Related Viruses Research Articles

Nanopore reads spanning the whole genome of arthropod-infecting large dsDNA viruses of the class Naldaviricetes enable assembly-free sequence analysis.

The nuclear arthropod-specific large DNA viruses comprise different phylogenetically related virus families (Baculoviridae, Nudiviridae, Hytrosaviridae and Nimaviridae) forming the class Naldaviricetes. One common characteristic of their large double-stranded circularly covalently closed DNA genomes is the size of up to hundreds of kilobase pairs, encoding up to hundreds of open reading frames. Until recently, analyzing such large viral dsDNA genomes was hampered by short read sequencing techniques, requiring bioinformatic assembly strategies to construct a consensus sequence as a descriptor of a population of haplotype sequences. The ideal aim for decoding large dsDNA genomes is an assembly-free method that allows the individual gnomes of a virus population to be sequenced in individual reads without fragmentation. Such full-length genome sequencing of single genome molecules is made possible by Nanopore sequencing, which is increasingly used to decode representatives of the Naldaviricetes. The present study discusses the impact of Nanopore sequencing on the characterization of viral NALDV populations. Full-length genome reads can be found in published sequence data from Bombyx mori nucleopolyhedrovirus (BmNPV; family Baculoviridae) and Oryctes rhinoceros nudivirus (OrNV; family Nudiviridae) isolates, providing insight into the future of deciphering populations of members of the order Lefavirales and class Naldaviricetes. Thirty years after the first sequencing of a baculovirus in 1994, a new era of sequencing of arthropod-infecting large dsDNA viruses has begun, which will allow assembly-free analysis of entire populations of large dsDNA viruses.

Outbreak of autochthonous dengue in Fano, Pesaro-Urbino Province - Marche region, Italy, September 2024.

Dengue is the most common arboviral disease globally. It is caused by four distinct but closely related Dengue viruses (DENV-1, -2, -3, and - 4) transmitted through bites of infected Aedes species mosquito vectors. In the last 50 years, incidence has increased 30-fold with increasing geographic expansion to new countries. Here we report the most important autochthonous Dengue epidemic in Italy ever recorded with a total of 86 confirmed cases occurring in September 2024 in Fano. They were caused by DENV-2. They were 61 Dengue fever, 21 Dengue with warning signs and no cases of severe Dengue. Our data underline the importance of considering Dengue not only as imported disease and rapidly testing all those patients with suggestive clinical pictures even if they have no travel history.

Overview of dengue diagnostic limitations and potential strategies for improvement.

Dengue is a viral infection caused by any one of the four related dengue virus (DENV) serotypes, 1-4. DENV is a single-stranded RNA virus belonging to the genus Flavivirus. Dengue can cause a range of symptoms, from mild to severe life-threatening illness. Currently, treatment for DENV is limited to supportive care, with better outcomes achieved through early diagnosis. The WHO has suggested that dengue mortality can be reduced to nearly zero by implementing appropriate clinical management strategies, such as early laboratory diagnosis. This calls for diagnostic approaches that combine high sensitivity and specificity, while also being suitable for point-of-care testing (POCT) in remote locations with minimal staff training and low testing costs. In this paper, we outline the limitations of existing confirmatory dengue diagnostic methods, such as ELISA and RT-PCR, which are time-consuming, expensive, and require skilled personnel. We also highlight alternative strategies toovercome these challenges. Additionally, the paper emphasizes the growing clinical demand for diagnosing severe dengue to reduce the risk of death, which must be addressed by next-generation dengue diagnostic approaches. We propose the adoption of alternative strategies, such as fluorescence immunoassay (FIA) and chemiluminescence immunoassay (CLIA), which have the potential to overcome the limitations of existing dengue diagnostic methods. Improvements in dengue diagnosis, with a specific focus on identifying severe dengue in POCT setting, can help achieve the goal of zero deaths from dengue.

