Viral Transport Research Articles

A-297 Usefulness of Non-Skin Samples in the PCR Diagnosis of Monkeypox

Abstract Background During 2022, human mpox cases were reported in several European countries. Until November 2022, in Spain, 7404 cases were detected, most of them linked to sexual intercourse. In that context, fast identification of cases and sources of infection were essential for an optimal management. Polymerase chain reaction (PCR) was the preferred laboratory test. Our objective was to evaluate the usefulness of sera and nasopharyngeal samples for MPXV detection, and its application in contact studies without skin or mucosal injuries. Methods In the Hospital Clínico Universitario of Zaragoza (Spain), 106 samples from 50 patients, 38 male and 12 female, aged between 11 to 62 years, with skin lesions suspected MPXV infection were analysed. Samples were collected between May and September 2022 in inactivating universal viral transport medium (Biocomma). Samples were obtained between 1 and 19 days of evolution. To perform the real-time PCR, the VIASURE Monkeypox Virus Real Time PCR Detection Kit and the VIASURE 48—Real Time PCR System were used. Nucleic acid extraction was performed with the automated nucleic acid extraction system MagLEAD12gC using the MagDEA®Dx SV reagents. Results Table 1 shows the results expressed in Ct values. Skin, anogenital and serum samples were the most efficient for MPXV diagnosis. Nevertheless, these data are influenced by the sample collection date. Conclusion Real-time PCR was positive in samples from all locations in 71,4% of the infected patients. Respiratory samples (pharyngeal and nasopharyngeal) were the ones that showed the highest number of false negative results, whereas there was only one skin sample and one serum sample with a negative result. This study shows that sera samples can be used in the diagnosis of MPXV infection because of ease of collection, applicability to contacts without lesions and the option of serology testing for other STI agents (HBV, HCV, HIV, Treponema pallidum…).

Validating Tools to Detect and Inactivate Monkeypox Virus in Human Milk.

Objectives: Breastfeeding and human milk (HM) improve maternal and infant morbidities and mortality. Therefore, monitoring the safety of breastfeeding and access to HM is of critical importance. In this study, we assessed tools to monitor the presence of monkeypox virus (MPXV) in HM and whether standard Holder pasteurization inactivates MPXV. Materials and Methods: Heat-inactivated MPXV was added to HM or viral transport media (VTM) and analyzed using both research and clinical MPXV quantitative polymerase chain reaction (qPCR) tests. Infectious MPXV was added to HM and was exposed to 1 cycle of freeze-thaw, incubation for 1 hour at room temperature, or conditions of Holder pasteurization (62.5°C for 30 minutes) followed by infectious unit quantification by plaque assay. Results: Research and clinical nucleic acid tests detect MPXV that was added to HM but with reduced sensitivity compared with equivalent samples in VTM at low virus inoculum. MPXV added to HM to achieve a starting concentration of 225,000 plaque forming units (pfu)/mL remains infectious after freeze-thaw or 1 hour storage at room temperature. However, Holder pasteurization reduced infectious virus below the limit of detection, >2,000-fold reduction in viral titer. Conclusion: MPXV can be detected when added to HM using a clinical laboratory-developed qPCR test without modification, but the detection limit is reduced compared with equivalent samples in VTM. MPXV remains viable in HM should the virus ever gain access to HM, but Holder pasteurization reduces infectious MPXV to below detection limits and can be used to reduce the risk of MPXV transmission to infants who receive pasteurized (donor) HM.

