Bidirectional transfer of human cytomegalovirus strains in donor and recipient seropositive lung transplant patients.

Donor and recipient human cytomegalovirus (HCMV) seropositive (D+R+) lung transplant recipients (LTRs) often harbor multiple strains of HCMV, likely due to transmitted donor (D) strains and reactivated recipient (R) strains. To date, the extent and timely occurrence of each likely source in shaping the post-transplantation (post-Tx) strain population is unknown. Here, we deciphered the D and R origin of the post-Tx HCMV strain composition in blood, bronchoalveolar lavage (BAL), and CD45+ BAL cell subsets. We investigated either D and/or R formalin-fixed paraffin-embedded blocks or fresh D lung tissue from four D+R+ LTRs obtained before transplantation. HCMV strains were characterized by short amplicon deep sequencing. In two LTRs, we show that the transplanted lung is reseeded by R strains within the first 6 months after transplantation, likely by infiltrating CD14+ CD163+/- alveolar macrophages. In three LTRs, we demonstrate both rapid D-strain dissemination and persistence in the transplanted lung for >1 year post-Tx. Broad inter-host diversity contrasts with intra-host genotype sequence stability upon transmission, during follow-up and across compartments. In D+R+ LTRs, HCMV strains of both, D and R origin can emerge first and dominate long-term in subsequent episodes of infection, indicating replication of both sources despite pre-existing immunity.

Near full-length genome analysis of HEV 4b subtype in pigs showed a similarity up to 99.944% compared to a patient in Guangdong province, China.

Hepatitis E virus (HEV) is a prevalent pathogen responsible for acute viral hepatitis, HEV genotypes 3 and 4 infections causing zoonotic infections. Currently, the nucleotide similarity analysis between humans and pigs for HEV genotype 4 is limited. In this study, stool samples from an HEV-infected patient who is a pig farmer and from pigs were collected to obtain the near full-length genome of HEV, phylogenetic trees were constructed for genotyping, and similarity of HEV sequences was analyzed. The results showed that HEV-RNA was detected in the stool samples from the patient and six pigs (6/30, 20.0%). Both HEV subtype in the patient and pigs was 4b. Additionally, similarity analysis showed that the range was 99.875%-99.944% between the patient and pigs at the nucleotide level. Four isolates of amino acid sequences (ORFs 1-3) from pigs were 100% identical to the patient. Phylogenetic tree and similarity analysis of an additional nine HEV sequences isolated from other patients in this region showed that the HEV sequence from the pig farmer had the closest relationship with the pigs from his farm rather than other sources of infection in this region. This study provides indirect evidences for HEV subtype 4b can be transmitted from pigs to humans at the nucleotide level. Further research is needed to explore the characteristics of different HEV subtypes.

Dynamics of peripheral T cell exhaustion and monocyte subpopulations in neurocognitive impairment and brain atrophy in chronic HIV infection.

BACKGROUND: HIV-associated neurocognitive disorders (HAND) is hypothesized to be a result of myeloid cell-induced neuro-inflammation in the central nervous system that may be initiated in the periphery, but the contribution of peripheral T cells in HAND pathogenesis remains poorly understood. METHODS: We assessed markers of T cell activation (HLA-DR + CD38+), immunosenescence (CD57 + CD28-), and immune-exhaustion (TIM-3, PD-1 and TIGIT) as well as monocyte subsets (classical, intermediate, and non-classical) by flow cytometry in peripheral blood derived from individuals with HIV on long-term stable anti-retroviral therapy (ART). Additionally, normalized neuropsychological (NP) composite test z-scores were obtained and regional brain volumes were assessed by magnetic resonance imaging (MRI). Relationships between proportions of immune phenotypes (of T-cells and monocytes), NP z-scores, and brain volumes were analyzed using Pearson correlations and multiple linear regression models. RESULTS: Of N = 51 participants, 84.3% were male, 86.3% had undetectable HIV RNA < 50 copies/ml, median age was 52 [47, 57] years and median CD4 T cell count was 479 [376, 717] cells/uL. Higher CD4 T cells expressing PD-1 + and/or TIM-3 + were associated with lower executive function and working memory and higher CD8 T cells expressing PD-1+ and/or TIM-3+ were associated with reduced brain volumes in multiple regions (putamen, nucleus accumbens, cerebellar cortex, and subcortical gray matter). Furthermore, higher single or dual frequencies of PD-1 + and TIM-3 + expressing CD4 and CD8 T-cells correlated with higher CD16 + monocyte numbers. CONCLUSIONS: This study reinforces evidence that T cells, particularly those with immune exhaustion phenotypes, are associated with neurocognitive impairment and brain atrophy in people living with HIV on ART. Relationships revealed between T-cell immune exhaustion and inflammatory in CD16+ monocytes uncover interrelated cellular processes likely involved in the immunopathogenesis of HAND.

