the dynamic COVID-zero policy. Following matching, 358 and 698 patients were assigned to the SARS-CoV-2-infected and uninfected groups, respectively. The laboratory and clinical outcomes of the two groups were compared. Results: : The average number of oocytes retrieved, mature oocyte rates, normal fertilization rates, abnormal fertilization rates, and cleavage rates did not differ significantly between the two groups. The blastocyst formation rates were considerably lower in the infected group than in the uninfected group. Stratification by time from SARS-CoV-2 infection to oocyte retrieval (≤ 30, 31-60, 61-90 and ≥90 days) revealed that both blastocyst formation and available blastocyst rates were significantly decreased when oocyte retrieval was performed 31-60 days after SARS-CoV-2 infection. However, after the first embryo transfer cycle, there were no significant differences in the rates of embryo implantation, biochemical pregnancy, clinical pregnancy or early abortion between the two matched cohorts. Conclusion: SARS-CoV-2 infection had no effect on clinical outcomes after the first embryo transfer cycle; however, the blastocyst formation rate was significantly lower in couples who underwent IVF treatment 31-60 days after SARS-CoV-2 infection, indicating that SARS-CoV-2 infection may still impair embryo developmental potential.

Zika virus (ZIKV) infection in pregnant women is associated with birth defects which are more prevalent and severe the earlier in pregnancy the infection occurs. Pregnant women at risk of possible ZIKV exposure (n = 154) were screened using ELISA for ZIKV IgM and IgG. Plaque reduction neutralization tests (PRNT) confirmed ZIKV infection in 9 cases (5.84%). Two cases of vertical ZIKV transmission were confirmed by PCR. One infant showed no signs of birth defects and had a normal developmental profile despite first trimester maternal infection. In the second case, pregnancy was terminated. Production of interferon-γ (IFN-γ) by peripheral blood mononuclear cells obtained from pregnant women and umbilical cord blood was measured using enzyme-linked immunospot assay (ELISpot) following stimulation with panels of synthetic peptides derived from the sequence of ZIKV proteins. This analysis revealed that among all peptide pools tested, those derived from the ZIKV envelope protein generated the strongest IFN-γ response.

Zika virus (ZIKV) is a widespread mosquito-borne pathogen. Phylogenetically, two lineages of the ZIKV are distinguished: African and Asian-American. The latter became the cause of the 2015-2016 pandemic with severe defeat to newborns. In West African countries the African lineage has been found, but there is evidence of the emergence of Asian-American lineage in Cape Verde and Angola. This highlights the need not only to monitor the ZIKV, but also to sequence the isolates. In this article, we present a case report of Zika fever in a pregnant woman from Guinea, identified in 2018. Viral RNA was detected by qRT-PCR in serum sample. In addition, seroconversion of anti-Zika IgM and IgG antibodies was detected in repeated blood samples. Subsequently, the virus was isolated in C6/36 cell line. The detected ZIKV belonged to the African lineage, the Nigerian sublineage. The strains with the closest sequences were isolated from mosquitoes in Senegal in 2011 and 2015. In addition, we conducted serological screening of 116 blood samples collected from patients presenting to the hospital of Faranah with fevers during the period 2018-2021. As a result, it was found that IgM-positive patients occurred each year, seroprevalence varied between 5.6% and 17.1%.