Number of kinesins engaged in axonal cargo transport: A novel biomarker for neurological disorders

Kinesin motor proteins play crucial roles in anterograde transport of cargo vesicles in neurons, moving them along axons from the cell body towards the synaptic region. Not only the transport force and velocity of single motor protein, but also the number of kinesin molecules involved in transporting a specific cargo, is pivotal for synapse formation. This collective transport by multiple kinesins ensures stable and efficient cargo transport in neurons. Abnormal increases or decreases in the number of engaged kinesin molecules per cargo could potentially act as biomarkers for neurodegenerative diseases such as Alzheimer's, Parkinson's, amyotrophic lateral sclerosis (ALS), spastic paraplegia, polydactyly syndrome, and virus transport disorders. We review here a model constructed using physical measurements to quantify the number of kinesin molecules associated with their cargo, which could shed light on the molecular mechanisms of neurodegenerative diseases related to axonal transport.

Volitional inspiration is mediated by two independent output channels in the primary motor cortex.

The diaphragm is a multifunctional muscle that mediates both autonomic and volitional inspiration. It is critically involved in vocalization, postural stability, and expulsive core-trunk functions, such as coughing, hiccups, and vomiting. In macaque monkeys, we used retrograde transneuronal transport of rabies virus injected into the left hemidiaphragm to identify cortical neurons that have multisynaptic connections with phrenic motoneurons. Our research demonstrates that representation of the diaphragm in the primary motor cortex (M1) is split into two spatially separate and independent sites. No cortico-cortical connections are known to exist between these two sites. One site is located dorsal to the arm representation within the central sulcus and the second site is lateral to the arm. The dual representation of the diaphragm warrants a revision to the somatotopic map of M1. The dorsal diaphragm representation overlaps with trunk and axial musculature. It is ideally situated to coordinate with these muscles during volitional inspiration and in producing intra-abdominal pressure gradients. The lateral site overlaps the origin of M1 projections to a laryngeal muscle, the cricothyroid. This observation suggests that the coordinated control of laryngeal muscles and the diaphragm during vocalization may be achieved, in part, by co-localization of their representations in M1. The neural organization of the two diaphragm sites underlies a new perspective for interpreting functional imaging studies of respiration and/or vocalization. Furthermore, our results provide novel evidence supporting the concept that overlapping output channels within M1 are a prerequisite for the formation of muscle synergies underlying fine motor control.

Noncanonical Rab9a action supports retromer-mediated endosomal exit of human papillomavirus during virus entry.

Rab GTPases play key roles in controlling intracellular vesicular transport. GTP-bound Rab proteins support vesicle trafficking. Here, we report that, unlike cellular protein cargos, retromer-mediated delivery of human papillomaviruses (HPV) into the retrograde transport pathway during virus entry is inhibited by Rab9a in its GTP-bound form. Knockdown of Rab9a inhibits HPV entry by modulating the HPV-retromer interaction and impairing retromer-mediated endosome-to-Golgi transport of the incoming virus, resulting in the accumulation of HPV in the endosome. Rab9a is in proximity to HPV as early as 3.5 h post-infection, prior to the Rab7-HPV interaction, and HPV displays increased association with retromer in Rab9a knockdown cells, even in the presence of dominant negative Rab7. Thus, Rab9a can regulate HPV-retromer association independently of Rab7. Surprisingly, excess GTP-Rab9a impairs HPV entry, whereas excess GDP-Rab9a reduces association between L2 and Rab9a and stimulates entry. These findings reveal that HPV and cellular proteins utilize the Rab9a host trafficking machinery in distinct ways during intracellular trafficking.

Pepper mild mottle virus can infect and traffick within Nicotiana benthamiana plants in non-virion forms

Virions are responsible for the long-distance transport of many viruses, such as Pepper mild mottle virus (PMMoV). Emerging evidence indicates viral traffic in the form of ribonucleoprotein complexes (RNP), yet comprehensive analysis is scarce. In this study, we inoculated plants with PMMoV-GFP, both with and without the coding sequence for the coat protein (CP). PMMoV-GFP was detected in systemic leaves, even in the absence of the CP, despite the presence of much smaller infection areas. Moreover, using leaf extracts from PMMoV-infected plants to perform a root-irrigation experiment, we confirmed that PMMoV can infect plants through root transmission. Diluting the leaf extracts significantly diminished infectivity, and attempts to compensate for the dilution of other components by adding virions above the original level proved ineffective. Our findings strongly indicate that PMMoV can infect and traffick within plants in non-virion forms. Future studies should aim to identify the specific forms involved.

