SARS-CoV-2 superinfection in CD14+ monocytes with latent human cytomegalovirus (HCMV) promotes inflammatory cascade.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of coronavirus disease 2019 (COVID-19), has posed significant challenges to global health. While much attention has been directed towards understanding the primary mechanisms of SARS-CoV-2 infection, emerging evidence suggests co-infections or superinfections with other viruses may contribute to increased morbidity and mortality, particularly in severe cases of COVID-19. Among viruses that have been reported in patients with SARS-CoV-2, seropositivity for Human cytomegalovirus (HCMV) is associated with increased COVID-19 risk and hospitalization. HCMV is a ubiquitous beta-herpesvirus with a seroprevalence of 60-90 % worldwide and one of the leading causes of mortality in immunocompromised individuals. The primary sites of latency for HCMV include CD14+ monocytes and CD34+ hematopoietic cells. In this study, we sought to investigate SARS-CoV-2 infection of CD14+ monocytes latently infected with HCMV. We demonstrate that CD14+ cells are susceptible and permissive to SARS-CoV-2 infection and detect subgenomic transcripts indicative of replication. To further investigate the molecular changes triggered by SARS-CoV-2 infection in HCMV-latent CD14+ monocytes, we conducted RNA sequencing coupled with bioinformatic differential gene analysis. The results revealed significant differences in cytokine-cytokine receptor interactions and inflammatory pathways in cells superinfected with replication-competent SARS-CoV-2 compared to the heat-inactivated and mock controls. Notably, there was a significant upregulation in transcripts associated with pro-inflammatory response factors and a decrease in anti-inflammatory factors. Taken together, these findings provide a basis for the heightened inflammatory response, offering potential avenues for targeted therapeutic interventions among HCMV-infected severe cases of COVID-19. SUMMARY: COVID-19 patients infected with secondary viruses have been associated with a higher prevalence of severe symptoms. Individuals seropositive for human cytomegalovirus (HCMV) infection are at an increased risk for severe COVID-19 disease and hospitalization. HCMV reactivation has been reported in severe COVID-19 cases with respiratory failure and could be the result of co-infection with SARS-CoV-2 and HCMV. In a cell culture model of superinfection, HCMV has previously been shown to increase infection of SARS-CoV-2 of epithelial cells by upregulating the human angiotensin-converting enzyme-2 (ACE2) receptor. In this study, we utilize CD14+ monocytes, a major cell type that harbors latent HCMV, to investigate co-infection of SARS-CoV-2 and HCMV. This study is a first step toward understanding the mechanism that may facilitate increased COVID-19 disease severity in patients infected with SARS-CoV-2 and HCMV.

Polyaniline/Titania Nanotube-Based Biosensor Strip for Sensitive and Specific Electrochemical Detection of SARS-CoV-2.

This study showcases the creation of a biosensor strip designed for the rapid, precise, and highly sensitive electrochemical detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). These biosensor strips were crafted by affixing a monoclonal antibody (mAb) specific to SARS-CoV-2 onto the surface of a commercially screen-printed carbon electrode (SPCE) modified with polyaniline-titania nanotubes (PANi-TNT). The transportable sensing device was constructed by pairing the mAb functionalized strip with a portable potentiostat wirelessly connected to either a Windows or Android device. Fast and specific conjugation between spike protein of SARS-CoV-2 and immobilized anti-SARS-CoV-2 triggered a change in the charge and electron mobility in the biosensing layer of the strip to produce detectable current during chronoamperometric scanning in the presence of a phosphate buffer solution (PBS). The excellent sensitivity and specificity of the sensor toward SARS-CoV-2 were detected as analytical analysis demonstrated linearity in the range of 80 to 200 copies/µL with a limit of detection of 25.59 copies/µL from the dose-response and standard fitted curve. Through experimental validation, the sensor strip's ability to specifically detect SARS-CoV-2 was established, distinguishing it from human coronavirus-OC43 (HCoV-OC43), HCoV-NL63, HCoV-229E, and adenovirus. The results from these tests indicate that these strips possess the potential for the future creation of dependable and easily transportable point-of-care diagnostic devices, enabling swift, sensitive, and precise detection of SARS-CoV-2 in the saliva or nasopharyngeal fluid of individuals infected with the virus.

