Effective process development towards intensified processing for gene delivery applications using Hepatitis B core Antigen (HBcAg) virus-like particles (VLPs) relies on analytical methods for the absolute quantification of HBcAg VLP proteins and bound nucleic acids. We investigated a silica spin column (SC)-based extraction procedure, including proteinase K lysis and silica chromatography, for the absolute quantification of different species of nucleic acids bound to HBcAg VLPs analyzed by dye-based fluorescence assays. This revealed load-dependent nucleic acid recoveries of the silica-SC-based extraction. We also developed a reversed-phase high-performance liquid chromatography (RP-HPLC) method to separate and quantify the HBcAg proteins and the bound nucleic acids simultaneously without prior sample treatment by dissociation reagents. The method demonstrated sufficient linearity, accuracy, and precision coefficients and is suited for determining absolute protein and nucleic acid concentrations and HBcAg protein purities at various purification stages. Both the silica-SC-based extraction and the RP-based extraction presented overcome the limitations of analytical techniques, which are restricted to relative or qualitative analyses for HBcAg VLPs with bound nucleic acids. In combination with existing analytics, the methods for an absolute quantification of HBcAg VLPs and bound nucleic acids presented here are required to evaluate downstream purification steps, such as the removal of host cell-derived nucleic acids, concurrent protein loss, and efficient loading with therapeutic nucleic acids. Hence, the methods are key for effective process development when using HBcAg VLP as potential gene delivery vehicles.

The causative agent of COVID-19, SARS-CoV-2, gains access to cells through interactions of the receptor-binding domain (RBD) on the viral S protein with angiotensin-converting enzyme 2 (ACE2) on the surface of human host cells. Systematic evolution of ligands by exponential enrichment (SELEX) was used to generate aptamers (nucleic acids selected for high binding affinity to a target) to the RBD made from 2'-fluoro-arabinonucleic acid (FANA). The best selected ~79 nucleotide aptamers bound the RBD (Arg319-Phe541) and the larger S1 domain (Val16-Arg685) of the 1272 amino acid S protein with equilibrium dissociation constants (KD,app) of ~10-20 nM, and binding half-life for the RBD, S1 domain, and full trimeric S protein of 53 ± 18, 76 ± 5, and 127 ± 7 min, respectively. Aptamers inhibited the binding of the RBD to ACE2 in an ELISA assay. Inhibition, on a per weight basis, was similar to neutralizing antibodies that were specific for RBD. Aptamers demonstrated high specificity, binding with about 10-fold lower affinity to the related S1 domain from the original SARS virus, which also binds to ACE2. Overall, FANA aptamers show affinities comparable to previous DNA aptamers to RBD and S1 protein and directly block receptor interactions while using an alternative Xeno-nucleic acid (XNA) platform.

Human norovirus is the leading cause of foodborne illness globally. One of the challenges in detecting noroviruses is the identification of a completely broadly reactive ligand; however, all detection ligands generated to date target the viral capsid, the outermost of which is the most variable region of the genome. The VPg is a protein covalently linked to the viral genome that is necessary for replication but hitherto remains underexplored as a target for detection or therapeutics. The purpose of this work was to generate nucleic acid aptamers against human norovirus (Norwalk) and cultivable surrogate (Tulane) VPgs for future use in detection and therapeutics. Eight rounds of positive-SELEX and two rounds of counter-SELEX were performed. Five and eight unique aptamer sequences were identified for Norwalk and Tulane VPg, respectively, all of which were predicted to be stable (∆G < -5.0) and one of which occurred in both pools. All candidates displayed binding to both Tulane and Norwalk VPg (positive:negative > 5.0), and all but two of the candidates displayed very strong binding (positive:negative > 10.0), significantly higher than binding to the negative control protein (p < 0.05). Overall, this work reports a number of aptamer candidates found to be broadly reactive and specific for in vitro-expressed VPgs across genus that could be used for future application in detection or therapeutics. Future work characterizing binding of the aptamer candidates against native VPgs and in therapeutic applications is needed to further evaluate their application.

The epitranscriptomic modification N6-methyladenosine (m6A) is a ubiquitous feature of the mammalian transcriptome. It modulates mRNA fate and dynamics to exert regulatory control over numerous cellular processes and disease pathways, including viral infection. Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation from the latent phase leads to the redistribution of m6A topology upon both viral and cellular mRNAs within infected cells. Here we investigate the role of m6A in cellular transcripts upregulated during KSHV lytic replication. Our results show that m6A is crucial for the stability of the GPRC5A mRNA, whose expression is induced by the KSHV latent-lytic switch master regulator, the replication and transcription activator (RTA) protein. Moreover, we demonstrate that GPRC5A is essential for efficient KSHV lytic replication by directly regulating NFκB signalling. Overall, this work highlights the central importance of m6A in modulating cellular gene expression to influence viral infection.