The Zika virus infection remodels the expression of the synaptotagmin-9 secretory protein.

The exact mechanisms involved in flaviviruses virions' release and the specific secretion of viral proteins, such as the Non Structural protein-1 (NS1), are still unclear. While these processes might involve vesicular transport to the cell membrane, NS1 from some flaviviruses was shown to participate in viral assembly and release. Here, we assessed the effect of the Zika virus (ZIKV) NS1 expression on the cellular proteome to identify trafficking-related targets that may be altered in the presence of the viral protein. We detected an increase in the synaptotagmin-9 (SYT9) secretory protein, which participates in the intracellular transport of protein-laden vesicles. We confirmed the effect of NS1 on SYT9 levels by transfection models while also detecting a significant subcellular redistribution of SYT9. We found that ZIKV prM-Env proteins, required for the viral particle release, also increased SYT9 levels and changed its localization. Finally, we demonstrated that ZIKV cellular infection raises SYT9 levels and promotes changes in its subcellular localization, together with a co-distribution with both Env and NS1. Altogether, the data suggest SYT9's implication in the vesicular transport of viral proteins or virions during ZIKV infection, showing for the first time the association of synaptotagmins with the flavivirus' life cycle.

Interferon-λ Activates a Differential Response in Peripheral Neurons That Is Effective against Alpha Herpesvirus Infections.

Alpha herpesviruses (α-HV) infect host mucosal epithelial cells prior to establishing a life-long latent infection in the peripheral nervous system. The initial spread of viral particles from mucosa to the nervous system and the role of intrinsic immune responses at this barrier is not well understood. Using primary neurons cultured in compartmentalized chambers, prior studies performed on Pseudorabies virus (PRV) have demonstrated that type I and type II interferons (IFNs) induce a local antiviral response in axons via distinct mechanisms leading to a reduction in viral particle transport to the neuronal nucleus. A new class of interferons known as type III IFNs has been shown to play an immediate role against viral infection in mucosal epithelial cells. However, the antiviral effects of type III IFNs within neurons during α-HV infection are largely unknown. In this study, we focused on elucidating the antiviral activity of type III IFN against PRV neuronal infection, and we compared the interferon-stimulated gene (ISGs) induction pattern in neurons to non-neuronal cells. We found that IFN pre-exposure of both primary neurons and fibroblast cells significantly reduces PRV virus yield, albeit by differential STAT activation and ISG induction patterns. Notably, we observed that type III IFNs trigger the expression of a subset of ISGs mainly through STAT1 activation to induce an antiviral state in primary peripheral neurons.

The Characteristics of Ocular Findings and the Presence of SARS-CoV-2 in the Tears of Coronavirus Disease 2019 Patients.