P-2346. Mpox-Specific Cellular and Humoral immunity in Mpox-Survivors Living With HIV

Abstract Background Mpox is an orthopoxvirus with a rodent reservoir that causes a disease similar to mild smallpox. In 2022, a sexually-transmitted global mpox outbreak infected over 90,000 individuals. Some immunocompromised patients experienced prolonged, severe mpox resulting in significant morbidity, amputations, and death. People living with HIV (PLWH) were at particularly high risk due to impaired cell-mediated immunity, as CD4 and CD8 T-cells help eradicate the virus from infected lesions. Mpox virion total binding titer in PLWH with or without history of mpox infection Methods We compared immune responses to mpox in PLWH who were either mpox-naïve (n = 10) versus mpox-survivors (n = 15). We measured total mpox antibody levels with a novel ELISA assay using lysed mpox virions. We characterized mpox and orthopoxvirus-specific cell-mediated immunity using intracellular cytokine staining with an enhanced protocol to allow detection of low-frequency cellular populations. Poxvirus-specific T-cell responses in PLWH as determined by intracellular cytokine stimulation using live vaccinia virus (MVA), conserved orthopoxvirus peptide pools (VCD4 and VCD8), and mpox-specific peptide pools (MCD4 and MCD8). Pools provided by Alba Grifoni and Alessandro Sette (La Jolla Institute). Interferon-gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin 2 (IL-2) were the predominant cytokines detected. Responders defined as having any CD4 or CD8 response for any of the 9 stimulations above the 90th percentile of the control group. Results Our assays differentiated well between the mpox-naïve and mpox-survivors. All mpox-survivors had binding titers of 10 to 1,000 times background with no antibodies detected in mpox-uninfected PLWH. Mpox-specific CD4 and CD8 responses of 0.01 to 0.3% were present in all infected individuals, comparable to immunocompetent mpox-survivors. The magnitude of mpox-specific T-cell responses correlated well with vaccinia-specific T-cell responses, demonstrating induction of broad anti-orthopoxvirus cell-mediated immunity. CD4 T-cell responses were TH1-type, with higher polyfunctionality in mpox-specific T-cells compared to HIV and CMV-specific T-cells. The magnitude of the IFNγ and TNFα CD4 and CD8 responses correlated with mpox-binding antibody titers, but did not predict cross-neutralization titers from vaccinia, a closely related virus that is used to vaccinate for smallpox and mpox. Host CD4 count did not correlate with the magnitude of mpox-specific cellular or humoral immunity. Conclusion These new assays effectively quantify and characterize mpox-specific immunity, with strong induction of orthopoxvirus-specific humoral and cellular immunity in immunocompromised hosts with HIV. Although PLWH are at increased risk for complications from mpox, their potent immune responses post-infection suggest that most are at reduced risk for severe complications from re-infection. Disclosures Samuel D. Stampfer, MD/PhD, Gilead: Stocks/Bonds (Public Company) Colleen F. Kelley, MD, MPH, Gilead: Grant/Research Support|Humanigen: Grant/Research Support|Moderna: Grant/Research Support|Novavax: Grant/Research Support|ViiV: Grant/Research Support

P-684. Wheezing, Fever, Snot, Oh My! Comparing Respiratory Syncytial Virus (RSV) and Human Metapneumovirus (HMPV) in Adults: Implications for Vaccine Development