Background The objectives of this study were to compare the dengue virus (DENV) infection, deaths, case-fatality ratio, as well as meteorological parameters between the first and and the recent decade (2000-2010 vs. 2011-2022) and to understand the trends, seasonality, and impact of change of temperature and rainfall pattern on transmission dynamics of Dengue in Bangladesh Methods For the period 2000-2022, dengue cases and death data from Bangladesh’s Ministry of Health and Family Welfare’s website, and meteorological data from the Bangladesh Meteorological Department were analyzed. Mann-Kendall and Sen’s slop tests were used for trends and variations and fitted a time series Poisson regression model to identify the impact of meteorological parameters on the incidence of dengue cases. A forecast of dengue cases was performed using an autoregressive integrated moving average model. Results Over the past 22 years, a total of 244,246 dengue cases were reported including 849 deaths (Case fatality ratio [CFR] =0.34%). The mean annual number of dengue cases increased eight-fold during the second decade, with 2216 cases during 2000-2011 vs. 18,321 during 2012-2022. The mean annual deaths have doubled (21 vs. 46) although the overall CFR had decreased to one-third (0.69 vs 0.24). Between the periods, the annual temperature increased by 0.49 °C, and rainfall decreased by 314 mm despite increasing unusual rainfall in the pre-and-post monsoon period. An increasing trend of dengue cases is observed with a much stiffer rise after 2018. Monthly mean temperature (Incidence risk ratio [IRR]: 1.26), first-lagged rainfall (IRR: 1.08), and second-lagged rainfall (IRR: 1.17) were significantly associated with monthly dengue incidence. Conclusions The increased local temperature and unusual rainfall might have contributed to the increased incidence of DENV infection in Bangladesh. Community engagement, vector control, and destruction of mosquito habitats are key to controlling dengue.

Background: As a highly popular choice for a household pet worldwide, domestic cats may carry pathogens and antimicrobial resistance genes (ARGs) that pose severe risks to human health. However, our knowledge about pathogens and ARGs carried by cats remains limited. We aimed to identify public health-associated pathogens and ARGs in the respiratory tracts of domestic cats. Methods: In this genomic surveillance study, zoonotic viruses were identified through meta-transcriptome sequencing of 1454 oropharyngeal-nasal swabs collected from domestic cats in 22 Chinese provinces; while bacterial species and ARGs were identified using metagenomic sequencing. Potential role of domestic cats in transmitting specific virus such as Zika virus (ZIKV) was assessed in laboratory. Findings: We identified DNAs or RNAs of 85 viral species that can infect humans. Of particular note are SARS-related coronavirus, monkeypox virus, and ZIKV. For the first time, we demonstrated that domestic cats can be infected with and transmit ZIKV via mosquito bites. We also identified a substantial number of bacterial species associated with public health, including the ESKAPE pathogens ( Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , and Enterobacter spp.) which showed a broad detection range. Additionally, a plentiful amount of ARGs, including those conferring resistance to the last resort antibiotics (carbapenems [ bla NDM , bla OXA , bla VIM ], colistin [ mcr ], and high-level tigecycline [ tetX3 , tetX4 , tetX5 , tetX6 ]), were also detected broadly. Interpretation: Although additional studies to isolate specific viruses (e.g., ZIKV) are needed, this study still provides comprehensive knowledge about the possible pathogens and ARGs carried by domestic cats from large geographic regions in China. It is important to monitoring domestic cats within the One Health approach to control these critical global public health issues. Funding: This work was supported by National Key Research and Development Program of China (2022YFD1800105, 2022YFD1801500, 2021YFC2600204), National Natural Science Foundation of China (32022082, 31972721), Natural Science Foundation of Hubei Province (2021CFA056), and Fundamental Research Funds for the Central Universities (Project 2662023PY005). Declaration of Interest: All authors declare no competing interests. Ethical Approval: This study was approved by the Animal Management and Ethics Committee of Huazhong Agricultural University. The reference ID number is 202303010001.