G-quadruplexes of KSHV oriLyt play important roles in promoting lytic DNA replication.

IMPORTANCE: Biological processes originating from the DNA and RNA can be regulated by the secondary structures present in the stretch of nucleic acids, and the G-quadruplexes are shown to regulate transcription, translation, and replication. In this study, we identified the presence of multiple G-quadruplex sites in the region (oriLyt) of Kaposi's sarcoma-associated herpesvirus (KSHV) DNA, which is essential for DNA replication during the lytic cycle. We demonstrated the roles of these G-quadruplexes through multiple biochemical and biophysical assays in controlling replication and efficient virus production. We demonstrated that KSHV achieves this by recruiting RecQ1 (helicase) at those G-quadruplex sites for efficient viral DNA replication. Analysis of the replicated DNA through nucleoside labeling and immunostaining showed a reduced initiation of DNA replication in cells with a pharmacologic stabilizer of G-quadruplexes. Overall, this study confirmed the role of the G-quadruplex in regulating viral DNA replication, which can be exploited for controlling viral DNA replication.

Significance of wastewater surveillance in detecting the prevalence of SARS-CoV-2 variants and other respiratory viruses in the community - A multi-site evaluation.

Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral genome in wastewater has proven to be useful for tracking the trends of virus prevalence within the community. The surveillance also provides precise and early detection of any new and circulating variants, which aids in response to viral outbreaks. Site-specific monitoring of SARS-CoV-2 variants provides valuable information on the prevalence of new or emerging variants in the community. We sequenced the genomic RNA of viruses present in the wastewater samples and analyzed for the prevalence of SARS-CoV-2 variants as well as other respiratory viruses for a period of one year to account for seasonal variations. The samples were collected from the Reno-Sparks metropolitan area on a weekly basis between November 2021 to November 2022. Samples were analyzed to detect the levels of SARS-CoV-2 genomic copies and variants identification. This study confirmed that wastewater monitoring of SARS-CoV-2 variants can be used for community surveillance and early detection of circulating variants and supports wastewater-based epidemiology (WBE) as a complement to clinical respiratory virus testing as a healthcare response effort. Our study showed the persistence of the SARS-CoV-2 virus throughout the year compared to a seasonal presence of other respiratory viruses, implicating SARS-CoV-2's broad genetic diversity and strength to persist and infect susceptible hosts. Through secondary analysis, we further identified antimicrobial resistance (AMR) genes in the same wastewater samples and found WBE to be a feasible tool for community AMR detection and monitoring.

Comparison of a Prototype SARS-CoV-2 Lateral Flow IMMUNOASSAY with the BinaxNOWTM COVID-19 Antigen CARD.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the COVID-19 pandemic. From the onset of the pandemic, rapid antigen tests have quickly proved themselves to be an accurate and accessible diagnostic platform. The initial (and still most commonly used antigen tests) for COVID-19 diagnosis were constructed using monoclonal antibodies (mAbs) specific to severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP). These mAbs are able to bind SARS-CoV-2 NP due to high homology between the two viruses. However, since first being identified in 2019, SARS-CoV-2 has continuously mutated, and a multitude of variants have appeared. These mutations have an elevated risk of leading to possible diagnostic escape when using tests produced with SARS-CoV-derived mAbs. Here, we established a library of 18 mAbs specific to SARS-CoV-2 NP and used two of these mAbs (1CV7 and 1CV14) to generate a prototype antigen-detection lateral flow immunoassay (LFI). A side-by-side analysis of the 1CV7/1CV14 LFI and the commercially available BinaxNOWTM COVID-19 Antigen CARD was performed. Results indicated the 1CV7/1CV14 LFI outperformed the BinaxNOWTM test in the detection of BA.2, BA.2.12.1, and BA.5 Omicron sub-variants when testing remnant RT-PCR positive patient nasopharyngeal swabs diluted in viral transport media.