Abstract Background RSV and HMPV cause acute respiratory infections (ARI) in children and adults. Two RSV vaccines for older adults are now licensed, and HMPV vaccines are in development. Limited data exist comparing clinical disease and impact of HMPV to RSV in older adults.Table 1.Demographic and Baseline Characteristics Methods We analyzed data from two studies of prospectively enrolled hospitalized adults with ARI from 2008-2012 and 2018-2023 and compared demographics, clinical characteristics and outcomes of HMPV and RSV. Similar inclusion/exclusion criteria and RT-PCR diagnosis were used for both studies, with addition of serology only in the first. All cases were adjudicated for bacterial coinfection and radiographic pneumonia by 1 pulmonary and 3 ID physicians. Comparisons used t-test or χ2.Table 2.Signs and Symptoms at Admission, Clinical Characteristics, and Outcomes Results Of 2,012 patients enrolled, 127 (6.3%) and 90 (4.5%) had RSV or HMPV, respectively: 5 patients with RSV/HMPV co-infection were excluded from analysis. Mean age (67 years), sex, race and ethnicity were similar (Table 1). HMPV patients were less likely to reside in congregate settings (7.8 v 14.9%, p=0.14) or have COPD (31.5 v 40.9%, p=0.16), CHF (17.8 v 26.0%, p=0.15), or active tobacco use (17.8 v 30.7%, p=0.03). Clinical presentation also differed with HMPV more associated with sore throat (50.0 v 33.9%, p=0.02), feverishness (64.4 v 48.0%, p=0.02), temperature >38oC (33.3 v 21.3%, p=0.05), and constitutional symptoms (75.6 v 66.1%, p=0.14). (Table 2) HMPV patients required less ICU care (5.6 v 12.6%, p=0.08) and mechanical ventilation (1.1 v 5.5%, p=0.09). Bacterial coinfection occurred equally, but HMPV patients judged as viral infection alone were more often adjudicated as pneumonia (35.6 v 22.8%, p=0.006). (Fig. 1).Figure 1.Adjudicated Radiographic Pneumonia with Microbiologic Diagnosis Conclusion Although RSV and HMPV are closely related viruses, we found differences in the clinical features of adults hospitalized with ARI. While HMPV accounted for about 3/4 the number of cases compared to RSV, HPMV patients were generally healthier and community-dwelling with fewer high-risk cardiopulmonary conditions. Notably, HMPV patients presented with more flu-like symptoms and were judged more often to have viral pneumonia. These results support development of an adult HMPV vaccine. Ongoing gene expression analyses may elucidate mechanisms underlying these differences. Disclosures Ann R. Falsey, MD, ADMA: Board Member|BioFire Diagnostics: Grant/Research Support|CyanVac: Grant/Research Support|GSK: Advisor/Consultant|GSK: Travel support|Janssen: Grant/Research Support|Moderna: Advisor/Consultant|Moderna: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Sanofi Pasteur: Advisor/Consultant|Sanofi Pasteur: Travel support|VaxCo: Grant/Research Support Angela R. Branche, MD, Cyanvac: Grant/Research Support|GSK: Advisor/Consultant|Merck: Grant/Research Support|Moderna: Grant/Research Support|Moderna: Honoraria|Novavax: Advisor/Consultant|Pfizer: Grant/Research Support|Sanofi: Honoraria|VaxCo: Grant/Research Support Edward E. Walsh, MD, Enanta: Advisor/Consultant|Enanta: Honoraria|GSK: Advisor/Consultant|Janssen: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support

Rapid and visual detection of hepatitis B virus using the ERA/Cas12f1_ge4.1-based lateral flow assay system.

Hepatitis B virus (HBV) is a major pathogen posing significant challenges to global public health, making early diagnosis critical for preventing severe liver diseases. We previously developed a fluorescent biosensor named PAM-dependent dsDNA Target-activated Cas12f1 Trans Reporter (PDTCTR). However, its reliance on specialized fluorescence equipment and lack of visual readout limited its application in resource-limited settings. To address these limitations, we developed a lateral flow assay (LFA) that integrates enzymatic recombinase amplification (ERA) with the Cas12f1_ge4.1 system. This approach enables the specific amplification of the HBV target gene through ERA and leverages the precise cleavage activity of Cas12f1_ge4.1 for enhanced signal amplification. The entire detection process is completed within 50 minutes, with results readily interpretable through visual inspection. The assay achieves a minimum detection limit of 100 copies per μL and demonstrates high specificity, showing no cross-reactivity with related viruses. In a validation study involving 71 clinical samples, the system achieved a sensitivity of 94.23%, specificity of 100%, and a kappa value of 0.90 compared to quantitative PCR (qPCR), indicating high reliability. This method thus shows promise as an effective tool for early HBV diagnosis, particularly suited for rapid, on-site detection in resource-constrained environments, and holds broad potential for diverse applications.

A Practical Synthetic Route to Cinnolines: Application to the Design and Synthesis of RSV NNI Inhibitor JNJ-8003 Analogues.