Clinical metagenomics is a broad-range agnostic detection method of pathogens, including novel microorganisms. A major limit is the low pathogen load compared to the high background of host nucleic acids. To overcome this issue, several solutions exist, such as applying a very high depth of sequencing, or performing a relative enrichment of viral genomes associated with capsids. At the end, the quantity of total nucleic acids is often below the concentrations recommended by the manufacturers of library kits, which necessitates to random amplify nucleic acids. Using a pool of 26 viruses representative of viral diversity, we observed a deep impact of the nature of sample (total nucleic acids versus RNA only), the reverse transcription, the random amplification and library construction method on virus recovery. We further optimized the two most promising methods and assessed their performance with fully characterized reference virus stocks. Good genome coverage and limit of detection lower than 100 or 1000 genome copies per mL of plasma, depending on the genome viral type, were obtained from a three million reads dataset. Our study reveals that optimized random amplification is a technique of choice when insufficient amounts of nucleic acid are available for direct libraries constructions.

Vaccinia virus (VACV) is a promising oncolytic agent because it exhibits many characteristic features of an oncolytic virus. However, its effectiveness is limited by the strong antiviral immune response induced by this virus. One possible approach to overcome this limitation is to develop deimmunized recombinant VACV. It is known that VACV p35 is a major protein for B- and T-cell immune response. Despite the relevance of p35, its epitope structure remains insufficiently studied. To determine neutralizing epitopes, a panel of recombinant p35 variants was designed, expressed, and used for mice immunization. Plaque-reduction neutralization tests demonstrated that VACV was only neutralized by sera from mice that were immunized with variants containing both N- and C- terminal regions of p35. This result was confirmed by the depletion of anti-p35 mice sera with recombinant p35 variants. At least nine amino acid residues affecting the immunogenic profile of p35 were identified. Substitutions of seven residues led to disruption of B-cell epitopes, whereas substitutions of two residues resulted in the recognition of the mutant p35 solely by non-neutralizing antibodies.

Parvovirus B19 (B19V), an ssDNA virus in the family Parvoviridae, is a human pathogenic virus, responsible for a wide range of clinical manifestations, still in need of effective and specific antivirals. DNA structures, including G-quadruplex (G4), have been recognised as relevant functional features in viral genomes, and small-molecule ligands binding to these structures are promising antiviral compounds. Bioinformatic tools predict the presence of potential G4 forming sequences (PQSs) in the genome of B19V, raising interest as targets for antiviral strategies. Predictions locate PQSs in the genomic terminal regions, in proximity to replicative origins. The actual propensity of these PQSs to form G4 structures was investigated by circular dichroism spectroscopic analysis on synthetic oligonucleotides of corresponding sequences. No signature of G4 structures was detected, and the interaction with the G4 ligand BRACO-19 (N,N'-(9-{[4-(dimethylamino)phenyl]amino}acridine-3,6-diyl)bis(3-pyrrolidin-1-ylpropanamide) did not appear consistent with the stabilisation of G4 structures. Any potential role of PQSs in the viral lifecycle was then assessed in an in vitro infection model system, by evaluating any variation in replication or expression of B19V in the presence of the G4 ligands BRACO-19 and pyridostatin. Neither showed a significant inhibitory activity on B19V replication or expression. Experimental challenge did not support bioinformatic predictions. The terminal regions of B19V are characterised by relevant sequence and symmetry constraints, which are functional to viral replication. Our experiments suggest that these impose a stringent requirement prevailing over the propensity of forming actual G4 structures.

The sandfly fever Toscana virus (TOSV, genus Phlebovirus, family Phenuiviridae) is endemic in Mediterranean countries. In Spain, phylogenetic studies of TOSV strains demonstrated that a genotype, different from the Italian, was circulating. This update reports 107 cases of TOSV neurological infection detected in Andalusia from 1988 to 2020, by viral culture, serology and/or RT-PCR. Most cases were located in Granada province, a hyperendemic region. TOSV neurological infection may be underdiagnosed since few laboratories include this virus in their portfolio. This work presents a reliable automated method, validated for the detection of the main viruses involved in acute meningitis and encephalitis, including the arboviruses TOSV and West Nile virus. This assay solves the need for multiple molecular platforms for different viruses and thus, improves the time to results for these syndromes, which require a rapid and efficient diagnostic approach.