Oral Administration of Limosilactobacillus reuteri KBL346 Ameliorates Influenza Virus A/PR8 Infection in Mouse.

Influenza virus infection is an important public-health concern because of its high transmissibility and potential for severe complications. To mitigate the severity and complications of influenza, probiotics containing Lactobacillus are used and generally recognized as safe. We evaluated the anti-influenza effect of Limosilactobacillus reuteri (L. reuteri) KBL346, isolated from the fecel sample of healthy South Koreans, in mice. BALB/c mice were orally administered live and heat-inactivated L. reuteri KBL346. After infection with influenza virus (A/Puerto Rico/8/34) 0.5 times the 50% lethal dose (LD50), body weight loss was improved and recovery was accelerated. Furthermore, L. reuteri KBL346 improved body weight loss and survival rate of mice infected with 4 times the LD50 of influenza virus. Heat-inactivated L. reuteri KBL346 reduced the viral titer in the lung and the plasma immunoglobulin G level. Expression levels of genes encoding inflammatory cytokines, such as interferon-γ and toll-like receptor 2 (Tlr2), were decreased in the lung tissues of mice administered L. reuteri KBL346. Live and heat-inactivated L. reuteri KBL346 increased the expression level of Adamts4, which promotes recovery after infection, and decreased that of Tlr2. The α-diversity of the gut microbiome was modulated by the administration of L. reuteri KBL346. In addition, the structure of the gut microbial community differed according to the degree of weight loss. L. reuteri KBL346 has the potential to alleviate disease severity and improve histopathological changes in mice infected with influenza A/PR8, suggesting its efficacy as a probiotic against influenza infection.

An in vitro antiviral evaluation of punicalagin toward influenza A virus.

Objective: Influenza complications are mild to serious, and can cause death in some cases. A great deal of attention has been paid in recent years to the development and use of new antiviral compounds to overcome drug resistance in certain strains of the influenza virus and treat the clinical implications. This study aimed to investigate the antiviral effect of punicalagin and its associated mechanism against influenza A (H1N1) virus in vitro. Materials and Methods: the ant-influenza activity of punicalagin was studied in Madin-Darby Canine Kidney (MDCK) cells using influenza virus A/Puerto Rico/8/34 (H1N1) (PR8) using Hemagglutinin assay (HA) and 50% tissue culture infective dose (TCID50). Then, the inhibition of haemagglutination, virucidal activity, inhibitory effect at different times, replication of viral RNA and expression of viral genes were investigated. Results: Punicalagin could inhibit influenza virus infection with 50% inhibitory concentration (IC50) of 3.98 µg/ml and selectivity index (SI) value of 6.1. Punicalagin decreased virus titers with an inhibitory effect on virus hemagglutination (p<0.05). Punicalagin also inhibited viral adsorption. The results of virus RNA replication and viral mRNA (NS1 and HA) expression after treatment with punicalagin showed significant suppression of viral mRNA expression but no effect on replication of viral RNA. Conclusion: The results of the present study indicated that punicalagin was effective against influenza infection most probably via inhibition of haemagglutination activity and virus binding.

Antiviral activity of temporin-1CEb analogues against gingival infection with herpes simplex virus type 1.