The manuscript describes the development of an efficient synthetic route to cinnolines, facilitating faster access to JNJ-8003 related Respiratory Syncytial Virus (RSV) non-nucleoside (NNI) inhibitors. Starting from correctly functionalized aryl halides, a Sonogashira reaction followed by SNAr reaction with hydrazine 1,2-dicabroxylate reagents provided dihydrocinnolines directly via in situ 6-endo-dig cyclization. The dihydrocinnolines were conveniently transformed to corresponding cinnolines in one step. Notably, this three-step route to cinnolines is more practical and safer than traditional methods that involve hazardous diazo intermediates. The methodology demonstrated a broad substrate scope. Strategic selection of a readily available aryl halide enabled the synthesis of diverse cinnolines that served as JNJ-8003 analogues through late-stage functionalization. Furthermore, by capitalizing the inherent reactivity of aryl halides toward SNAr reactions, we explored the synthesis of various heteroaromatic cinnolines. Given the extensive biological properties exhibited by cinnolines, our approach is poised to inspire further investigations in this field.

SARS-CoV-2 neutralizing antibody specificities differ dramatically between recently infected infants and immune-imprinted individuals.

The immune response to viral infection is shaped by past exposures to related virus strains, a phenomenon known as imprinting. For SARS-CoV-2, much of the population has been imprinted by a viral spike from an early strain, either through vaccination or infection during the early stages of the COVID-19 pandemic. As a consequence of this imprinting, infection with more recent SARS-CoV-2 strains primarily boosts cross-reactive antibodies elicited by the imprinting strain. Here we compare the neutralizing antibody specificities of imprinted individuals versus infants infected with a recent strain. Specifically, we use pseudovirus-based deep mutational scanning to measure how spike mutations affect neutralization by the serum antibodies of adults and children imprinted by the original vaccine versus infants with a primary infection by a XBB* variant. While the serum neutralizing activity of the imprinted individuals primarily targets the spike receptor-binding domain (RBD), serum neutralizing activity of infants only infected with XBB* mostly targets the spike N-terminal domain (NTD). In these infants, secondary exposure to the XBB* spike via vaccination shifts more of the neutralizing activity towards the RBD, although the specific RBD sites targeted are different than for imprinted adults. The dramatic differences in neutralization specificities among individuals with different exposure histories likely impact SARS-CoV-2 evolution.

Oral obeldesivir provides postexposure protection against Marburg virus in nonhuman primates.

The recent outbreak of Marburg virus (MARV) in Rwanda underscores the need for effective countermeasures against this highly fatal pathogen, with case fatality rates reaching 90%. Currently, no vaccines or approved treatments exist for MARV infection, distinguishing it from related viruses like Ebola. Our research demonstrates that the oral drug obeldesivir (ODV), a nucleoside analog prodrug, shows promising antiviral activity against filoviruses in vitro and offers significant protection in animal models. In this study with cynomolgus macaques (n=6), a 10-day regimen of once-daily ODV, initiated 24 hours post-exposure, provided 80% protection against a thousand-fold lethal MARV challenge, delaying viral replication and disease onset. Transcriptome analysis revealed that early adaptive responses correlated with successful outcomes. Compared to intravenous options, oral antivirals like ODV offer logistical advantages in outbreak settings, enabling easier administration and broader contact coverage. Our findings support ODV's potential as a broad-spectrum, oral postexposure prophylaxis for filoviruses.

A Review on Reliable and Standardized Animal Models to Study the Pathogenesis of Schmallenberg Virus in Ruminant Natural Host Species.

In the late summer of 2011, the Netherlands reported a cluster of reduced milk yield, fever, and diarrhea in dairy cattle. In March 2012, congenital malformations appeared, and Schmallenberg virus (SBV) was identified, becoming one of the few orthobunyaviruses distributed in Europe. Initially, little was known about the pathogenesis and epidemiology of these viruses in the European context, so assumptions were largely extrapolated from related viruses and other regions worldwide. To study SBV's pathogenesis and its ability to cross the placental barrier, standardized and repeatable models that mimic clinical signs observed in the field are essential. This review discusses some of the latest experimental designs for infectious disease challenges involving SBV, covering infectious doses, routes of infection, inoculum preparation, and origin. Special attention is given to the placental crossing associated with SBV.

Rapid on-site detection of crop RNA viruses using CRISPR/Cas13a.