Inactivation of SARS-CoV-2 and Other Human Coronaviruses Aided by Photocatalytic One-Dimensional Titania Nanotube Films as a Self-Disinfecting Surface.

SARS-CoV-2 and its variants that continue to emerge have necessitated the implementation of effective disinfection strategies. Developing self-disinfecting surfaces can be a potential route for reducing fomite transmissions of infectious viruses. We show the effectiveness of TiO2 nanotubes (T_NTs) on photocatalytic inactivation of human coronavirus, HCoV-OC43, as well as SARS-CoV-2. T_NTs were synthesized by the anodization process, and their impact on photocatalytic inactivation was evaluated by the detection of residual viral genome copies (quantitative real-time quantitative reverse transcription polymerase chain reaction) and infectious viruses (infectivity assays). T_NTs with different structural morphologies, wall thicknesses, diameters, and lengths were prepared by varying the time and applied potential during anodization. The virucidal efficacy was tested under different UV-C exposure times to understand the photocatalytic reaction's kinetics. We showed that the T_NT presence boosts the inactivation process and demonstrated complete inactivation of SARS-CoV-2 as well as HCoV-OC43 within 30 s of UV-C illumination. The remarkable cyclic stability of these T_NTs was revealed through a reusability experiment. The spectroscopic and electrochemical analyses have been reported to correlate and quantify the effects of the physical features of T_NT with photoactivity. We anticipate that the proposed one-dimensional T_NT will be applicable for studying the surface inactivation of other coronaviruses including SARS-CoV-2 variants due to similarities in their genomic structure.

Detecting SARS-CoV-2 variants in wastewater and their correlation with circulating variants in the communities.

Detection of SARS-CoV-2 viral load in wastewater has been highly informative in estimating the approximate number of infected individuals in the surrounding communities. Recent developments in wastewater monitoring to determine community prevalence of COVID-19 further extends into identifying SARS-CoV-2 variants, including those being monitored for having enhanced transmissibility. We sequenced genomic RNA derived from wastewater to determine the variants of coronaviruses circulating in the communities. Wastewater samples were collected from Truckee Meadows Water Reclamation Facility (TMWRF) from November 2020 to June 2021. SARS-CoV-2 variants resulting from wastewater were compared with the variants detected in infected individuals' clinical specimens (nasal/nasopharyngeal swabs) during the same period and found conclusively in agreement. Therefore, wastewater monitoring for SARS-CoV-2 variants in the community is a feasible strategy as a complementary tool to clinical specimen testing in the latter's absence.

Accuracy of 2 Rapid Antigen Tests During 3 Phases of SARS-CoV-2 Variants.