Introduction: Oral herpes infections caused by herpes simplex virus type 1 (HSV-1) are one of the most common in the human population. Recently, they have been classified as an increasing problem in immunocompromised patients and those suffering from chronic inflammation of the oral mucosa and gums. Treatment mainly involves nucleoside analogues, such as acyclovir and its derivatives, which reduce virus replication and shedding. As drug-resistant strains of herpes emerge rapidly, there is a need for the development of novel anti-herpes agents. The aim of the study was to design an antiviral peptide, based on natural compounds, non-toxic to the host, and efficient against drug-resistant HSV-1. Here, we designed a lysine-rich derivative of amphibian temporin-1CEb conjugated to peptides penetrating the host cell membrane and examined their activity against HSV-1 infection of oral mucosa. Methods: We assessed the antiviral efficiency of the tested compound in simple 2D cell models (VeroE6 and TIGKs cells) and a 3D organotypic model of human gingiva (OTG) using titration assay, qPCR, and confocal imaging. To identify the molecular mechanism of antiviral activity, we applied the Azure A metachromatic test, and attachment assays techniques. Toxicity of the conjugates was examined using XTT and LDH assays. Results: Our results showed that temporin-1CEb analogues significantly reduce viral replication in oral mucosa. The mechanism of peptide analogues is based on the interaction with heparan sulfate, leading to the reduce attachment of HSV-1 to the cell membrane. Moreover, temporin-1CEb conjugates effectively penetrate the gingival tissue being effective against acyclovir-resistant strains. Collectively, we showed that temporin-1CEb can be regarded as a novel, naturally derived antiviral compound for HSV-1 treatment.

Unraveling the role of γδ T cells in the pathogenesis of an oncogenic avian herpesvirus.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that causes deadly lymphomas in chickens. In chickens, up to 50% of all peripheral T cells are gamma delta (γδ) T cells. Until now, their role in MDV pathogenesis and tumor formation remains poorly understood. To investigate the role of γδ T cells in MDV pathogenesis, we infected recently generated γδ T cell knockout chickens with very virulent MDV. Strikingly, disease and tumor incidence were highly increased in the absence of γδ T cells, indicating that γδ T cells play an important role in the immune response against MDV. In the absence of γδ T cells, virus replication was drastically increased in the thymus and spleen, which are potential sites of T cell transformation. Taken together, our data provide the first evidence that γδ T cells play an important role in the pathogenesis and tumor formation of this highly oncogenic herpesvirus.IMPORTANCEGamma delta (γδ) T cells are the most abundant T cells in chickens, but their role in fighting pathogens remains poorly understood. Marek's disease virus (MDV) is an important veterinary pathogen, that causes one of the most frequent cancers in animals and is used as a model for virus-induced tumor formation. Our study revealed that γδ T cells play a crucial role in combating MDV, as disease and tumor incidence drastically increased in the absence of these cells. γδ T cells restricted virus replication in the key lymphoid organs, thereby decreasing the likelihood of causing tumors and disease. This study provides novel insights into the role of γδ T cells in the pathogenesis of this highly oncogenic virus.

Host cells reprogram lipid droplet synthesis through YY1 to resist PRRSV infection.

Metabolism in host cells can be modulated after viral infection, favoring viral survival or clearance. Here, we report that lipid droplet (LD) synthesis in host cells can be modulated by yin yang 1 (YY1) after porcine reproductive and respiratory syndrome virus (PRRSV) infection, resulting in active antiviral activity. As a ubiquitously distributed transcription factor, there was increased expression of YY1 upon PRRSV infection both in vitro and in vivo. YY1 silencing promoted the replication of PRRSV, whereas YY1 overexpression inhibited PRRSV replication. PRRSV infection led to a marked increase in LDs, while YY1 knockout inhibited LD synthesis, and YY1 overexpression enhanced LD accumulation, indicating that YY1 reprograms PRRSV infection-induced intracellular LD synthesis. We also showed that the viral components do not colocalize with LDs during PRRSV infection, and the effect of exogenously induced LD synthesis on PRRSV replication is nearly lethal. Moreover, we demonstrated that YY1 affects the synthesis of LDs by regulating the expression of lipid metabolism genes. YY1 negatively regulates the expression of fatty acid synthase (FASN) to weaken the fatty acid synthesis pathway and positively regulates the expression of peroxisome proliferator-activated receptor gamma (PPARγ) to promote the synthesis of LDs, thus inhibiting PRRSV replication. These novel findings indicate that YY1 plays a crucial role in regulating PRRSV replication by reprogramming LD synthesis. Therefore, our study provides a novel mechanism of host resistance to PRRSV and suggests potential new antiviral strategies against PRRSV infection.IMPORTANCEPorcine reproductive and respiratory virus (PRRSV) has caused incalculable economic damage to the global pig industry since it was first discovered in the 1980s. However, conventional vaccines do not provide satisfactory protection. It is well known that viruses are parasitic pathogens, and the completion of their replication life cycle is highly dependent on host cells. A better understanding of host resistance to PRRSV infection is essential for developing safe and effective strategies to control PRRSV. Here, we report a crucial host antiviral molecule, yin yang 1 (YY1), which is induced to be expressed upon PRRSV infection and subsequently inhibits virus replication by reprogramming lipid droplet (LD) synthesis through transcriptional regulation. Our work provides a novel antiviral mechanism against PRRSV infection and suggests that targeting YY1 could be a new strategy for controlling PRRSV.

