Viral DNA Research Articles

CoCoNuTs are a diverse subclass of Type IV restriction systems predicted to target RNA

A comprehensive census of McrBC systems, among the most common forms of prokaryotic Type IV restriction systems, followed by phylogenetic analysis, reveals their enormous abundance in diverse prokaryotes and a plethora of genomic associations. We focus on a previously uncharacterized branch, which we denote coiled-coil nuclease tandems (CoCoNuTs) for their salient features: the presence of extensive coiled-coil structures and tandem nucleases. The CoCoNuTs alone show extraordinary variety, with three distinct types and multiple subtypes. All CoCoNuTs contain domains predicted to interact with translation system components, such as OB-folds resembling the SmpB protein that binds bacterial transfer-messenger RNA (tmRNA), YTH-like domains that might recognize methylated tmRNA, tRNA, or rRNA, and RNA-binding Hsp70 chaperone homologs, along with RNases, such as HEPN domains, all suggesting that the CoCoNuTs target RNA. Many CoCoNuTs might additionally target DNA, via McrC nuclease homologs. Additional restriction systems, such as Type I RM, BREX, and Druantia Type III, are frequently encoded in the same predicted superoperons. In many of these superoperons, CoCoNuTs are likely regulated by cyclic nucleotides, possibly, RNA fragments with cyclic termini, that bind associated CARF (CRISPR-Associated Rossmann Fold) domains. We hypothesize that the CoCoNuTs, together with the ancillary restriction factors, employ an echeloned defense strategy analogous to that of Type III CRISPR-Cas systems, in which an immune response eliminating virus DNA and/or RNA is launched first, but then, if it fails, an abortive infection response leading to PCD/dormancy via host RNA cleavage takes over.

CoCoNuTs are a diverse subclass of Type IV restriction systems predicted to target RNA.

A comprehensive census of McrBC systems, among the most common forms of prokaryotic Type IV restriction systems, followed by phylogenetic analysis, reveals their enormous abundance in diverse prokaryotes and a plethora of genomic associations. We focus on a previously uncharacterized branch, which we denote coiled-coil nuclease tandems (CoCoNuTs) for their salient features: the presence of extensive coiled-coil structures and tandem nucleases. The CoCoNuTs alone show extraordinary variety, with three distinct types and multiple subtypes. All CoCoNuTs contain domains predicted to interact with translation system components, such as OB-folds resembling the SmpB protein that binds bacterial transfer-messenger RNA (tmRNA), YTH-like domains that might recognize methylated tmRNA, tRNA, or rRNA, and RNA-binding Hsp70 chaperone homologs, along with RNases, such as HEPN domains, all suggesting that the CoCoNuTs target RNA. Many CoCoNuTs might additionally target DNA, via McrC nuclease homologs. Additional restriction systems, such as Type I RM, BREX, and Druantia Type III, are frequently encoded in the same predicted superoperons. In many of these superoperons, CoCoNuTs are likely regulated by cyclic nucleotides, possibly, RNA fragments with cyclic termini, that bind associated CARF (CRISPR-Associated Rossmann Fold) domains. We hypothesize that the CoCoNuTs, together with the ancillary restriction factors, employ an echeloned defense strategy analogous to that of Type III CRISPR-Cas systems, in which an immune response eliminating virus DNA and/or RNA is launched first, but then, if it fails, an abortive infection response leading to PCD/dormancy via host RNA cleavage takes over.

Far-UVC (222 nm) irradiation effectively inactivates ssRNA, dsRNA, ssDNA, and dsDNA viruses as compared togermicidal UVC (254 nm).