Aedes aegypti is the primary vector of several arboviruses, including dengue virus (DENV), chikungunya virus (CHIKV), yellow fever virus (YFV), and Zika virus (ZIKV). This vector is widespread globally in tropical and subtropical areas, but also found in temperate areas. Kenya experienced its first chikungunya outbreaks in Lamu County in 2004 and later in Mandera: 2016, and Mombasa: 2017. While there is yet to be a report of Zika outbreaks in Kenya, sero-surveillance studies indicate low-level transmission of this virus in coastal and northern parts of the country. Despite the presence of Ae. aegypti in Kisumu and Busia counties in sufficient densities, and free movement of people between the coast and the two western Kenya counties, no outbreaks of either disease have been reported in these regions. To investigate this phenomenon, we collected Ae. aegypti mosquitoes from county headquarter towns near railway stations connecting the coast and western Kenya and reared them under controlled laboratory conditions. The mosquitoes were then assessed for genetic variability using CO1 genes as well as their efficiency to transmit viruses using Laboratory colonies (F 1 ) of the field mosquitoes challenged with an infectious blood meal containing CHIKV and ZIKV. Genetic analysis revealed the presence of both Ae. aegypti subspecies, ( Ae. aegypti aegypti [ Aaa ] and Ae. aegypti formosus [ Aaf ]) in the two western Kenya counties, with Aaf being dominant (19:8 for Kisumu samples and 25:6 for Busia samples). Additionally, pairwise comparison revealed minimal genetic differentiation (0.62%) between the study populations, with a high genetic variation (99.38%) observed within each population, indicating significant diversity within individual populations. Ae. aegypti populations from Kisumu and Busia counties exhibited competence for CHIKV, with infection, dissemination, and transmission rates of 55.2%, 85.5%, and 27.1% for Kisumu; and 57.8%, 71.8%, and 25% for Busia populations, respectively. There was no significant difference in vector competence between these two populations. Interestingly, neither population was competent for ZIKV. In conclusion, the data shows that the Ae. aegypti populations in the two cities were homogeneous. This could explain the observed similarity in vector competence for CHIKV and ZIKV. Author Summary Our study investigated the genetic variability and vector competence of Ae. aegypti mosquito populations in Kisumu and Busia Counties to CHIKV and ZIKV; revealing the presence and even distribution of both Aaa and Aaf subspecies. We also found that the Ae. aegypti populations from the two counties were not genetically differentiated. Furthermore, our study revealed that the Ae. aegypti mosquitoes from Kisumu and Busia counties were competent for CHIKV but may be refractory to ZIKV infection. These findings highlight the importance of continued monitoring of Ae. aegypti populations and their potential for arboviral disease transmission in the region.

Background: To characterize neurodevelopmental abnormalities in children up to 36 months of age with congenital Zika virus exposure. Methods: From the U.S. Zika Pregnancy and Infant Registry, a national surveillance system to monitor pregnancies with laboratory evidence of Zika virus infection, pregnancy outcomes and presence of Zika associated birth defects (ZBD) were reported among infants with available information. Neurologic sequelae and developmental delay were reported among children with ≥ 1 follow-up exam after 14 days of age or with ≥ 1 visit with development reported, respectively. Results: Among 2,248 infants, 10.1% were born preterm, and 10.5% were small-for-gestational age. Overall, 122 (5.4%) had any ZBD; 91.8% of infants had brain abnormalities or microcephaly, 23.0% had eye abnormalities, and 14.8% had both. Of 1,881 children ≥ 1 follow-up exam reported, neurologic sequelae were more common among children with ZBD (44.6%) vs. without ZBD (1.5%). Of children with ≥ 1 visit with development reported, 46.8% (51/109) of children with ZBD and 7.4% (129/1739) of children without ZBD had confirmed or possible developmental delay. Conclusion: Understanding the prevalence of developmental delays and healthcare needs of children with congenital Zika virus exposure can inform health systems and planning to ensure services are available for affected families.

The intracellular Gram-negative bacterium Coxiella burnetii replicates within macrophages and causes a zoonotic disease known as Q fever. In murine macrophages, the cytokine tumor necrosis factor (TNF) is critical for restriction of intracellular C. burnetii replication. Here, we show that TNF collaborates with type I interferon (IFN) signaling for maximal control of C. burnetii . We found that TNF and type I IFN upregulate the expression of the metabolic enzyme immune responsive gene 1 (IRG1), also known as cis-aconitate decarboxylase 1 (ACOD1), and that IRG1 is required to restrict C. burnetii T4SS translocation and replication within macrophages. Further, we show that itaconic acid, the metabolic product of IRG1, restricts C. burnetii replication both intracellularly and in axenic culture. These data reveal that TNF and type I IFN upregulate the IRG1-itaconate pathway to restrict intracellular C . burnetii replication within murine macrophages.