Thorough QT/QTc study to evaluate the effect of a single supratherapeutic dose of islatravir on QTc interval prolongation in healthy adults.

Islatravir is a deoxynucleoside analog being developed for the treatment of HIV-1 infection. Clinical studies are being conducted to evaluate islatravir, administered in combination with other antiretroviral therapies, at doses of 0.25 mg once daily and 2 mg once weekly. In multiple previous clinical studies, islatravir was generally well tolerated, with no clear trend in cardiac adverse events. A trial was conducted to evaluate the effect of islatravir on cardiac repolarization. A randomized, double-blind, active- and placebo-controlled phase 1 trial was conducted, in which a single dose of islatravir 0.75 mg, islatravir 240 mg (supratherapeutic dose), moxifloxacin 400 mg (active control), or placebo was administered. Continuous 12-lead electrocardiogram monitoring was performed before dosing through 24 hours after dosing. QT interval measurements were collected, and safety and pharmacokinetics were evaluated. Sixty-three participants were enrolled, and 59 completed the study. Fridericia's QT correction for heart rate was inadequate; therefore, a population-specific correction was applied (QTcP). The placebo-corrected change from baseline in QTcP (ΔΔQTcP) interval at the observed geometric mean maximum plasma concentration associated with islatravir 0.75 mg and islatravir 240 mg was <10 ms at all time points. Assay sensitivity was confirmed because the use of moxifloxacin 400 mg led to a ΔΔQTcP >10 ms. The pharmacokinetic profile of islatravir was consistent with that of previous studies, and islatravir was generally well tolerated. Results from the current trial suggest that single doses of islatravir as high as 240 mg do not lead to QTc interval prolongation.

Simultaneous quantitation of neutralizing antibodies against all four dengue virus serotypes using optimized reporter virus particles.

Serum-neutralizing antibody titers are a critical measure of vaccine immunogenicity and are used to determine flavivirus seroprevalence in study populations. An effective dengue virus (DENV) vaccine must confer simultaneous protection against viruses grouped within four antigenic serotypes. Existing flavivirus neutralization assays, including the commonly used plaque/focus reduction neutralization titer (PRNT/FRNT) assay, require an individual assay for each virus, serotype, and strain and easily become a labor-intensive and time-consuming effort for large epidemiological studies or vaccine trials. Here, we describe a multiplex reporter virus particle neutralization titer (TetraPlex RVPNT) assay for DENV that allows simultaneous quantitative measures of antibody-mediated neutralization of infection against all four DENV serotypes in a single low-volume clinical sample and analyzed by flow cytometry. Comparative studies confirm that the neutralization titers of antibodies measured by the TetraPlex RVPNT assay are similar to FRNT/PRNT assay approaches performed separately for each viral strain. The use of this high-throughput approach enables the careful serological study in DENV endemic populations and vaccine recipients required to support the development of a safe and effective tetravalent DENV vaccine. IMPORTANCE: As a mediator of protection against dengue disease and a serological indicator of prior infection, the detection and quantification of neutralizing antibodies against DENV is an important "gold standard" tool. However, execution of traditional neutralizing antibody assays is often cumbersome and requires repeated application for each virus or serotype. The optimized RVPNT assay described here is high-throughput, easily multiplexed across multiple serotypes, and targets reporter viral particles that can be robustly produced for all four DENV serotypes. The use of this transformative RVPNT assay will support the expansion of neutralizing antibody datasets to answer research and public health questions often limited by the more cumbersome neutralizing antibody assays and the need for greater quantities of test serum.