Importance: Variants of SARS-CoV-2 have sequence variations in the viral genome that may alter the accuracy of rapid diagnostic tests. Objective: To assess the analytical and clinical accuracy of 2 rapid diagnostic tests for detecting SARS-CoV-2 during 3 phases of variants. Design, Setting, and Participants: This diagnostic study included participants aged 18 years or older who reported onset of COVID-19-like symptoms within the prior 5 days and were tested at multiple COVID-19 testing locations in King County, Washington, from February 17, 2021, to January 11, 2022, during 3 distinct phases of SARS-CoV-2 infection (pre-Delta, Delta, and Omicron). Interventions: Two anterior nasal swab specimens were collected from each participant-1 for onsite testing by the SCoV-2 Ag Detect Rapid Self-Test and 1 for reverse transcriptase-polymerase chain reaction (RT-PCR) testing. Main Outcomes and Measures: The analytical limit of detection of the 2 rapid diagnostic tests (SCoV-2 Ag Detect Rapid Self-Test and BinaxNOW COVID-19 Ag Card) was assessed using Omicron (B.1.1.529/BA.1), Delta (B.1.617.2), and a wild-type (USA-WA1/2020) variant. Diagnostic sensitivity and specificity of clinical testing for the rapid antigen tests were compared with that of RT-PCR testing. Results: A total of 802 participants were enrolled (mean [SD] age, 37.3 [13.3] years; 467 [58.2%] female), 424 (52.9%) of whom had not received COVID-19 vaccination and presented a median of 2 days (IQR, 1-3 days) from symptom onset. Overall, no significant differences were found in the analytical limit of detection or clinical diagnostic accuracy of rapid antigen testing across SARS-CoV-2 variants. The estimated limit of detection for both rapid nucleocapsid antigen tests was at or below a 50% tissue culture infectious dose of 62.5, and the positive percent agreement of the SCoV-2 Ag Detect Rapid Self-Test ranged from 81.2% (95% CI, 69.5%-89.9%) to 90.7% (95% CI, 77.9%-97.4%) across the 3 phases of variants. The diagnostic sensitivity increased for nasal swabs with a lower cycle threshold by RT-PCR, which correlates with a higher viral load. Conclusions and Relevance: In this diagnostic study, analytical and clinical performance data demonstrated accuracy of 2 rapid antigen tests among adults with COVID-19 symptoms across 3 phases of SARS-CoV-2 variants. The findings suggest that home-based rapid antigen testing programs may be an important intervention to reduce global SARS-CoV-2 transmission.

Detecting SARS-CoV-2 Variants in Wastewater and Their Correlation With Circulating Variants in the Communities.

Detection of SARS-CoV-2 viral load in wastewater has been highly informative in estimating the approximate number of infected individuals in the surrounding communities. Recent developments in wastewater monitoring to determine community prevalence of COVID-19 further extends into identifying SARS-CoV-2 variants, including those being monitored for having enhanced transmissibility. We sequenced genomic RNA derived from wastewater to determine the variants of coronaviruses circulating in the communities. Wastewater samples were collected from Truckee Meadows Water Reclamation Facility (TMWRF) from November 2021 to June 2021 were analyzed for SARS-CoV-2 variants and were compared with the variants detected in the clinical specimens (nasal/nasopharyngeal swabs) of infected individuals during the same period. The comparison was found to be conclusively in agreement. Therefore, wastewater monitoring for SARS-CoV-2 variants in the community is a feasible strategy both as a complementary tool to clinical specimen testing and in the latter's absence.

Screening of SARS-CoV-2 antivirals through a cell-based RNA-dependent RNA polymerase (RdRp) reporter assay.

COVID-19 (Coronavirus Disease 2019) caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus-2) continues to pose an international public health threat and thus far, has resulted in greater than 6.4 million deaths worldwide. Vaccines are critical tools to limit COVID-19 spread, but antiviral drug development is an ongoing global priority due to fast-spreading COVID-19 variants that may elude vaccine efficacies. The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is an essential enzyme of viral replication and transcription machinery complex. Therefore, the RdRp is an attractive target for the development of effective anti-COVID-19 therapeutics. In this study, we developed a cell-based assay to determine the enzymatic activity of SARS-CoV-2 RdRp through a luciferase reporter system. The SARS-CoV-2 RdRp reporter assay was validated using known inhibitors of RdRp polymerase, remdesivir along with other anti-virals including ribavirin, penciclovir, rhoifolin, 5'CT, and dasabuvir. Dasabuvir (an FDA-approved drug) exhibited promising RdRp inhibitory activity among these inhibitors. Anti-viral activity of dasabuvir was also tested on the replication of SARS-CoV-2 through infection of Vero E6 cells. Dasabuvir inhibited the replication of SARS-CoV-2, USA-WA1/2020 as well as B.1.617.2 (delta variant) in Vero E6 cells in a dose-dependent manner with EC50 values 9.47 µM and 10.48 µM, for USA-WA1/2020 and B.1.617.2 variants, respectively. Our results suggest that dasabuvir can be further evaluated as a therapeutic drug for COVID-19. Importantly, this system provides a robust, target-specific, and high-throughput screening compatible (z- and z'-factors of >0.5) platforms that will be a valuable tool for screening SARS-CoV-2 RdRp inhibitors.