Neurologic complications of Zika virus (ZIKV) infection across the lifespan have been described during outbreaks in Southeast Asia, South America, and Central America since 2016. In the adult CNS ZIKV tropism for neurons is tightly linked to its effects, with neuronal loss within the hippocampus during acute infection and protracted synapse loss during recovery, which is associated with cognitive deficits. The effects of ZIKV on cortical networks have not been evaluated. Although animal behavior assays have been used previously to model cognitive impairment, in vivo brain imaging can provide orthogonal information regarding the health of brain networks in real time, providing a tool to translate findings in animal models to humans. In this study, we use widefield optical imaging to measure cortical functional connectivity (FC) in mice during acute infection with, and recovery from, intracranial infection with a mouse-adapted strain of ZIKV. Acute ZIKV infection leads to high levels of myeloid cell activation, with loss of neurons and presynaptic termini in the cerebral cortex and associated loss of FC primarily within the somatosensory cortex. During recovery, neuron numbers, synapses and FC recover to levels near those of healthy mice. However, hippocampal injury and impaired spatial cognition persist. The magnitude of activated myeloid cells during acute infection predicted both recovery of synapses and the degree of FC recovery after recovery from ZIKV infection. These findings suggest that a robust inflammatory response may contribute to the health of functional brain networks after recovery from infection. Significance Statement Determining the long-term cognitive impact of infections is clinically challenging. We found that the degree of myeloid cell activation correlated with the degree of recovery of functional connectivity after recovery from ZIKV encephalitis. Using functional cortical connectivity, we demonstrate that interhemispheric cortical connectivity is decreased in individuals with acute ZIKV encephalitis. This correlates with decreased presynaptic terminals in the somatosensory cortex. During recovery from ZIKV infection, presynaptic terminals recover, which is associated with recovered interhemispheric connectivity. This suggests a role for activated myeloid cells in maintenance of cognition and further supports the contribution of synapses in the cortex to functional networks in the brain, which can be detected by widefield optical imaging. These findings also suggest neuroinflammation may play a neuroprotective role in addition to aiding in local virologic control.

Background: Sexual and reproductive health (SRH) literacy allows young adults to make informed decisions about health outcomes. In Peru, roughly one fifth of the population lives in rural areas, and little is known about where young adults in rural areas get their SRH information. The aim of this study was to identify what motivates and influences young adults to seek information and care related to SRH in three rural communities in the highlands of Northern Peru. Methods: : Five gender-stratified focus group discussions with a total of 24 participants, and nine follow-up interviews were conducted to generate in-depth narrative data and triangulate data from the target group. Participants were women and men aged 18-24. The focus group discussions and interviews explored sources of reproductive health information, the role of informal social networks, barriers to care, and primary health concerns of the target population. Results: : Main findings include 1) The two greatest perceived SRH risks were unwanted pregnancy and abnormal discharge; 2) There appears to be limited concern about HIV or other sexually transmitted infections in the narratives; 3) There is a low quality of information concerning SRH, with discrepancies between the genders; 4) A broad spectrum of sources for SRH information were cited, including Internet, traditional healers, and specialized care; and varied by gender and life experience; 5) Having trust in a person was the primary variable associated with use of services and/or access to information for both men and women. However, men reported more embarrassment around seeking services and information, whereas women faced more physical barriers. Conclusions: : There is a lack of SRH information among young adults in some communities in the northern highlands of Peru. Both schools and health centers were noted as being trusted and established information sources for all genders so could be a key resource to explore as a way to disseminate information.