Investigating the role of killer cell immunoglobulin-like receptors and human leukocyte antigen genetic variants in hepatitis C virus infection.

The genetic diversity of killer cell immunoglobulin-like receptors (KIRs) and human leukocyte antigen (HLA) genes influences the host's immune response to viral pathogens. This study aims to explore the impact of five single nucleotide polymorphisms (SNPs) in KIR3DL2 and HLA-A genes on hepatitis C virus (HCV) infection. A total of 2251 individuals were included in the case-control study. SNPs including KIR3DL2 rs11672983, rs3745902, rs1654644, and HLA-A rs3869062, rs12202296 were genotyped. By controlling various confounding factors using a modified logistic regression model, as well as incorporating stratified analysis, joint effects analysis, and multidimensional bioinformatics analysis, we analyzed the relationship between SNPs and HCV infection. The logistic regression analysis showed a correlation between KIR3DL2 rs11672983 AA, KIR3DL2 rs3745902 TT, and increased HCV susceptibility (p < 0.01). Stratified analysis indicated that KIR3DL2 rs1654644 and HLA-A rs3869062 also heightened HCV susceptibility in certain subgroups. A linear trend of rising HCV infection rates was observed when combining KIR3DL2 rs11672983 AA and KIR3DL2 rs3745902 TT (ptrend = 0.007). Bioinformatics analysis suggested these SNPs' regulatory potential and their role in altering messenger RNA secondary structure, implying their functional relevance in HCV susceptibility. Our findings indicate that KIR3DL2 rs11672983 AA and KIR3DL2 rs3745902 TT are significantly associated with increased susceptibility to HCV infection.

A community-based participatory research approach to evaluating and improving hepatitis C risk, knowledge, and stigma associations among people who inject substances in Indiana.

OBJECTIVES: To explore hepatitis C risk, knowledge, and stigma among individuals who inject substances in South Central Indiana. DESIGN: A cross-sectional study design was employed using a community-based participatory research approach. The community partner was a grassroots harm reduction organization. SAMPLE: Participants in this study were at least 18 years of age, current residents of Indiana, and self-identified as injection substance users (n = 179). MEASUREMENTS: The survey measured hepatitis C risk, knowledge, and stigma, as well as differences in hepatitis C risk scores among key demographic characteristics. RESULTS: Most participants identified as male (n = 106, 59%), White (n = 139, 78%), and straight (n = 143, 80%). People of color reported lower hepatitis C knowledge than White participants. Women had significantly lower hepatitis C knowledge compared with men. LGBTQ participants reported increased hepatitis C risk compared with straight participants. Increased frequency of substance use was associated with decreased stigma. Unhoused participants demonstrated significantly lower hepatitis C knowledge compared with housing-secure participants. CONCLUSIONS: Our findings increase understanding that knowledge and risk around hepatitis C are associated with demographic characteristics. Results underscore the need for tailored public health interventions to increase hepatitis C knowledge, reduce stigma, and improve testing and treatment among vulnerable populations.

Reconstruction of the historic time course of blood-borne virus contamination of clotting factor concentrates, 1974-1992.