Ultraviolet-C (UVC) irradiation is being used as an effective approach for the disinfection of pathogenic viruses present in air, surfaces, and water. Recently, far-UVC radiation (222 nm) emitted by KrCl* (krypton-chloride) excimer lamps have been recommended for disinfecting high-risk public spaces to reduce the presence and transmission of infectious viruses owing to limited human health exposure risks as compared to germicidal UVC (254 nm). In this study, the UVC inactivation performances of individual filtered KrCl* excimer lamp (222 nm) and germicidal UVC lamp (254 nm) were determined against four viruses, bacteriophages MS2, Phi6, M13, and T4, having different genome compositions (ssRNA, dsRNA, ssDNA and dsDNA, respectively) and shapes (i.e., spherical (Phi6), linear (M13), and icosahedral (MS2 and T4)). Here, the disinfection efficacies of filtered KrCl* excimer lamp (222 nm) and germicidal UVC lamp (254 nm) were evaluated for highly concentrated virus droplets that mimic the virus-laden droplets released from the infected person and deposited on surfaces as fomites. Filtered KrCl* excimer (222 nm) showed significantly better inactivation against all viruses having different genome compositions and structures compared to germicidal UVC (254 nm). The obtained sensitivity against the filtered KrCl* excimer (222 nm) was found to be in the order, T4 > M13 > Phi6 > MS2 whereas for the germicidal UVC (254 nm) it was T4 > M13 > MS2 > Phi6. These results provide a strong basis to promote the use of filtered KrCl* excimer lamps (222 nm) in disinfecting contagious viruses and to limit the associated disease spread in public places and other high-risk areas.

Circulating tumor cells: a valuable indicator for locally advanced nasopharyngeal carcinoma.

Advancements in nasopharyngeal carcinoma (NPC) treatment have led to a focus on personalized treatment. Circulating tumor cells (CTCs) are important for liquid biopsies and personalized treatment but are not being fully utilized. This study examined how pre- and post-treatment CTC counts, EMT subtypes, clinical characteristics, and patient prognosis are related in order to support the use of liquid biopsy in managing NPC. This retrospective study included 141 patients with locally advanced NPC. All patients underwent CanPatrol™ CTC detection pre- and post-treatment and were categorized into EMT subtypes: epithelial type, mixed type, and mesenchymal type. This study analyzed CTC enumeration, EMT subtypes, and their associations with clinical characteristics and survival outcomes. The results indicated a positive correlation between the pre-treatment detection rate of CTCs and N stage (P < 0.01), alongside a positive correlation with the TNM clinical stage (P = 0.02). Additionally, the detection rate of mesenchymal CTCs post-treatment is positively associated with the N stage (P = 0.02). The enumeration of CTCs pre- and post-treatment is negatively correlated with prognosis and has statistical significance. Additionally, an investigation into the EMT subtypes of CTCs revealed a significant association between the presence of mesenchymal CTCs pre- and post-treatment and decreased overall survival (OS) (P < 0.05). Furthermore, T stage, N stage, TNM clinical stage, and Epstein-Barr virus (EBV) DNA were also significantly correlated with OS. The study found that mesenchymal CTCs pre- and post-treatment, as well as the number of CTCs, were linked to a poor prognosis.

The efficacy and safety of adding PD-1 blockade to induction chemotherapy and concurrent chemoradiotherapy (IC-CCRT) for locoregionally advanced nasopharyngeal carcinoma: an observational, propensity score-matched analysis

BackgroundDespite the success of PD-1 blockade in recurrent/metastatic nasopharyngeal carcinoma (NPC), its effect for locoregionally advanced NPC (LANPC) remains unclear. This study aimed to evaluate the benefit of adding PD-1 blockade to the current standard treatment (gemcitabine and cisplatin IC <induction chemotherapy> plus cisplatin CCRT <concurrent chemoradiotherapy>) for LANPC patients.MethodsFrom January 2020 to November 2022, 347 patients with non-metastatic high-risk LANPC (stage III-IVA, excluding T3-4N0) were included. Of the 347 patients, 268 patients were treated with standard treatment (IC-CCRT), and 79 received PD-1 blockade plus IC-CCRT (PD-1 group). For the PD-1 group, PD-1 blockade was given intravenously once every 3 weeks for up to 9 cycles (3 induction and 6 adjuvant). The primary endpoint was disease-free survival (DFS) (i.e. freedom from local/regional/distant failure or death). The propensity score matching (PSM) with the ratio of 1:2 was performed to control confounding factors.ResultsAfter PSM analysis, 150 patients receiving standard treatment and 75 patients receiving additional PD-1 blockade remained in the current analysis. After three cycles of IC, the PD-1 group had significantly higher rates of complete response (defined as disappearance of all target lesions; 24% vs. 9%; P = 0.006) and complete biological response (defined as undetectable cell-free Epstein-Barr virus DNA, cfEBV DNA; 79% vs. 65%; P = 0.046) than that in the standard group. And the incidence of grade 3–4 toxicity during IC was 47% in the PD-1 group and 41% in the standard group, with no significant difference (P = 0.396). During follow-up period, additional PD-1 blockade to standard treatment improved 3-year DFS from 84 to 95%, with marginal statistical significance (HR, 0.28; 95%CI, 0.06-1.19; P = 0.064).ConclusionAdditiaonl PD-1 blockade to gemcitabine and cisplatin IC and adjuvant treatment results in significant improvement in tumor regression, cfEBV DNA clearance, superior DFS, and comparable toxicity profiles in high-risk LANPC patients.