Viruses can trigger glomerulonephritis (GN) development. Hepatitis viruses, especially Hepatitis C virus and Hepatitis B viruses, are examples of the viruses that trigger GN initiation or progression. However, the proof of a correlation between GN and Hepatitis E virus infection is not clear. Some studies confirmed the development of GN during acute or chronic HEV infections, mainly caused by genotype 3. While others reported that there is no relation between HEV exposure and GN development. A recent study showed that a reduced glomerular filtration rate was developed in 16% of acute HEV genotype 1 (HEV-1) infections that returned to normal during recovery. HEV-1 is endemic in Egypt with a high seroprevalence among villagers and pregnant women. There is no available data about a link between HEV and GN in Egypt.

The importance of COVID-19 surveillance from wastewater continues to grow since case-based surveillance in the general population has been scaled back world-wide. In Berlin, Germany, quantitative and genomic wastewater monitoring for SARS-CoV-2 is performed in three wastewater treatment plants (WWTP) covering 84% of the population since December 2021. The SARS-CoV-2 Omicron sublineage JN.1 (B.2.86.1.1), was first identified from wastewater on 22 October 2023 and rapidly became the dominant sublineage. This change was accompanied by a parallel and still ongoing increase in the notification-based 7-day-hospitalization incidence of COVID-19 and COVID-19 ICU utilization, indicating increasing COVID-19 activity in the (hospital-prone) population and a higher strain on the healthcare system. In retrospect, unique mutations of JN.1 could be identified in wastewater as early as September 2023 but were of unknown relevance at the time. The timely detection of new sublineages in wastewater therefore depends on the availability of new sequences from GISAID and updates to Pango lineage definitions and Nextclade. We show that genomic wastewater surveillance provides timely public health evidence on a regional level, complementing the existing indicators.

On 17 September 2019, the first outbreak of African swine fever in a pig farm was confirmed in South Korea. By 9 October, 14 outbreaks of ASF in domestic pigs had been diagnosed in 4 cities/counties. We isolated viruses from all infected farms and performed genetic characterization. The phylogenetic analysis showed that all of fourteen ASFV isolates in South Korea belong to genotype II and serogroup 8. Additionally, all isolates had an intergenic region (IGR) II variant with additional tandem repeat sequences (TRSs) between the I73R and I329L genes and showed characteristics of central variable region (CVR) 1 of the B602L gene and IGR 1 of MGF 505 9R/10R genes. These are identical to the genetic characteristics of some European isolates and Chinese isolates.

The physical entry of virus particles into cells triggers an innate immune response that is dependent on both calcium and nucleic acid sensors, with particles containing RNA or DNA genomes detected by RNA or DNA sensors, respectively. While membrane fusion in the absence of viral nucleic acid causes an innate immune response that is dependent on calcium, the involvement of nucleic acid sensors is poorly understood. Here, we used lipoplexes containing purified reovirus p14 fusion protein as a model of exogenous or fusion from without and a cell line expressing inducible p14 protein as a model of endogenous or fusion from within to examine cellular membrane fusion sensing events. We show that the cellular response to membrane fusion in both models is dependent on calcium, IRF3 and IFN. The method of sensing fusion, however, differs between fusion from without and fusion from within. Exogenous p14 lipoplexes are detected by RIG-I-like RNA sensors, whereas fusion by endogenous p14 requires both RIG-I and STING to trigger an IFN response. The source of nucleic acid that is sensed appears to be cellular in origin. Future studies will investigate the source of endogenous nucleic acids recognized following membrane fusion events.

Wild aquatic birds are the primary natural reservoir for influenza A viruses (IAVs). In this study, an A(H9N9) influenza A virus (A/duck/Bangladesh/44493/2020) was identified via routine surveillance in free-range domestic ducks in Bangladesh. Phylogenetic analysis of hemagglutinin showed that the H9N9 virus belonged to the Y439-like lineage. The HA gene had the highest nucleotide identity to A/Bean Goose (Anser fabalis)/South Korea/KNU 2019-16/2019 (H9N2). The other seven gene segments clustered within the Eurasian lineage.