Factor VIII and IX clotting factor concentrates manufactured from pooled plasma have been identified as potent sources of virus infection in persons with hemophilia (PWHs) in the 1970s and 1980s. To investigate the range and diversity of viruses over this period, we analysed 24 clotting factor concentrates for several blood-borne viruses. Nucleic acid was extracted from 14 commercially produced clotting factors and 10 from nonremunerated donors, preserved in lyophilized form (expiry dates: 1974-1992). Clotting factors were tested by commercial and in-house quantitative PCRs for blood-borne viruses hepatitis A, B, C and E viruses (HAV, HBV, HCV, HEV), HIV- types 1/2, parvoviruses B19V and PARV4, and human pegiviruses types 1 and 2 (HPgV-1,-2). HCV and HPgV-1 were the most frequently detected viruses (both 14/24 tested) primarily in commercial clotting factors, with frequently extremely high viral loads in the late 1970s-1985 and a diverse range of HCV genotypes. Detection frequencies sharply declined following introduction of virus inactivation. HIV-1, HBV, and HAV were less frequently detected (3/24, 1/24, and 1/24 respectively); none were positive for HEV. Contrastingly, B19V and PARV4 were detected throughout the study period, even after introduction of dry heat treatment, consistent with ongoing documented transmission to PWHs into the early 1990s. While hemophilia treatment is now largely based on recombinant factor VIII/IX in the UK and elsewhere, the comprehensive screen of historical plasma-derived clotting factors reveals extensive exposure of PWHs to blood-borne viruses throughout 1970s-early 1990s, and the epidemiological and manufacturing parameters that influenced clotting factor contamination.

Exploring Barmah Forest virus pathogenesis: molecular tools to investigate non-structural protein 3 nuclear localization and viral genomic determinants of replication.

Barmah Forest virus (BFV) is a mosquito-borne virus that causes arthralgia with accompanying rash, fever, and myalgia in humans. The virus is mainly found in Australia and has caused outbreaks associated with significant health concerns. As the sole representative of the Barmah Forest complex within the genus Alphavirus, BFV is not closely related genetically to other alphaviruses. Notably, basic knowledge of BFV molecular virology has not been well studied due to a lack of critical investigative tools such as an infectious clone. Here we describe the construction of an infectious BFV cDNA clone based on Genbank sequence and demonstrate that the clone-derived virus has in vitro and in vivo properties similar to naturally occurring virus, BFV field isolate 2193 (BFV2193-FI). A substitution in nsP4, V1911D, which was identified in the Genbank reference sequence, was found to inhibit virus rescue and replication. T1325P substitution in nsP2 selected during virus passaging was shown to be an adaptive mutation, compensating for the inhibitory effect of nsP4-V1911D. The two mutations were associated with changes in viral non-structural polyprotein processing and type I interferon (IFN) induction. Interestingly, a nuclear localization signal, active in mammalian but not mosquito cells, was identified in nsP3. A point mutation abolishing nsP3 nuclear localization attenuated BFV replication. This effect was more prominent in the presence of type I interferon signaling, suggesting nsP3 nuclear localization might be associated with IFN antagonism. Furthermore, abolishing nsP3 nuclear localization reduced virus replication in mice but did not significantly affect disease.IMPORTANCEBarmah Forest virus (BFV) is Australia's second most prevalent arbovirus, with approximately 1,000 cases reported annually. The clinical symptoms of BFV infection include rash, polyarthritis, arthralgia, and myalgia. As BFV is not closely related to other pathogenic alphaviruses or well-studied model viruses, our understanding of its molecular virology and mechanisms of pathogenesis is limited. There is also a lack of molecular tools essential for corresponding studies. Here we describe the construction of an infectious clone of BFV, variants harboring point mutations, and sequences encoding marker protein. In infected mammalian cells, nsP3 of BFV was located in the nuclei. This finding extends our understanding of the diverse mechanisms used by alphavirus replicase proteins to interact with host cells. Our novel observations highlight the complex synergy through which the viral replication machinery evolves to correct mutation errors within the viral genome.

Characterization of human tibrovirus envelope glycoproteins.