Optimization of immune checkpoint inhibitor treatment planning for relapsed or refractory extranodal NK/T cell lymphoma.

Pembrolizumab (anti-programmed cell death-ligand 1 inhibitor) is a promising salvage therapeutic option for relapsed/refractory extranodal NK/T-cell lymphoma (R/R ENKTL). However, the appropriate duration of pembrolizumab use in R/R ENKTL patients and the optimal timing for administering pembrolizumab remain undetermined. We collected and analyzed clinical information on R/R ENKTL 58 patients who received pembrolizumab to evaluate the optimal treatment durations and clinical information for considering treatment interruption. Treatment outcomes were assessed by 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) and Epstein Barr virus DNA (EBV DNA) every 3months. Nineteen (32.8%) patients had been treated with more than three chemotherapies before pembrolizumab administration. The best response rate towards the first try of pembrolizumab was 38.9% (31.5% complete response rate (CR), 7.4% partial response (PR)). During the 41.8-month median follow-up duration, the median progression-free survival (PFS) was 3.1months, and the median overall survival (OS) was 7.1months. The failure group, which was characterized by Deaville score (DS) 3-4 and circulating EBV detection, or DS 5 with/without EBV detection, had the worst PFS (p < 0.001) and OS (p < 0.001), followed by the high (DS 1-2 and EBV detection, or DS 3-4 and EBV not detected) and low-risk groups (DS 1-2 and EBV not detected). Among the 21 patients who achieved the best response at the first pembolizumab try, the patients who received planned 24 cycles presented better PFS than those who received incomplete cycles (57.6 months vs 20.9 months, P-value = 0.012). Among 13 patients who received avelumab or pembrolizumab in advance, a few who responded to the second trial of pembrolizumab administration had over one year of chemotherapy vacation. Determining the discontinuation or continuation of pembrolizumab would be considered in selected cases assessed by PET-CT and EBV monitoring. Disruption of pembrolizumab treatment may be advisable for the low-risk group(DS 1-2 and EBV not detected), whereas continuation could be warranted for the high-risk group (DS 1-2 and EBV detection, or DS 3-4 and EBV not detected). Moreover, it might be critical to maintain over 24 cycles to improve the survival outcome of R/R ENKTL.

Association of HBeAg decline rate from mid-pregnancy to delivery with HBeAg seroconversion after delivery in hepatitis B virus-infected mothers.