ABSTRACT The emergence of viral infections with global impact highlights the urgent need for broad-spectrum antivirals. In this study, we evaluated the effect of palmitoylation inhibitors [2-bromopalmitate (2-BP), cerulenin, and 2-fluoro palmitic acid (2-FPA)] and the enhancer palmostatin B on the replication of human coronaviruses (hCoV-229E, hCoV-Oc43) and murine hepatitis virus (MHV-A59) at non-cytotoxic concentrations. The results demonstrated that 2-BP strongly suppressed MHV-A59 replication, while cerulenin and 2-FPA only moderately inhibited viral replication. Palmostatin B significantly enhanced viral replication. Notably, 2-BP exhibited superior efficacy. Interestingly, palmostatin B failed to rescue the inhibitory effects of 2-BP but effectively rescued cerulenin and 2-FPA, suggesting additional biological activities of 2-BP beyond palmitoylation inhibition. Furthermore, we discovered that 2-BP specifically disrupted lipid droplets (LDs), and this LD disruption was correlated with viral replication inhibition. Based on our findings, we conclude that the inhibitory effects of 2-BP on viral replication primarily stem from LD disruption rather than palmitoylation inhibition. Therefore, we revealed the crucial role of LDs in the viral replication. Our study provides insights into the development of wide-spectrum antiviral strategies.

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. G3BP1/2 are prominent interactors of the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the functional consequences of the G3BP1-N interaction in the context of viral infection remain unclear. Here we used structural and biochemical analyses to define the residues required for G3BP1-N interaction, followed by structure-guided mutagenesis of G3BP1 and N to selectively and reciprocally disrupt their interaction. We found that mutation of F17 within the N protein led to selective loss of interaction with G3BP1 and consequent failure of the N protein to disrupt stress granule assembly. Introduction of SARS-CoV-2 bearing an F17A mutation resulted in a significant decrease in viral replication and pathogenesis in vivo, indicating that the G3BP1-N interaction promotes infection by suppressing the ability of G3BP1 to form stress granules.

Rolling circle amplification (RCA) is a widely used DNA amplification method that uses circular template DNA as input and produces multimeric, linear single or double stranded DNA. Circle-to-circle amplification (C2CA) has further expanded this method by implementing product re-circularization using restriction and ligation, leading to a higher amplification yield, and enabling the generation of circular products. However, C2CA is a multistep, non-isothermal method, requiring multiple fluid manipulations and thereby compromises several advantages of RCA. Here, we improved C2CA to implement a one-pot, single step, isothermal reaction at temperatures ranging from 25 to 37°C. Our C2CAplus method is simple, robust, and produces large quantities of product DNA that can be seen with the naked eye.

A promising candidate for arbovirus control and prevention relies on replacing arbovirus-susceptible Aedes aegypti populations with mosquitoes that have been colonized by the intracellular bacterium Wolbachia and thus have a reduced capacity to transmit arboviruses. This reduced capacity to transmit arboviruses is mediated through a phenomenon referred to as pathogen blocking. Pathogen blocking has primarily been proposed as a tool to control dengue virus (DENV) transmission, however it works against a range of viruses, including Zika virus (ZIKV). Despite years of research, the molecular mechanisms underlying pathogen blocking still need to be better understood. Here, we used RNA-seq to characterize mosquito gene transcription dynamics in Ae. aegypti infected with the w Mel strain of Wolbachia that are being released by the World Mosquito Program in Medellín, Colombia. Comparative analyses using ZIKV-infected, uninfected tissues, and mosquitoes without Wolbachia revealed that the influence of w Mel on mosquito gene transcription is multifactorial. Importantly, because Wolbachia limits, but does not completely prevent, replication of ZIKV and other viruses in coinfected mosquitoes, there is a possibility that these viruses could evolve resistance to pathogen blocking. Therefore, to understand the influence of Wolbachia on within-host ZIKV evolution, we characterized the genetic diversity of molecularly barcoded ZIKV virus populations replicating in Wolbachia -infected mosquitoes and found that within-host ZIKV evolution was subject to weak purifying selection and, unexpectedly, loose anatomical bottlenecks in the presence and absence of Wolbachia . Together, these findings suggest that there is no clear transcriptional profile associated with Wolbachia -mediated ZIKV restriction, and that there is no evidence for ZIKV escape from this restriction in our system. Author Summary When Wolbachia bacteria infect Aedes aegypti mosquitoes, they dramatically reduce the mosquitoes’ susceptibility to infection with a range of arthropod-borne viruses, including Zika virus (ZIKV). Although this pathogen-blocking effect has been widely recognized, its mechanisms remain unclear. Furthermore, because Wolbachia limits, but does not completely prevent, replication of ZIKV and other viruses in coinfected mosquitoes, there is a possibility that these viruses could evolve resistance to Wolbachia -mediated blocking. Here, we use host transcriptomics and viral genome sequencing to examine the mechanisms of ZIKV pathogen blocking by Wolbachia and viral evolutionary dynamics in Ae. aegypti mosquitoes. We find complex transcriptome patterns that do not suggest a single clear mechanism for pathogen blocking. We also find no evidence that Wolbachia exerts detectable selective pressures on ZIKV in coinfected mosquitoes. Together our data suggest that it may be difficult for ZIKV to evolve Wolbachia resistance, perhaps due to the complexity of the pathogen blockade mechanism.