Tibroviruses are novel rhabdoviruses detected in humans, cattle, and arthropods. Four tibroviruses are known to infect humans: Bas-Congo virus (BASV), Ekpoma virus 1 (EKV-1), Ekpoma virus 2, and Mundri virus. However, since none of them has been isolated, their biological properties are largely unknown. We aimed to characterize the human tibrovirus glycoprotein (G), which likely plays a pivotal role in viral tropism and pathogenicity. Human tibrovirus Gs were found to share some primary structures and display 14 conserved cysteine residues, although their overall amino acid homology was low (29%-48%). Multiple potential glycosylation sites were found on the G molecules, and endoglycosidase H- and peptide-N-glycosidase F-sensitive glycosylation was confirmed. AlphaFold-predicted three-dimensional (3D) structures of human tibrovirus Gs were overall similar. Membrane fusion mediated by these tibrovirus Gs was induced by acidic pH. The low pH-induced conformational change that triggers fusion was reversible. Virus-like particles (VLPs) were produced by transient expression of Gs in cultured cells and used to produce mouse antisera. Using vesicular stomatitis Indiana virus pseudotyped with Gs, we found that the antisera to the respective tibrovirus VLPs showed limited cross-neutralizing activity. It was also found that human C-type lectins and T-cell immunoglobulin mucin 1 acted as attachment factors for G-mediated entry into cells. Interestingly, BASV-G showed the highest ability to utilize these molecules. The viruses infected a wide range of cell lines with preferential tropism for human-derived cells whereas the preference of EKV-1 was unique compared with the other human tibroviruses. These findings provide fundamental information to understand the biological properties of the human tibroviruses. IMPORTANCE: Human tibroviruses are poorly characterized emerging rhabdoviruses associated with either asymptomatic infection or severe disease with a case fatality rate as high as 60% in humans. However, the extent and burden of human infection as well as factors behind differences in infection outcomes are largely unknown. In this study, we characterized human tibrovirus glycoproteins, which play a key role in virus-host interactions, mainly focusing on their structural and antigenic differences and cellular tropism. Our results provide critical information for understanding the biological properties of these novel viruses and for developing appropriate preparedness interventions such as diagnostic tools, vaccines, and effective therapies.

Graphene Multiplexed Sensor for Point-of-Need Viral Wastewater-Based Epidemiology.

Wastewater-based epidemiology (WBE) can help mitigate the spread of respiratory infections through the early detection of viruses, pathogens, and other biomarkers in human waste. The need for sample collection, shipping, and testing facilities drives up the cost of WBE and hinders its use for rapid detection and isolation in environments with small populations and in low-resource settings. Given the ubiquitousness and regular outbreaks of respiratory syncytial virus, SARS-CoV-2, and various influenza strains, there is a rising need for a low-cost and easy-to-use biosensing platform to detect these viruses locally before outbreaks can occur and monitor their progression. To this end, we have developed an easy-to-use, cost-effective, multiplexed platform able to detect viral loads in wastewater with several orders of magnitude lower limit of detection than that of mass spectrometry. This is enabled by wafer-scale production and aptamers preattached with linker molecules, producing 44 chips at once. Each chip can simultaneously detect four target analytes using 20 transistors segregated into four sets of five for each analyte to allow for immediate statistical analysis. We show our platform's ability to rapidly detect three virus proteins (SARS-CoV-2, RSV, and Influenza A) and a population normalization molecule (caffeine) in wastewater. Going forward, turning these devices into hand-held systems would enable wastewater epidemiology in low-resource settings and be instrumental for rapid, local outbreak prevention.

Foals of mares vaccinated for Hendra virus have a suboptimal response to HeV vaccination.

Hendra virus (HeV) is lethal to horses and a zoonotic threat to humans in Australia, causing severe neurological and/or respiratory disease with high mortality. An equine vaccine has been available since 2012. Foals acquire antibodies from their dams by ingesting colostrum after parturition, therefore it is assumed that foals of mares vaccinated against HeV will have passive HeV antibodies circulating during the first several months of life until they are actively vaccinated. However, no studies have yet examined passive or active immunity against HeV in foals. Here, we investigated anti-HeV antibody levels in vaccinated mares and their foals. Testing for HeV neutralising antibodies is cumbersome due to the requirement for Biosafety level 4 (BSL-4) containment to conduct virus neutralisation tests (VNT). For this study, a subset of samples was tested for HeV G-specific antibodies by both an authentic VNT with infectious HeV and a microsphere-based immunoassay (MIA), revealing a strong correlation. An indicative neutralising level was then applied to the results of a larger sample set tested using the MIA. Mares had high levels of HeV-specific neutralising antibodies at the time of parturition. Foals acquired high levels of maternal antibodies which then waned to below predictive protective levels in most foals by 6 months old when vaccination commenced. Foals showed a suboptimal response to vaccination, suggesting maternal antibodies may interfere with active vaccination. The correlation analysis between the authentic HeV VNT and HeV MIA will enable further high throughput serological studies to inform optimal vaccination protocols for both broodmares and foals.