There is still controversy about whether to continue antiviral therapy (AVT) after delivery, especially for pregnant women in the immune tolerance (IT) phase. In this study, a retrospective cohort study was conducted to explore the relationship between hepatitis B e antigen (HBeAg) decline rate (%) from mid-pregnancy to delivery and HBeAg seroconversion postpartum among patients using nucleos(t)ide analogs (NAs) to prevent mother-to-child transmission (MTCT), with the goal of identifying the ideal candidates for postpartum AVT continuation. This retrospective cohort study included 151 postpartum women. Univariate and multivariable logistic regression analyses were conducted to assess the association between the HBeAg decline rate (%) from mid-pregnancy to delivery and HBeAg seroconversion postpartum. Receiver operating characteristic (ROC) analysis was utilized to evaluate the predictive capacity of the HBeAg decline rate (%) and determine the optimal cut-off point. The univariate analysis revealed a significant association between the HBeAg decline rate (%) and HBeAg seroconversion postpartum (OR 1.068, 95% CI: 1.034-1.103, p < .001). In the multivariate regression analysis, adjusting for age, hepatitis B surface antigen (HBsAg) titre (log10 IU/mL) at mid-pregnancy, HBeAg titre (log10 S/CO) at mid-pregnancy, and hepatitis B virus (HBV) DNA load decline rate (%) from mid-pregnancy to delivery, the HBeAg decline rate(%) remained significantly associated with HBeAg seroconversion postpartum (OR 1.050, 95% CI: 1.015-1.093, p = .009). Then HBeAg decline rate (%) was treated as a categorical variable (tertiles) for sensitivity analysis. In the three distinct models, taking Tertile1 as a reference, women in Tertile3 still had a 4.201-fold (OR 4.201, 95% CI: 1.382-12.773, p = .011) higher risk of developing HBeAg seroconversion (p for trend <.05) after adjusting above covariates. The area under the curve (AUC) was 0.723 (95% CI: 0.627-0.819). The optimal cut-off value was 5.43%, with a sensitivity of 0.561, specificity of 0.791, and Youden's index of 0.352.A higher HBeAg decline rate (%) from mid-pregnancy to delivery independently correlated with an increased risk of HBeAg seroconversion postpartum. This decline rate can serve as a valuable clinical indicator for predicting HBeAg seroconversion.

Novel betaherpesviruses and gammaherpesviruses in bats from central China

Herpesviruses are large double-stranded DNA viruses that cause infections in animals and humans with a characteristic of latent infectious within specific tissues. Bats are natural hosts of variety human-infecting viruses and recently have been described as hosts for herpesviruses in several countries around the world. In this study we collected 140 insectivorous bats in the neighboring urban areas of Wuhan City, Hubei Province in the central China between 2020 and 2021. Nested PCR targeting the dpol gene sequence indicated that a total of 22 individuals (15.7% of the sample) tested positive for herpesvirus with 4 strains belonging to the genus Betaherpesvirus and the remaining 18 strains classified as Gammahersvirus. Furthermore, the herpesvirus prevalence in Rhinolophus pusillus was higher at 26.3%, compared to 8.4% in Myotis davidii. The RP701 strain from R. pusillus was the predominant gammaherpesvirus strain detected in bats, accounting for 94.4% (17/18) of all strains. The variations in γ-herpesviruses genomic sequences was evident in phylogenetic tree, where RP701 strain was clustered together with ruminant γ-herpesviruses, while MD704 strain formed a distinct clade with a hedgehog γ-herpesvirus. Four betaherpesviruses exclusively identified from M. davidii, with nucleotide identities ranging from 79.7 to 82.6% compared to known betaherpesviruses. Our study provided evidence that M. davidii can sever as natural host for β-herpesviruses, which extended the host species range. In conclusion, we found that bats from central China harbored novel β-herpesviruses and γ-herpesviruses which were phylogenetically related to ruminant γ-herpesvirus and hedgehog γ-herpesvirus. Our study indicates that bats are natural hosts of β- and γ-herpesviruses and further studies are needed to determine whether there is cross-species transmission of herpesviruses between bats and other animals, or humans.

Roles Played by DOCK11, a Guanine Nucleotide Exchange Factor, in HBV Entry and Persistence in Hepatocytes

HBV infection is challenging to cure due to the persistence of viral covalently closed circular viral DNA (cccDNA). The dedicator of cytokinesis 11 (DOCK11) is recognized as a guanine nucleotide exchange factor (GEF) for CDC42 that has been reported to be required for HBV persistence. DOCK11 is expressed in both the cytoplasm and nucleus of human hepatocytes and is functionally associated with retrograde trafficking proteins Arf-GAP with GTPase domain, ankyrin repeat, and pleckstrin homology domain-containing protein 2 (AGAP2), and ADP-ribosylation factor 1 (ARF1), together with the HBV capsid, in the trans-Golgi network (TGN). This opens an alternative retrograde trafficking route for HBV from early endosomes (EEs) to the TGN and then to the endoplasmic reticulum (ER), thereby avoiding lysosomal degradation. DOCK11 also facilitates the association of cccDNA with H3K4me3 and RNA Pol II for activating cccDNA transcription. In addition, DOCK11 plays a crucial role in the host DNA repair system, being essential for cccDNA synthesis. This function can be inhibited by 10M-D42AN, a novel DOCK11-binding peptide, leading to the suppression of HBV replication both in vitro and in vivo. Treatment with a combination of 10M-D42AN and entecavir may represent a promising therapeutic strategy for patients with chronic hepatitis B (CHB). Consequently, DOCK11 may be seen as a potential candidate molecule in the development of molecularly targeted drugs against CHB.