Inactivation of Human Coronavirus by FATHHOME's Dry Sanitizer Device: Rapid and Eco-Friendly Ozone-Based Disinfection of SARS-CoV-2.

The pandemic of SARS-CoV-2/COVID-19 was reported in December 2019 in Wuhan, China. Pertaining to its high transmissibility and wide host adaptability, this unique human coronavirus spread across the planet inflicting 115 million people and causing 2.5 million deaths (as of March 3rd, 2021). Limited or negligible pre-existing immunity to multiple SARS-CoV-2 variants has resulted in severe morbidity and mortality worldwide, as well as a record-breaking surge in the use of medical-surgical supplies and personal protective equipment. In response to the global need for effective sterilization techniques, this study evaluated the virucidal efficacy of FATHHOME's self-contained, ozone-based dry-sanitizing device, by dose and time response assessment. We tested inactivation of human coronavirus, HCoV-OC43, a close genetic model of SARS-CoV-2, on porous (N95 filtering facepiece respirator/FFR) and nonporous (glass) surfaces. We started our assays with 20 ppm-10 min ozone exposure, and effectively reduced 99.8% and 99.9% of virus from glass and N95 FFR surfaces, respectively. Importantly, the virus was completely inactivated, below the detection limit (over 6-log10 reduction) with 25 ppm-15 min ozone exposure on both tested surfaces. As expected, a higher ozone exposure (50 ppm-10 min) resulted in faster inactivation of HCoV-OC43 with 100% inactivation from both the surfaces, with no residual ozone present after completion of the 5-min post exposure recapture cycle and no measurable increase in ambient ozone levels. These results confirmed that FATHHOME's device is suitable for rapid decontamination of SARS-CoV-2- from worn items, frequently touched items, and PPE including N95 FFRs, face shields, and other personal items.

Inactivation of Human Coronavirus by Titania Nanoparticle Coatings and UVC Radiation: Throwing Light on SARS-CoV-2.

The newly identified pathogenic human coronavirus, SARS-CoV-2, led to an atypical pneumonia-like severe acute respiratory syndrome (SARS) outbreak called coronavirus disease 2019 (abbreviated as COVID-19). Currently, nearly 77 million cases have been confirmed worldwide with the highest numbers of COVID-19 cases in the United States. Individuals are getting vaccinated with recently approved vaccines, which are highly protective in suppressing COVID-19 symptoms but there will be a long way before the majority of individuals get vaccinated. In the meantime, safety precautions and effective disease control strategies appear to be vital for preventing the virus spread in public places. Due to the longevity of the virus on smooth surfaces, photocatalytic properties of "self-disinfecting/cleaning" surfaces appear to be a promising tool to help guide disinfection policies for controlling SARS-CoV-2 spread in high-traffic areas such as hospitals, grocery stores, airports, schools, and stadiums. Here, we explored the photocatalytic properties of nanosized TiO2 (TNPs) as induced by the UV radiation, towards virus deactivation. Our preliminary results using a close genetic relative of SAR-CoV-2, HCoV-NL63, showed the virucidal efficacy of photoactive TNPs deposited on glass coverslips, as examined by quantitative RT-qPCR and virus infectivity assays. Efforts to extrapolate the underlying concepts described in this study to SARS-CoV-2 are currently underway.

Functionalized TiO2 Nanotube-Based Electrochemical Biosensor for Rapid Detection of SARS-CoV-2.