Plant viruses are destructive pathogens for various crop species. Rapid, sensitive, and specific detection is crucial for the effective containment of emerging and resistance-breaking viruses. CRISPR/Cas has been established as a new tool for plant virus identification. However, its application for direct detection of viruses in the field is still limited. In this study, we present a CRISPR/Cas13a-based method for rapid detection of different viruses directly from RNA of several crop species, including tomato, cucumber and rapeseed. This method was used to identify the emerging tomato brown rugose fruit virus (ToBRFV), a prominent pathogen in tomato cultivation, and distinguish it from closely related viruses in infected tomato plants. ToBRFV could be identified in a 100-fold dilution and early during infection, prior to the onset of viral symptoms. Finally, we developed a user-friendly, extraction-free, 15-minute protocol for on-site virus detection using a portable fluorescent viewer and a mobile phone camera. This protocol was successfully applied for ToBRFV identification in several commercial greenhouses. These results demonstrate that CRISPR/Cas13a is a robust technology for on-site detection of multiple viruses in different crop plants. This method could be swiftly adapted to identify newly emerging pests, which threaten global food security.

The Nucleocapsid (N) Proteins of Different Human Coronaviruses Demonstrate a Variable Capacity to Induce the Formation of Cytoplasmic Condensates.

To date, seven human coronaviruses (HCoVs) have been identified. Four of these viruses typically manifest as a mild respiratory disease, whereas the remaining three can cause severe conditions that often result in death. The reasons for these differences remain poorly understood, but they may be related to the properties of individual viral proteins. The nucleocapsid (N) protein plays a crucial role in the packaging of viral genomic RNA and the modification of host cells during infection, in part due to its capacity to form dynamic biological condensates via liquid-liquid phase separation (LLPS). In this study, we investigated the capacity of N proteins derived from all HCoVs to form condensates when transiently expressed in cultured human cells. Some of the transfected cells were observed to contain cytoplasmic granules in which most of the N proteins were accumulated. Notably, the N proteins of SARS-CoV and SARS-CoV-2 showed a significantly reduced tendency to form cytoplasmic condensates. The condensate formation was not a consequence of overexpression of N proteins, as is typical for LLPS-inducing proteins. These condensates contained components of stress granules (SGs), indicating that the expression of N proteins caused the formation of SGs, which integrate N proteins. Thus, the N proteins of two closely related viruses, SARS-CoV and SARS-CoV-2, have the capacity to antagonize SG induction and/or assembly, in contrast to all other known HCoVs.

Gastrointestinal Shedding of Rubulaviruses from Egyptian Rousette Bats: Temporal Dynamics and Spillover Implications

Bats are recognized as reservoirs for diverse paramyxoviruses, some of which are closely related to known human pathogens or directly implicated in zoonotic transmission. The emergence of the zoonotic Sosuga virus (SOSV) from Egyptian rousette bats (ERBs), which caused an acute febrile illness in a reported human case in Africa, has increased the focus on the zoonotic potential of the Rubulavirinae subfamily. Previous studies identified human parainfluenza virus 2 (HPIV2)- and mumps (MuV)-related viruses in ERBs from South Africa, with HPIV2-related viruses restricted to gastrointestinal samples, an underexplored target for rubulavirus biosurveillance, suggesting that sample-type bias may have led to their oversight. To address this, we performed a longitudinal analysis of population-level fecal samples from an ERB maternity roost for rubulavirus RNA, employing a broadly reactive hemi-nested RT-PCR assay targeting the polymerase gene. We detected HPIV2- and MuV-related viruses in addition to numerous pararubulaviruses, highlighting significant viral diversity. Temporal analysis of three major clades revealed peaks in rubulavirus shedding that correlated with seasonal environmental changes and host reproductive cycles, although shedding patterns varied between clades. These findings identify specific periods of increased risk for the spillover of bat-associated rubulaviruses to humans, providing critical information for developing targeted mitigation strategies to minimize zoonotic transmission risk within the local community.

A RNA Dodecahedral Cage Inside a Human Virus Plays a Dual Biological Role in Virion Assembly and Genome Release Control.