Insights into phage-bacteria interaction in cold seep Gigantidas platifrons through metagenomics and transcriptome analyses

Viruses are crucial for regulating deep-sea microbial communities and biogeochemical cycles. However, their roles are still less characterized in deep-sea holobionts. Bathymodioline mussels are endemic species inhabiting cold seeps and harboring endosymbionts in gill epithelial cells for nutrition. This study unveiled a diverse array of viruses in the gill tissues of Gigantidas platifrons mussels and analyzed the viral metagenome and transcriptome from the gill tissues of Gigantidas platifrons mussels collected from a cold seep in the South Sea. The mussel gills contained various viruses including Baculoviridae, Rountreeviridae, Myoviridae and Siphovirdae, but the active viromes were Myoviridae, Siphoviridae, and Podoviridae belonging to the order Caudovirales. The overall viral community structure showed significant variation among environments with different methane concentrations. Transcriptome analysis indicated high expression of viral structural genes, integrase, and restriction endonuclease genes in a high methane concentration environment, suggesting frequent virus infection and replication. Furthermore, two viruses (GP-phage-contig14 and GP-phage-contig72) interacted with Gigantidas platifrons methanotrophic gill symbionts (bathymodiolin mussels host intracellular methanotrophic Gammaproteobacteria in their gills), showing high expression levels, and have huge different expression in different methane concentrations. Additionally, single-stranded DNA viruses may play a potential auxiliary role in the virus–host interaction using indirect bioinformatics methods. Moreover, the Cro and DNA methylase genes had phylogenetic similarity between the virus and Gigantidas platifrons methanotrophic gill symbionts. This study also explored a variety of viruses in the gill tissues of Gigantidas platifrons and revealed that bacteria interacted with the viruses during the symbiosis with Gigantidas platifrons. This study provides fundamental insights into the interplay of microorganisms within Gigantidas platifrons mussels in deep sea.

The Interplay between KSHV Infection and DNA-Sensing Pathways

During viral infection, the innate immune system utilizes a variety of specific intracellular sensors to detect virus-derived nucleic acids and activate a series of cellular signaling cascades that produce type Ⅰ IFNs and proinflammatory cytokines and chemokines. Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic double-stranded DNA virus that has been associated with a variety of human malignancies, including Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman disease. Infection with KSHV activates various DNA sensors, including cGAS, STING, IFI16, and DExD/H-box helicases. Activation of these DNA sensors induces the innate immune response to antagonize the virus. To counteract this, KSHV has developed countless strategies to evade or inhibit DNA sensing and facilitate its own infection. This review summarizes the major DNA-triggered sensing signaling pathways and details the current knowledge of DNA-sensing mechanisms involved in KSHV infection, as well as how KSHV evades antiviral signaling pathways to successfully establish latent infection and undergo lytic reactivation.