Advances in stem cell technologies, revolutionising regenerative therapies and advanced in vitro testing, require novel cell manufacturing pipelines able to cope with scale up and parallelisation. Microdroplet technologies, which have transformed single cell sequencing and other cell-based assays, are attractive in this context, but the inherent soft mechanics of liquid-liquid interfaces is typically thought to be incompatible with the expansion of induced pluripotent stem cells (iPSCs), and their differentiation. In this work, we report the design of protein nanosheets stabilising liquid-liquid interfaces and enabling the adhesion, expansion and retention of stemness by iPSCs. We use microdroplet microfluidic chips to control the formulation of droplets with defined dimensions and size distributions and demonstrate that these sustain high expansion rates, with excellent retention of stem cell marker expression. We further demonstrate that iPSCs cultured in such conditions retain the capacity to differentiate into cardiomyocytes and demonstrate such process on droplets. This work provides clear evidence that local nanoscale mechanics, associated with interfacial viscoelasticity, provides strong cues able to regulate and maintain pluripotency, as well as to support commitment in defined differentiation conditions. Microdroplet technologies appear as attractive candidates to transform cell manufacturing pipelines, bypassing significant hurdles paused by solid substrates and microcarriers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has been identified as the causative agent for the COVID-19 pandemic. Herein, we report the development of a point-of-care diagnostic (POC) test strip for rapid and specific diagnosis of surface spike protein (S-protein) of SARS-CoV2 virus, as a target analyte for the virus diagnosis. The test detects changes in the physicochemical properties of gold nanoparticles with the utility of a DNA aptamer that is specifically selected for binding to the receptor-binding domain (RBD) of S-protein of SARS-CoV-2. Structural changes in aptamer enables the aggregation/disaggregation of gold nanoparticles in the absence/presence of the target analyte, respectively. Lateral flow assays were utilized to visualize results and stratify positive samples (containing viral S-protein) from negative samples (blank) with high specificity and sensitivity (1 to 10 nM) within about 2 minutes. The proposed sensing technology was successfully developed for rapid quantification of SARS-CoV2 S-protein; however, it can be further adapted to detect variants, viral DNA/RNA, and other targets, enabling the development of POC devices for multiplexing assays.