The COronaVIrus Disease (COVID-19) is a newly emerging viral disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Rapid increase in the number of COVID-19 cases worldwide led the WHO to declare a pandemic within a few months after the first case of infection. Due to the lack of a prophylactic measure to control the virus infection and spread, early diagnosis and quarantining of infected as well as the asymptomatic individuals are necessary for the containment of this pandemic. However, the current methods for SARS-CoV-2 diagnosis are expensive and time consuming, although some promising and inexpensive technologies are becoming available for emergency use. In this work, we report the synthesis of a cheap, yet highly sensitive, cobalt-functionalized TiO2 nanotubes (Co-TNTs)-based electrochemical sensor for rapid detection of SARS-CoV-2 through sensing the spike (receptor binding domain (RBD)) present on the surface of the virus. A simple, low-cost, and one-step electrochemical anodization route was used for synthesizing TNTs, followed by an incipient wetting method for cobalt functionalization of the TNTs platform, which was connected to a potentiostat for data collection. This sensor specifically detected the S-RBD protein of SARS-CoV-2 even at very low concentration (range of 14 to 1400 nM (nano molar)). Additionally, our sensor showed a linear response in the detection of viral protein over the concentration range. Thus, our Co-TNT sensor is highly effective in detecting SARS-CoV-2 S-RBD protein in approximately 30 s, which can be explored for developing a point of care diagnostics for rapid detection of SARS-CoV-2 in nasal secretions and saliva samples.

KSHV ORF59 and PAN RNA Recruit Histone Demethylases to the Viral Chromatin during Lytic Reactivation.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes multiple malignancies in immunocompromised individuals. KSHV primarily establishes a lifelong latency in infected humans during which only a subset of viral genes is expressed while most of the viral genome remains transcriptionally silent with condensed chromatin. However, during the lytic phase, the viral genome undergoes dramatic changes in chromatin landscape leading to a transcriptionally active state with the expression of most of the viral genes and production of progeny virions. Multiple cellular and viral factors influence the epigenetic gene regulation and transitioning of virus from latency to the lytic state. We have earlier shown that KSHV ORF59, viral processivity factor, binds to a protein arginine methyl transferase 5 (PRMT5) to alter the histone arginine methylation during reactivation. Additionally, ORF59 has been shown to interact with most abundantly expressed KSHV long noncoding polyadenylated nuclear RNA (PAN RNA), which associates with the viral epigenome during reactivation. Interestingly, PAN RNA interacts with UTX and JMJD3, cellular H3K27me3 demethylases, and removes the repressive marks on the chromatin. In this study, we report that the recruitment of histone demethylases to the viral chromatin is facilitated by the expression of ORF59 protein and PAN RNA. Using biochemical and localization assays including co-immunoprecipitation and immunofluorescence, we demonstate ORF59 localizes with UTX and JMJD3. Our results confirm that PAN RNA enhances the interaction of ORF59 with the chromatin modifying enzymes UTX and JMJD3.

The group A Streptococcus accessory protein RocA: regulatory activity, interacting partners and influence on disease potential.

The group A Streptococcus (GAS) causes diseases that range from mild (e.g. pharyngitis) to severely invasive (e.g. necrotizing fasciitis). Strain- and serotype-specific differences influence the ability of isolates to cause individual diseases. At the center of this variability is the CovR/S two-component system and the accessory protein RocA. Through incompletely defined mechanisms, CovR/S and RocA repress the expression of more than a dozen immunomodulatory virulence factors. Alleviation of this repression is selected for during invasive infections, leading to the recovery of covR, covS or rocA mutant strains. Here, we investigated how RocA promotes CovR/S activity, identifying that RocA is a pseudokinase that interacts with CovS. Disruption of CovS kinase or phosphatase activities abolishes RocA function, consistent with RocA acting through the modulation of CovS activity. We also identified, in conflict with a previous study, that the RocA regulon includes the secreted protease-encoding gene speB. Finally, we discovered an inverse correlation between the virulence of wild-type, rocA mutant, covS mutant and covR mutant strains during invasive infection and their fitness in an ex vivo upper respiratory tract model. Our data inform on mechanisms that control GAS disease potential and provide an explanation for observed strain- and serotype-specific variability in RocA function.