Global Re-emergence of Monkeypox: A Synoptic Review

AbstractAs the coronavirus disease 2019 pandemic continues to rage, the unprecedented manifold increase in monkeypox (mpox) cases throughout the world is raising qualms about a possible pandemic. As of January 2024, the disease has been reported in around 116 countries, with nearly 92,500 confirmed cases and 170 deaths. In this mini-review, we have endeavored to cover multiple aspects of the mpox disease. Mpox virus is an enveloped double-stranded DNA virus that belongs to the Orthopoxvirus genus. The disease is endemic in certain African countries. Until recently, however, the disease was rarely reported in Europe and the United States. In contrast to the previous outbreaks outside Africa, reported cases in the 2022 outbreak did not travel to the endemic areas. Superspreading events at mass gatherings, multiple-partner sexual encounters, and international travel were major drivers of the recent global outbreak of mpox. The 2022 mpox virus may have undergone accelerated evolution. It diverges from the related 2018 to 2019 viruses by around 50 single-nucleotide polymorphisms, some of which brought about amino acid changes in immunogenic surface glycoprotein B21. Differential diagnosis for mpox could be quite challenging since it can masquerade as a wide variety of illnesses. Worse still, some patients may be asymptomatic or show subtle symptoms. The infection is confirmed by conventional or real-time polymerase chain reaction on lesion material. Although there is no specific therapy approved for mpox infections, two antivirals (tecovirimat and brincidofovir) and vaccinia immune globulin may be used. Vaccines also provide protection against mpox when properly administered prior to exposure. Finally, the implementation of preventive measures is of paramount importance, especially in regions where mpox transmission is widespread and among high-risk populations.

A critical review of diagnostic and prognostic markers of chronic hepatitis B infection

Abstract A major worldwide health concern, chronic hepatitis B necessitates precise prognostic and diagnostic indicators for clinical guidance. This article highlights the clinical importance and current issues of the major markers used in both the detection and prognosis of chronic hepatitis B. An important indicator of an ongoing and persistent infection is the hepatitis B surface antigen. Hepatitis B virus DNA quantification monitoring aids in assessing viral load and hepatic cancer risk. While limited evidence of liver damage is provided by alanine aminotransferase levels, the hepatitis B core antibody verifies acute infection. Seroconversion to the hepatitis B e antibody is linked to a lower risk of disease development, and the hepatitis B e antigen status is a critical prognostic factor. Treatment choices are guided by a biopsy of the liver or minimally invasive liver fibrosis detection. Genotypes of the hepatitis B virus and host variables influence the prognosis by adding to the disease’s variability. Noninvasive techniques to evaluate the severity of the disease are provided by serum markers of fibrosis, such as the fibrosis score based on four criteria and the aspartate aminotransferase-to-platelet ratio index. The requirement for indicators that distinguish between distinct viral phases and increase specificity in evaluating liver damage is one of the challenges facing chronic hepatitis B research. Even though it is quite difficult to find reliable biomarkers for resistance especially when it comes to hepatocellular cancer risk estimation, there are advanced methods, which include imaging and omics that can help in improving the accuracy of the diagnostics and prognosis. Interventions early point that improve patient outcomes are made possible using diagnostics and prognostics as they are quite effective in managing the complicated landscape of chronic hepatitis B. Key in addressing these challenges today and improving the diagnostic and prognostic markers in the future, particularly those that would support the development of successful treatment plans for people living with chronic hepatitis B virus (HBV), are scientific research, technological advances and collaborations.

Experimental Inoculation of Pigs with Monkeypox Virus Results in Productive Infection and Transmission to Sentinels

Monkeypox virus (MPXV) is a re-emerging zoonotic poxvirus responsible for producing skin lesions in humans. Endemic in sub-Saharan Africa, the 2022 outbreak with a clade IIb strain has resulted in ongoing sustained transmission of the virus worldwide. MPXV has a relatively wide host range, with infections reported in rodent and non-human primate species. However, the susceptibility of many domestic livestock species remains unknown. Here, we report on a susceptibility/transmission study in domestic pigs that were experimentally inoculated with a 2022 MPXV clade IIb isolate or served as sentinel contact control animals. Several principal-infected and sentinel contact control pigs developed minor lesions near the lips and nose starting at day 12 through 18 days post-challenge (DPC). No virus was isolated or viral DNA was detected from the lesions; however, MPXV antigen was detected by IHC in tissue from a pustule of a principal infected pig. Viral DNA and infectious virus were detected in nasal and oral swabs up to 14 DPC, with peak titers observed at 7 DPC. Viral DNA was also detected in nasal tissues or skin collected from two principal-infected animals at 7 DPC post-mortem. Furthermore, all principal-infected and sentinel control animals enrolled in the study seroconverted. In conclusion, we provide the first evidence that domestic pigs are susceptible to experimental MPXV infection and can transmit the virus to contact animals.