Pacific oyster mortality syndrome (POMS), which is caused by Ostreid herpesvirus 1 (OsHV-1), causes economic losses in Pacific oyster (Crassostrea gigas) aquaculture in many countries. Reducing the mortality in disease outbreaks requires changing the host, pathogen and environment interactions to favor the host. Survivors of natural exposure to OsHV-1 are able to survive subsequent outbreaks. This has been replicated under laboratory conditions, suggesting the existence of an immune response. The aim of the present study is to compare the effects of prior exposure to infectious OsHV-1, heat-inactivated OsHV-1 and the chemical anti-viral immune stimulant poly I:C on mortality following exposure to virulent OsHV-1. All treatments were administered by intramuscular injection. Oysters were maintained at 18 °C for 14 days; then, the temperature was increased to 22 °C and the oysters were challenged with virulent OsHV-1. Heat-inactivated OsHV-1, infectious OsHV-1 and poly I:C all induced significant protection against mortality, with the hazard of death being 0.41, 0.18 and 0.02, respectively, compared to the controls, which had no immune priming. The replication of OsHV-1 on first exposure was not required to induce a protective response. While the underlying mechanisms for protection remain to be elucidated, conditioning for resistance to POMS by prior exposure to inactivated or infectious OsHV-1 may have practical applications in oyster farming but requires further development to optimize the dose and delivery mechanism and evaluate the duration of protection.

The localization of viral nucleic acids in the cell is essential for understanding the infectious cycle. One of the strategies developed for this purpose is the use of nucleotide analogs such as bromodeoxyuridine (BrdU, analog to thymine) or bromouridine (BrU, analog of uridine), which are incorporated into the nucleic acids during replication or transcription. In adenovirus infections, BrdU has been used to localize newly synthesized viral genomes in the nucleus, where it is key to distinguish between host and viral DNA. Here, we describe our experience with methodological variations of BrdU labeling to localize adenovirus genomes in fluorescence and electron microscopy. We illustrate the need to define conditions in which most of the newly synthesized DNA corresponds to the virus and not the host, and the amount of BrdU provided is enough to incorporate to the new DNA molecules without hampering the cell metabolism. We hope that our discussion of problems encountered and solutions implemented will help other researches interested in viral genome localization in infected cells.

The distribution of Bluetongue virus (BTV) in Europe can be represented by two distinct and interconnected epidemiological systems (episystems), each characterized by different ecological characteristics and vector species. This study investigated the vector competence of Italian populations of Culicoides imicola and Culicoides obsoletus/scoticus to some representative BTV strains after artificial oral infection. The BTV strains were selected according to their ability to spread to one or both episystems and included BTV-4 ITA, responsible of the recent Italian and French BTV-4 outbreaks; the BTV-2 strain which caused the first BTV incursion in Italy, Corsica, and Balearic Islands; BTV-4 MOR, responsible for the epidemic in Morocco; and BTV-8, the strain which spread through Europe between 2006 and 2008. Blood-soaked cotton pledgets and Hemotek membrane feeder using Parafilm® membrane were used to artificially feed midges. For each population/strain, recovery rates (positive/tested heads) were evaluated using serogroup- and serotype-specific RT-PCR. The trial demonstrated that, except for the Abruzzo population of C. obsoletus/C. scoticus, which was refractory to BTV-4 MOR infection, all the investigated Culicoides populations are susceptible to the selected BTV strains and that, if prompt vaccination programs and restriction measures had not been implemented, BTV-2 and BTV-4 MOR could have spread all over Europe.

Enterovirus A71 (EV71), coxsackievirus A16 (CVA16), and coxsackievirus B3 (CVB3) are pathogenic members of the Picornaviridae family that cause a range of diseases, including severe central nervous system complications, myocarditis, and pancreatitis. Despite the considerable public health impact of these viruses, no approved antiviral treatments are currently available. In the present study, we confirmed the potential of saucerneol, a compound derived from Saururus chinensis, as an antiviral agent against EV71, CVA16, and CVB3. In the in vivo model, saucerneol effectively suppressed CVB3 replication in the pancreas and alleviated virus-induced pancreatitis. The antiviral activity of saucerneol is associated with increased mitochondrial ROS (mROS) production. In vitro inhibition of mROS generation diminishes the antiviral efficacy of saucerneol. Moreover, saucerneol treatment enhanced the phosphorylation of STING, TBK-1, and IRF3 in EV71- and CVA16-infected cells, indicating that its antiviral effects were mediated through the STING/TBK-1/IRF3 antiviral pathway, which was activated by increased mROS production. Saucerneol is a promising natural antiviral agent against EV71, CVA16, and CVB3 and has potential against virus-induced pancreatitis and myocarditis. Further studies are required to assess its safety and efficacy, which is essential for the development of effective antiviral strategies against these viruses.