Human rhinoviruses (RV) are among the most frequent human pathogens. As major causative agents of common colds they originate serious socioeconomic problems and huge expenditure every year, and they also exacerbate severe respiratory diseases. No anti-rhinoviral drugs or vaccines are available so far. Antiviral drug design may benefit from an understanding of the role during the infectious cycle of the interactions in the virion between the capsid and the viral nucleic acid. The genomic RNA inside the human RV virion forms a dodecahedral cage made of 30 double-stranded RNA elements that interact with equivalent sites at the capsid inner wall. RNA dodecahedral cages also occur in distantly related insect and plant viruses. However, the functional role(s) of the interactions between any dodecahedral cage and the capsid remained to be established. Here we describe an extensive structure-function mutational analysis of the capsid-RNA dodecahedral cage interface in the RV virion, to dissect the role of the interactions between the capsid and the cage-forming RNA duplexes in: i) infection by RV; ii) virus biological fitness; iii) virion assembly; iv) virion stability; and v) viral RNA uncoating. The results reveal that the capsid-bound dsRNA dodecahedral cage in the human RV virion is a multifunctional structural element. Two structurally overlapping subsets of RNA duplex-capsid interactions promote virus infectivity and biological fitness by respectively facilitating virion assembly or restraining the untimely, unproductive uncoating of the viral RNA genome. These results provide new insights into virion morphogenesis and genome uncoating, and have implications for antiviral drug design.

Efficacy assessment of antiretroviral drugs against equine infectious anemia virus in vitro.

Equine infectious anemia virus (EIAV) is an equine lentivirus related to human immunodeficiency virus type 1 (HIV-1). Both viruses are related among the Retroviridae family, but their clinical manifestations are different as EIAV causes a long persistent infection with no progressive immune dysfunction in most cases. Today, no treatment is approved against EIAV, contrary to HIV-1, manageable through antiretroviral therapy, known as HAART (highly active antiretroviral therapy) or cART (combination antiretroviral therapy). No information about the efficacy of antiretroviral drugs against EIAV is available in the literature. This study evaluates the in vitro antiviral effect of eighteen FDA-approved antiretroviral compounds from different drug families, in an equine cells in vitro infection model with EIAV reference strain. Equine dermal cells, as well as equine peripheral blood mononuclear cells were treated with non-cytotoxic drug concentrations and infected with EIAV. Relative virus release in culture supernatants was assessed through relative quantification of viral RNA via RTqPCR and viral DNA comprising proviral integration in the cell genome was assessed through qPCR of infected cells, both after nucleic acid extractions. Out of eighteen tested drugs, thirteen showed a significant antiviral effect against EIAV in vitro, an interesting discovery showing the similarities between HIV-1 and EIAV and opening a possibility to treat equine infectious anemia to avoid the disease spread.

Epidemiology of Tomato Brown Rugose Fruit Virus (ToBRFV) in Commercial Greenhouses.

The tomato brown rugose fruit virus (ToBRFV) poses a considerable threat to tomato production worldwide. Substantial experimental evidence supports the role of infected seeds as a contamination route, but the epidemiologic portrait of the virus has received less attention. This study reports the first survey of ToBRFV prevalence in commercial greenhouses. The aim was to examine the distribution of the virus in relation to greenhouse size and management practices in Québec (Canada). Plant samples collected at three production stages in 31 commercial greenhouses were subjected to ToBRFV detection and genome sequencing. The virus was detected in seven commercial greenhouses (11 positive samples out of 311 analyzed). Retrieved partial genome sequences formed a cluster with ToBRFV variants from Canada and Mexico, suggesting cross-border propagation through commercial trades. There was no link between greenhouse features and ToBRFV diagnosis, indicating that no specific profile is more susceptible to infection than others.

Establishment of a New Real-Time Molecular Assay for the Detection of Babanki Virus in Africa.