Characterizing Glycosylation of Adeno-Associated Virus Serotype 9 Capsid Proteins Generated from HEK293 Cells through Glycopeptide Mapping and Released Glycan Analysis

Recombinant adeno-associated viral (AAV) vectors have emerged as prominent gene delivery vehicles for gene therapy. AAV capsid proteins determine tissue specificity and immunogenicity and play important roles in receptor binding, the escape of the virus from the endosome, and the transport of the viral DNA to the nuclei of target cells. Therefore, the comprehensive characterization of AAV capsid proteins is necessary for a better understanding of the vector assembly, stability, and transduction efficiency of AAV gene therapies. Glycosylation is one of the most common post-translational modifications (PTMs) and may affect the tissue tropism of AAV gene therapy. However, there are few studies on the characterization of the N- and O-glycosylation of AAV capsid proteins. In this study, we identified the N- and O-glycosylation sites and forms of AAV9 capsid proteins generated from HEK293 cells using liquid chromatography–tandem mass spectrometry (LC-MS)-based glycopeptide mapping and identified free N-glycans released from AAV9 capsid proteins by PNGase F using hydrophilic interaction (HILIC) LC-MS and HILIC LC-fluorescence detection (FLD) methods. This study demonstrates that AAV9 capsids are sprinkled with sugars, including N- and O-glycans, albeit at low levels. It may provide valuable information for a better understanding of AAV capsids in supporting AAV-based gene therapy development.

Presence of Epstein-Barr virus in cerebrospinal fluid is associated with increased mortality in HIV-negative cryptococcal meningitis.

Cryptococcus neoformans is the most common cause of fungal meningitis and is associated with a high mortality. The clinical significance of concurrent Epstein-Barr virus (EBV) in the cerebrospinal fluid (CSF) of human immunodeficiency virus (HIV)-negative patients with cryptococcal meningitis (CM) remains unclear. A retrospective cohort study was performed by analyzing CSF samples from 79 HIV-negative Chinese Han patients with confirmed CM. We identified CSF viral DNA in these patients by metagenomic next-generation sequencing (mNGS) and compared 10-week survival rates among those with and without EBV DNA in CSF. Of 79 CSF samples tested, 44.3% (35/79) had detectable viral DNA in CSF, while 55.7% (44/79) were virus-negative. The most frequent viral pathogen was EBV, which was detected in 22.8% (18/79) patients. The median number of CSF-EBV DNA reads was 4 reads with a range from 1 to 149 reads. The 10-week mortality were 22.2% (4/18) in those with positive-CSF-EBV and 2.3% (1/44) in those with negative-CSF-virus (hazard ratio 8.20, 95% confidence interval [CI] 1.52-81.80; p=0.014), which remained significant after a multivariate adjustment for the known risk factors of mortality (adjusted hazard ratio 8.15, 95% CI 1.14-92.87; p=0.037). mNGS can identify viruses that coexist in CSF of HIV-negative patients with CM. EBV DNA is most commonly found together with Cryptococcus neoformans in CSF and its presence is associated with increased mortality in HIV-negative CM patients.

Partial Alleviation of Homologous Superinfection Exclusion of SeMNPV Latently Infected Cells by G1 Phase Infection and G2/M Phase Arrest