Advanced nucleic acid-based technologies are powerful research tools for novel virus discovery but need to be standardized for broader applications such as virus detection in biological products and clinical samples. We have used well-characterized retrovirus stocks to evaluate the limit of detection (LOD) for broad-range PCR with electrospray ionization mass spectrometry (PCR/ESI-MS or PLEX-ID), RT-PCR assays, and virus microarrays. The results indicated that in the absence of background cellular nucleic acids, PLEX-ID and RT-PCR had a similar LOD for xenotropic murine retrovirus-related virus (XMRV; 3.12 particles per µL) whereas sensitivity of virus detection was 10-fold greater using virus microarrays. When virus was spiked into a background of cellular nucleic acids, the LOD using PLEX-ID remained the same, whereas virus detection by RT-PCR was 10-fold less sensitive, and no virus could be detected by microarrays. Expected endogenous retrovirus (ERV) sequences were detected in cell lines tested and known species-specific viral sequences were detected in bovine serum and porcine trypsin. A follow-up strategy was developed using PCR amplification, nucleotide sequencing, and bioinformatics to demonstrate that an RD114-like retrovirus sequence that was detected by PLEX-ID in canine cell lines (Madin-Darby canine kidney (MDCK) and Cf2Th canine thymus) was due to defective, endogenous gammaretrovirus-related sequences.

In July 2018, a novel avian influenza virus (A/Mandarin duck/South Korea/KNU18-12/2018(H11N9)) was isolated from Mandarin ducks in South Korea. Phylogenetic and molecular analyses were conducted to characterize the genetic origins of the H11N9 strain. Phylogenetic analysis indicated that eight gene segments of strain H11N9 belonged to the Eurasian lineages. Analysis of nucleotide sequence similarity of both the hemagglutinin (HA) and neuraminidase (NA) genes revealed the highest homology with A/duck/Kagoshima/KU57/2014 (H11N9), showing 97.70% and 98.00% nucleotide identities, respectively. Additionally, internal genes showed homology higher than 98% compared to those of other isolates derived from duck and wild birds. Both the polymerase acidic (PA) and polymerase basic 1 (PB1) genes were close to the H5N3 strain isolated in China; whereas, other internal genes were closely related to that of avian influenza virus in Japan. A single basic amino acid at the HA cleavage site (PAIASR↓GLF), the lack of a five-amino acid deletion (residue 69-73) in the stalk region of the NA gene, and E627 in the polymerase basic 2 (PB2) gene indicated that the A/Mandarin duck/South Korea/KNU18-12/2018(H11N9) isolate was a typical low-pathogenicity avian influenza. In vitro viral replication of H11N9 showed a lower titer than H1N1 and higher than H9N2. In mice, H11N9 showed lower adaptation than H1N1. The novel A/Mandarin duck/South Korea/KNU18-12/2018(H11N9) isolate may have resulted from an unknown reassortment through the import of multiple wild birds in Japan and Korea in approximately 2016-2017, evolving to produce a different H11N9 compared to the previous H11N9 in Korea (2016). Further reassortment events of this virus occurred in PB1 and PA in China-derived strains. These results indicate that Japanese- and Chinese-derived avian influenza contributes to the genetic diversity of A/Mandarin duck/South Korea/KNU18-12/2018(H11N9) in Korea.

Teschovirus encephalomyelitis is a sporadic disease associated with Teschovirus A (PTV) serotype 1 and, less frequently, other serotypes. In recent years, the number of cases submitted to the Iowa State University Veterinary Diagnostic Laboratory with a history of posterior paresis has increased. Submission histories from various regions of the United States suggest a trend for clinical disease to persist in herds and affect a wider age-range of pigs than historically reported. Polioencephalitis and/or myelitis was consistently present and PTV was detected in affected neural tissue by PCR in a portion of cases. Sequencing from two clinical cases identified PTV-2 and PTV-11. To assess neuropathogenicity of these isolates, 5-week-old cesarean derived and colostrum-deprived pigs were assigned to three groups: negative control (n = 4), PTV-2-inoculated (n = 7), and PTV-11-inoculated (n = 7). Three PTV-2-inoculated pigs developed mild incoordination of the hind limbs, one of which progressed to posterior ataxia. While all PTV-11-inoculated pigs showed severe neurological signs consistent with Teschovirus encephalomyelitis, no evidences of neurological signs were observed in sham-inoculated animals. All PTV-2- and PTV-11-inoculated pigs had microscopic lesions consistent with Teschovirus encephalomyelitis. To our knowledge, this is the first description of PTV-11 and experimental study demonstrating the neuropathogenicity of PTV-11 in the United States.