Zika Virus Transmission Through Blood Tissue Barriers.

The recent Zika virus (ZIKV) epidemic in the Americas and the Caribbean revealed a new deadly strain of the mosquito-borne virus, which has never been associated with previous outbreaks in Asia. For the first time, widespread ZIKV infection was shown to cause microcephaly and death of newborns, which was most likely due to the mutation acquired during the large outbreak recorded in French Polynesia in 2013-2014. Productive ZIKV replication and persistence has been demonstrated in placenta and fetal brains. Possible association between ZIKV and microcephaly and fetal death has been confirmed using immunocompetent mouse models in vitro and in vivo. Having crossed the placenta, ZIKV directly targets neural progenitor cells (NPCs) in developing human fetus and triggers apoptosis. The embryonic endothelial cells are exceptionally susceptible to ZIKV infection, which causes cell death and tissue necrosis. On the contrary, ZIKV infection does not affect the adult brain microvascular cell morphology and blood-brain barrier function. ZIKV is transmitted primarily by Aedes mosquito bite and is introduced into the placenta/blood through replication at the site of the entry. Also, virus can be transmitted through unprotected sex. Although, multiple possible routes of virus infection have been identified, the exact mechanism(s) utilized by ZIKV to cross the placenta still remain largely unknown. In this review, the current understanding of ZIKV infection and transmission through the placental and brain barriers is summarized.

Transcriptome Profiling Reveals Pro-Inflammatory Cytokines and Matrix Metalloproteinase Activation in Zika Virus Infected Human Umbilical Vein Endothelial Cells.

The deformities in the newborns infected with Zika virus (ZIKV) present a new potential public health threat to the worldwide community. Although ZIKV infection is mainly asymptomatic in healthy adults, infection during pregnancy can cause microcephaly and other severe brain defects and potentially death of the fetus. The detailed mechanism of ZIKV-associated damage is still largely unknown; however, it is apparent that the virus crosses the placental barrier to reach the fetus. Endothelial cells are the key structural component of the placental barrier. Endothelium integrity as semi-permeable barrier is essential to control the molecules and leukocytes trafficking across the placenta. Damaged endothelium or disruption of adherens junctions could compromise endothelial barrier integrity causing leakage and inflammation. Endothelial cells are often targeted by viruses, including the members of the Flaviviridae family such as dengue virus (DENV) and West Nile virus (WNV); however, little is known about the effects of ZIKV infection of endothelial cell functions. Our transcriptomic data have demonstrated that the large number of cytokines is affected in ZIKV-infected endothelial cells, where significant changes in 13 and 11 cytokines were identified in cells infected with PRVABC59 and IBH30656 ZIKV strains, respectively. Importantly, these cytokines include chemokines attracting mononuclear leukocytes (monocytes and lymphocytes) as well as neutrophils. Additionally, changes in matrix metalloproteinase (MMPs) were detected in ZIKV-infected cells. Furthermore, we for the first time showed that ZIKV infection of human umbilical vein endothelial cells (HUVECs) increases endothelial permeability. We reason that increased endothelial permeability was due to apoptosis of endothelial cells caused by caspase-8 activation in ZIKV-infected cells.

KSHV lytic proteins K-RTA and K8 bind to cellular and viral chromatin to modulate gene expression.

The oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) has two distinct life cycles with lifelong latent/non-productive and a sporadic lytic-reactivating/productive phases in the infected immune compromised human hosts. The virus reactivates from latency in response to various chemical or environmental stimuli, which triggers the lytic cascade and leads to the expression of immediate early gene, i.e. Replication and Transcription Activator (K-RTA). K-RTA, the latent-to-lytic switch protein, activates the expression of early (E) and late (L) lytic genes by transactivating multiple viral promoters. Expression of K-RTA is shown to be sufficient and essential to switch the latent virus to enter into the lytic phase of infection. Similarly, the virus-encoded bZIP family of protein, K8 also plays an important role in viral lytic DNA replication. Although, both K-RTA and K8 are found to be the ori-Lyt binding proteins and are required for lytic DNA replication, the detailed DNA-binding profile of these proteins in the KSHV and host genomes remains uncharacterized. In this study, using chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq) assay, we performed a comprehensive analysis of K-RTA and K8 binding sites in the KSHV and human genomes in order to identify specific DNA binding sequences/motifs. We identified two novel K-RTA binding motifs, (i.e. AGAGAGAGGA/motif RB and AGAAAAATTC/motif RV) and one K8 binding motif (i.e. AAAATGAAAA/motif KB), respectively. The binding of K-RTA/K8 proteins with these motifs and resulting transcriptional modulation of downstream genes was further confirmed by DNA electrophoretic gel mobility shift assay (EMSA), reporter promoter assay, Chromatin Immunoprecipitation (ChIP) assay and mRNA quantitation assay. Our data conclusively shows that K-RTA/K8 proteins specifically bind to these motifs on the host/viral genomes to modulate transcription of host/viral genes during KSHV lytic reactivation.

The DNase Activity of Kaposi's Sarcoma-Associated Herpesvirus SOX Protein Serves an Important Role in Viral Genome Processing during Lytic Replication.

The Kaposi's sarcoma-associated herpesvirus (KSHV) alkaline exonuclease SOX, encoded by open reading frame 37 (ORF37), is a bifunctional early-lytic-phase protein that possesses alkaline 5'-to-3' DNase activity and promotes host shutoff at the mRNA level during productive lytic infection. While the SOX protein is well characterized for drastically impairing cellular gene expression, little is known about the impact of its DNase activity on the KSHV genome and life cycle and the biology of KSHV infections. Here, we introduced a previously described DNase-inactivating Glu129His (Q129H) mutation into the ORF37 gene of the viral genome to generate ORF37-Q129H recombinant virus (the Q129H mutant) and investigated the effects of loss or inactivation of DNase activity on viral genome replication, cleavage, and packaging. For the first time, we provide experimental evidence that the DNase activity of the SOX protein does not affect viral latent/lytic DNA synthesis but is required for cleavage and processing of the KSHV genome during lytic replication. Interestingly, the Q129H mutation severely impaired intranuclear processing of progeny virions compared to the wild-type ORF37, as assessed by pulsed-field and Gardella gel electrophoresis, electron microscopy, and single-molecule analysis of replicating DNA (SMARD) assays. Complementation with ORF37-wt (wild type) or BGLF5 (the KSHV protein homolog in Epstein-Barr virus) in 293L/Q129H cells restored the viral genome encapsidation defects. Together, these results indicated that ORF37's proposed DNase activity is essential for viral genome processing and encapsidation and, hence, can be targeted for designing antiviral agents to block KSHV virion production.IMPORTANCE Kaposi's sarcoma (KS)-associated herpesvirus is the causative agent of multiple malignancies, predominantly in immunocompromised individuals, including HIV/AIDS patients. Reduced incidence of KS in HIV/AIDS patients receiving antiherpetic drugs to block lytic replication confirms the role of lytic DNA replication and gene products in KSHV-mediated tumorigenesis. Herpesvirus lytic replication results in the production of complex concatemeric DNA, which is cleaved into unit length viral DNA for packaging into the infectious virions. The conserved herpesviral alkaline exonucleases play an important role in viral genome cleavage and packaging. Here, by using the previously described Q129H mutant virus that selectively lacks DNase activity but retains host shutoff activity, we provide experimental evidence confirming that the DNase function of the KSHV SOX protein is essential for viral genome processing and packaging and capsid maturation into the cytoplasm during lytic replication in infected cells. This led to the identification of ORF37's DNase activity as a potential target for antiviral therapeutics.