Viral infection can regulate the cell cycle, thereby promoting viral replication. Hijacking and altering the cell cycle are important for the virus to establish and maintain a latent infection. Previously, Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV)-latently infected P8-Se301-C1 cells, which grew more slowly than Se301 cells and interfered with homologous SeMNNPV superinfection, were established. However, the effects of latent and superinfection with baculoviruses on cell cycle progression remain unknown. In this study, the cell cycle profiles of P8-Se301-C1 cells and SeMNPV or Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected P8-Se301-C1 cells were characterized by flow cytometry. The results showed that replication-related genes MCM4, PCNA, and BAF were down-regulated (p &lt; 0.05) in P8-Se301-C1 cells, and the S phase of P8-Se301-C1 cells was longer than that of Se301 cells. P8-Se301-C1 cells infected with SeMNPV did not arrest in the G2/M phase or affect the expression of Cyclin B and cyclin-dependent kinase 1 (CDK1). Furthermore, when P8-Se301-C1 cells were infected with SeMNPV after synchronized treatment with hydroxyurea and nocodazole, light microscopy and qRT-PCR analysis showed that, compared with unsynchronized cells and S and G2/M phase cells, SeMNPV-infected P8-Se301-C1 cells in G1 phase induced G2/M phase arrest, and the amount of virus adsorption and intracellular viral DNA replication were significantly increased (p &lt; 0.05). In addition, budded virus (BV) production and occlusion body (OB)-containing cells were both increased at 120 h post-infection (p &lt; 0.05). The expression of Cyclin B and CDK1 was significantly down-regulated at 48 h post-infection (p &lt; 0.05). Finally, the arrest of SeMNPV-infected G1 phase cells in the G2/M phase increased BV production (p &lt; 0.05) and the number of OB-containing cells. In conclusion, G1 phase infection and G2/M arrest are favorable to SeMNPV proliferation in P8-Se301-C1 cells, thereby alleviating the homologous superinfection exclusion. The results contribute to a better understanding of the relationship between baculoviruses and insect cell cycle progression and regulation.

Co-prevalence of Human Parvovirus 4 and Primate Erythroparvovirus 1 (B19) in Sickle Cell Disease and Healthy Populations

Co-prevalence of Human Parvovirus 4 and Primate Erythroparvovirus 1 (B19) in Sickle Cell Disease and Healthy Populations

PSI-5 Evaluation of EhV (surrogate for African swine fever virus) inactivation in corn- and soybean-based feed ingredients and diets at various times and temperatures during storage

Abstract African swine fever virus (ASFV) is a member of the nucleocytoplasmic large DNA viruses (NCLDVs) and remains stable in a variety of environments, including animal feed ingredients as shown in previous laboratory experiments and simulations. Emiliania huxleyi virus (EhV) is another member of NCLDV, which has a restricted host range limited to a species of marine algae called Emiliania huxleyi. This algal NCLDV has many similar morphological and physical characteristics to ASFV which makes it a safe and reliable surrogate for studying ASFV survival and inactivation in feed ingredient matrices in-situ under various time and temperature storage conditions. The objective of the present study was to investigate the inactivation of ASFV in various feedstuffs under a wide range of time and temperature conditions during storage using EhV as a surrogate. Corn- and soybean-based feed ingredients including corn, corn fermented protein, distiller dried grains with solubles, extruded soybean meal, high protein distiller’s grains, solvent-extracted soybean meal, soybean hulls, complete feed, and a positive control (no matrix) were inoculated with EhV (1 × 108 viruses/mL). Negative controls were inoculated with medium containing no EhV for each matrix. To assess the effects of different times and temperatures of storage, all samples were stored at temperatures of 4, 24, and 34 °C for 1, 5, 60, and 120 d. For each timepoint and temperature combination, samples were evaluated for virus presence (qPCR) and viability using a previously validated viability qPCR (v-qPCR) method. On average, we observed a 0.2 log reduction in viable viral DNA content over the 120-d period, with a 0.1 log reduction across varying temperatures. No reductions in EhV viability exceeding 1 log were detected for any matrix and the positive control at any time and temperature combination evaluated. No EhV was recovered from the negative control samples. Given that the experimental error was calculated to be within the range of 1 log, we concluded that storing EhV at 4, 24, or 34 °C for up to 120 d did not result in the inactivation of EhV in any of the ingredients, complete feed, or the control with no matrix. These results demonstrate that ASFV-like NCLDVs is extremely resilient and can maintain viability in various feed matrices during long-term storage and suggest that extending storage time alone may not be an effective mitigation practice against African swine fever virus.

Reactivation of Multidrug-Resistant HSV-1 in a Post-Allogenic Hematopoietic Stem Cell Transplant Patient: Dynamic detection of the rare A605 V mutation by next generation sequencing

Abstract We present an immunocompromised patient with a multi-resistant HSV-1 reactivation with a rare mutation (A605 V) in the viral DNA polymerase gene. Next Generation Sequencing suggests the presence of multiple drug-resistant strains before treatment and altered ratios during treatment, affecting the clinical response to aciclovir and foscarnet.