Evaluation of dried blood spots for Epstein-Barr virus nucleic acid testing.

Epstein-Barr virus (EBV) is a ubiquitous and oncogenic virus that is associated with various malignancies and non-malignant diseases and EBV DNA detection is widely used for the diagnosis and prognosis prediction for these diseases. The dried blood spots (DBS) sampling method holds great potential as an alternative to venous blood samples in geographically remote areas, for individuals with disabilities, or for newborn blood collection. Therefore, the objective of this study was to assess the viability of detecting EBV DNA load from DBS. Matched whole blood and DBS samples were collected for EBV DNA extraction and quantification detection. EBV DNA detection in DBS presented a specificity of 100 %. At different EBV DNA viral load in whole blood, the sensitivity of EBV DNA detection in DBS was 38.78 % (≥1 copies/mL), 43.18 % (≥500 copies/mL), 58.63 % (≥1000 copies/mL), 71.43 % (≥2000 copies/mL), 82.35 % (≥4000 copies/mL), and 92.86 % (≥5000 copies/mL), respectively. These results indicated that the sensitivity of EBV DNA detection in DBS increased with elevating viral load. Moreover, there was good correlation between EBV DNA levels measured in whole blood and DBS, and on average, the viral load measured in whole blood was about 6-fold higher than in DBS. Our research firstly demonstrated the feasibility of using DBS for qualitative and semi-quantitative detection of EBV DNA for diagnosis and surveillance of EBV-related diseases.

Protection against the H1N1 influenza virus using self-assembled nanoparticles formed by lumazine synthase and bearing the M2e peptide.

There is an urgent need for influenza vaccines that offer broad cross-protection. The highly conserved ectodomain of the influenza matrix protein 2 (M2e) is a promising candidate; however, its low immunogenicity can be addressed. In this study, we developed influenza vaccines using the Lumazine synthase (LS) platform. The primary objective of this study was to determine the protective potential of M2e proteins expressed on Lumazine synthase (LS) nanoparticles. M2e-LS proteins, produced through the E. coli system, spontaneously assemble into nanoparticles. The study investigated the efficacy of the M2e-LS nanoparticle vaccine in mice. Mice immunized with M2e-LS nanoparticles exhibited significantly higher levels of intracellular cytokines than those receiving soluble M2e proteins. The M2e-LS protein exhibited robust immunogenicity and provided 100% protection against cross-clade influenza.

Combined Effect of Basic Antiherpetic Drugs with a New Inhibitor of the Terminase Complex of Herpes Simplex Virus Type 1 in Vero Cell Cultures.

Abstract-Carriers of herpes simplex virus type 1 (HSV-1) account for more than 90% of the global population. Infection manifests itself in the formation of blisters and ulcers on the face or genitals and can cause blindness, encephalitis, and generalized infection. All first- and second-line modern antiherpetic drugs selectively inhibit viral DNA polymerase. The purine-benzoxazine conjugate LAS-131 ((S)-4-[6-(purin-6-yl)aminohexanoyl]-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine), which we have described earlier, uses the large subunit of the HSV-1 terminase complex as a biotarget and selectively inhibits HSV-1 reproduction in vitro. Basically new results were for the first time obtained to characterize the combined effect on human herpesvirus infection for LAS-131 used in combination with practically significant antiviral compounds, including the nucleoside analogs acyclovir (ACV), penciclovir (PCV), ganciclovir (GCV), brivudine (BVdU), iododeoxyuridine (IdU), and adenine arabinoside (Ara-A); the nucleoside phosphonate analog cidofovir (CDV); and the pyrophosphate analog foscarnet (FOS). A cytopathic effect (CPE) inhibition assay showed that the drug concentration that inhibited the virus-induced CPE by 50% decreased by a factor of 2 (an additive effect, FOS) or more (a synergistic effect; ACV, PCV, GCV, IdU, BVdU, Ara-A, and CDV) when the drugs were used in combination with LAS-131. Nonpermissive conditions for HSV-1 reproduction were thus created at lower drug concentrations, opening up new real possibilities to control human herpesvirus infection.