Babanki virus is a subtype of the Sindbis virus, a widespread arthropod-borne alphavirus circulating in Eurasia, Africa, and Oceania. Characterized by rashes and arthritis, clinical infections due to Sindbis were mainly reported in Africa, Australia, Asia, and Europe. However, its sub-type, Babanki virus, was reported in Northern Europe and Africa, where its epidemiology potential remains poorly understood. The diagnosis of alphaviruses is mainly based on serological testing and conventional PCR methods, which have considerable limits. In this study, we developed a real-time qRT-PCR assay for the detection of Babanki virus. The analytical sensitivity and specificity of the newly established assay were evaluated using in vitro standard RNA and related viruses relevant to the African context, respectively. In addition, its diagnostic sensitivity was assessed using a subset of Babanki virus-positive and -negative mosquito pools collected from the field. The new real-time qRT-PCR assay exhibited a 100% specificity, a 95% detection limit of 1 RNA molecule/reaction, and a diagnostic sensitivity of up to 120 pfu/reaction. This newly established assay could be useful not only for the detection of Babanki virus during epidemics but also in future experimental and surveillance studies focusing on their epidemiology and pathogenicity.

Exclusion of Superinfection or Enhancement of Superinfection in Pestiviruses-APPV Infection Is Not Dependent on ADAM17.

Some viruses can suppress superinfections of their host cells by related or different virus species. The phenomenon of superinfection exclusion can be caused by inhibiting virus attachment, receptor binding and entry, by replication interference, or competition for host cell resources. Blocking attachment and entry not only prevents unproductive double infections but also stops newly produced virions from re-entering the cell post-exocytosis. In this study, we investigated the exclusion of superinfections between the different pestivirus species. Bovine and porcine cells pre-infected with non-cytopathogenic pestivirus strains were evaluated for susceptibility to subsequent superinfection using comparative titrations. Our findings revealed significant variation in exclusion potency depending on the pre- and superinfecting virus species, as well as the host cell species. Despite this variability, all tested classical pestivirus species reduced host cell susceptibility to subsequent infections, indicating a conserved entry mechanism. Unexpectedly, pre-infection with atypical porcine pestivirus (APPV) increased host cell susceptibility to classical pestiviruses. Further analysis showed that APPV can infect SK-6 cells independently of ADAM17, a critical attachment factor for the classical pestiviruses. These results indicate that APPV uses different binding and entry mechanisms than the other pestiviruses. The observed increase in the susceptibility of cells post-APPV infection warrants further investigation and could have practical implications, such as aiding challenging pestivirus isolation from diagnostic samples.

Recombination across distant coronavirid species and genera is a rare event with distinct genomic features.

Understanding the evolutionary events that led to SARS-CoV-2 emergence, spillover, and spread is crucial to prevent, or at least be prepared for, the same type of occurrence in the future. Given that SARS-CoV-2 has some characteristics not found in other closely related viruses, we aimed to systematically assess how likely these unique features may have been acquired through recombination. We found that, although recombination is a frequent phenomenon among betacoronaviruses, it is mostly limited to closely related members of the same species. Therefore, we conclude that the most likely scenario involved feature acquisition from recombination with a closely related virus that was circulating in a geographically overlapping area or through a different biological process, but not recombination from a virus of a different species, genus, or subgenus.

Immunoinformatic approach to design T cell epitope-based chimeric vaccine targeting multiple serotypes of dengue virus

The global dengue outbreak is a significant public health concern, with the World Health Organization recording over 3 million cases and a 0.04% case fatality rate until July 2023. The infection rate is anticipated to rise in vulnerable regions worldwide. While live-attenuated vaccines are the current standard, their effectiveness in certain populations is debated. Furthermore, the presence of four closely related dengue virus serotypes can lead to antibody-dependent enhancement, compromising vaccine efficacy. In response, we propose the development of a therapeutic subunit-vaccine based on epitopes from all four serotypes to induce robust cross-protective cellular immunity. Our approach involves designing a multi-epitope chimeric immunogen using the envelope protein of the dengue virus. MHC-I and MHC-II binding T-cell epitopes were selected based on their antigen processing criteria. The most potent and immunodominant epitopes for each serotype, considering immunogenicity, population coverage, and prediction scores, were combined using AAY linker peptides to create a stable multi-epitope polypeptide. Predicted to be both antigenic and non-allergenic, the protein design exhibits a stable and soluble tertiary structure with a half-life of 4.4 h in mammalian systems. In addition, we employed an agonist to toll-like receptor-4 at the N-terminal of the vaccine design to induce downstream immunostimulatory response, validated through docking and molecular dynamics simulations. This multi-epitope construct shows promise in eliciting an effective cellular immune response against all dengue virus serotypes.