ABSTRACT Antibodies targeting the so-called envelope dimer epitope (EDE) cross-neutralize Zika virus (ZIKV) and all four dengue virus (DENV) serotypes and have thus inspired an epitope-focused vaccine design against these flaviviruses. There are two EDE antibody subclasses (EDE1, EDE2) distinguished by their dependence on viral envelope (E) protein N -linked glycosylation at position N153 (DENV) or N154 (ZIKV) for binding. Here, we determined how E glycosylation affects neutralization by EDE and other broadly neutralizing antibodies. Consistent with structural studies, mutations abolishing the N153/N154 glycosylation site increased DENV and ZIKV sensitivity to neutralization by EDE1 antibodies. Surprisingly, these mutations also increased sensitivity to EDE2 antibodies although they occurred at predicted contact sites. Despite preserving the glycosylation site motif (N-X-S/T), substituting the threonine at ZIKV E residue 156 with a serine resulted in loss of glycan occupancy accompanied with increased neutralization sensitivity to EDE antibodies. For DENV, the presence of a serine instead of a threonine at E residue 155 retained glycan occupancy, but nonetheless increased sensitivity to EDE antibodies, in some cases to a similar extent as mutation at N153, which abolishes glycosylation. E glycosylation site mutations also increased ZIKV and DENV sensitivity to other broadly neutralizing antibodies, but had limited effects on ZIKV-or DENV-specific antibodies. Thus, E protein glycosylation is context-dependent and modulates the potency of broadly neutralizing antibodies in a manner not predicted by existing structures. Manipulating E protein glycosylation could be a novel strategy for engineering vaccine antigens to elicit antibodies that broadly neutralize ZIKV and DENV. IMPORTANCE Antibodies that can potently cross-neutralize Zika (ZIKV) and dengue (DENV) viruses are attractive to induce via vaccination to protect against these co-circulating flaviviruses. Structural studies have shown that viral envelope protein glycosylation is important for binding by one class of these so-called broadly neutralizing antibodies, but less is known about the determinants of neutralization. Here, we investigated how envelope protein glycosylation impacts broadly neutralizing antibody potency. By characterizing a panel of ZIKV and DENV variants encoding envelope protein glycosylation site mutations, we found that glycan occupancy was not always predicted by an intact N-X-S/T sequence motif. Moreover, envelope protein glycosylation status alters the neutralization potency of broadly neutralizing antibodies in a manner unexpected from their predicted binding mechanism as determined by existing structures. We highlight the complex role and determinants of envelope protein glycosylation that should be considered in the design of vaccine antigens to elicit broadly neutralizing antibodies.

Background RNA editing is a process that increases transcriptome diversity, often through Adenosine Deaminases Acting on RNA (ADARs) that catalyze the deamination of adenosine to inosine. ADAR editing plays an important role in regulating brain function and immune activation, and is dynamically regulated during brain development. Additionally, the ADAR1 p150 isoform is induced by interferons in viral infection and plays a role in antiviral immune response. However, the question of how virus-induced ADAR expression affects host transcriptome editing remains largely unanswered. This question is particularly relevant in the context of congenital infections, given the dynamic regulation of ADAR editing during brain development, the importance of this editing for brain function, and subsequent neurological symptoms of such infections, including microcephaly, sensory issues, and other neurodevelopmental abnormalities. Here, we begin to address this question, examining ADAR expression in publicly available datasets of congenital infections of human cytomegalovirus (HCMV) microarray expression data, as well as mouse cytomegalovirus (MCMV) and mouse/ human induced pluripotent neuroprogenitor stem cell (hiNPC) Zika virus (ZIKV) RNA-seq data. Results We found that in all three datasets, ADAR1 was overexpressed in infected samples compared to uninfected samples. In the RNA-seq datasets, editing rates were also analyzed. In all mouse infections cases, the number of editing sites was significantly increased in infected samples, albeit this was not the case for hiNPC ZIKV samples. Mouse ZIKV samples showed altered editing of well-established protein-recoding sites such as Gria3, Grik5, and Nova1, as well as editing sites that may impact miRNA binding. Conclusions Our findings provide evidence for changes in ADAR expression and subsequent dysregulation of ADAR editing of host transcriptomes in congenital infections. These changes in editing patterns of key neural genes have potential significance in the development of neurological symptoms, thus contributing to neurodevelopmental abnormalities. Further experiments should be performed to explore the full range of editing changes that occur in different congenital infections, and to confirm the specific functional consequences of these editing changes.