Purpose The purpose of the study is to observe the characteristics of ocular manifestationsin coronavirus disease 2019 (COVID-19)patients and to analyzethe presence of severe acute respiratory syndrome coronavirus 2 ribonucleic acid (SARS-CoV-2 RNA) in the tears of patients with moderate-to-severe COVID-19. Material and methods We conductedthis prospective cross-sectional study from February to June 2021 at the AllIndia Institute of Medical Sciences, one of the tertiary eye care centers in Nagpur, India. The study included confirmed COVID-19 patients based on real-time reverse transcription-polymerase chain reaction (RT-PCR) nasopharyngeal swabs, whether or not the patients exhibited ocular symptoms. We recorded detailed information regarding the patients' history, including demographic profile, ocular symptoms, systemic symptoms, and radiologic findings. We collected ocular samples within 48 hours of collecting naso-oropharyngeal samples from the patients' eyes. We used conjunctival swabs to obtain tear samples, which we then placed in viral transport media (VTM) for cold chain transportation to the microbiology department. We performed RT-PCR on the tear samples to detect the presence of the SARS-CoV-2virus. Result We included 40 patients in the study, with 26 (65%) classified as having moderate COVID-19, six (15%) classified as having severe COVID-19, and the remaininghaving mild COVID-19. Out ofthe 40 patients, five (12%) tested positive for SARS-CoV-2 in the tear sample using RT-PCR, seven (17%) exhibited ocular signsand symptoms, and only one tested positive for SARS-CoV-2 in their tears. The ocular manifestations observed in COVID-19 patients included dry eye, conjunctivitis (including conjunctival hyperemia and epiphora), and lid edema. Notably, we detected a positive COVID-19 tear sample in patients both with and without ocular symptoms. Conclusion Limited reports have focused on ocular involvement in patients with COVID-19. However, our study demonstrates the detection of SARS-CoV-2 in conjunctival swabs from confirmed COVID-19 patients, albeit with a lower positivity rate. Despite the low prevalence of the virus found in tears, there is a potential risk of transmission through ocular routes. It is noteworthy that we observed a COVID-19-positive tear sample in patients with and without ocular symptoms. Therefore, it is important to consider the possibility of ocular transmission even in the absence of ocular manifestations. Medical personnel should takecareful precautionsduring ocular examinations of patients diagnosed with COVID-19 to minimize the risk of transmission.

Associating bovine herpesvirus 1 envelope glycoprotein gD with activated phospho-PLC-γ1(S1248).

Phospholipase C gamma 1 (PLC-γ1) may locate at distinct subcellular locations, such as cytosol, plasma membrane, and nucleus for varied biological functions. Bovine herpesvirus 1 (BoHV-1) productive infection activates PLC-γ1 signaling, as demonstrated by increased protein levels of phosphorylated-PLC-γ1 at Ser1248 [p-PLC-γ1(S1248)], which benefits virus productive infection. Here, for the first time, we reported that Golgi apparatus also contains activated p-PLC-γ1(S1248). And BoHV-1 productive infection at later stages (24 hpi) increased the accumulation of p-PLC-γ1(S1248) in the Golgi apparatus, where p-PLC-γ1(S1248) forms highlighted puncta observed via a confocal microscope. Coimmunoprecipitation studies demonstrated that the Golgi p-PLC-γ1(S1248) is specifically associated with the viral protein gD but not gC. In addition, we found that p-PLC-γ1(S1248) is consistently associated with both the plasma membrane-associated virions and the released virions. When the virus-infected cells were treated with PLC-γ1-specific inhibitor, U73122, for a short duration of 4 hours prior to the endpoint of virus infection, we found that the viral protein gD was trapped in the Golgi apparatus, suggesting that the PLC-γ1 signaling may facilitate trafficking of progeny virions out of this organelle. These findings provide a novel insight into the interplay between PLC-γ1 signaling and BoHV-1 replication. IMPORTANCE Bovine herpesvirus 1 (BoHV-1) productive infection increases protein levels of phosphorylated-phospholipase C gamma 1 at Ser1248 [p-PLC-γ1(S1248)]. However, whether it causes any variations to p-PLC-γ1(S1248) localization is not well understood. Here, for the first time, we found that partial p-PLC-γ1(S1248) is residing in the Golgi apparatus, where the accumulation is enhanced by virus infection. p-PLC-γ1(S1248) is consistently associated with virions, partially via binding to gD, in both the Golgi apparatus and cytoplasm membranes. Surprisingly, it also associates with the released virions. Of note, this is the first evidenced BoHV-1 virion-bound host protein. It seems that p-PLC-γ1(S1248) works as an escort during trafficking of progeny virions out of Golgi apparatus to the plasma membranes as well as releasing outside of the cell membranes. Furthermore, we showed that the activated p-PLC-γ1(S1248) is potentially implicated in the transport of virions out of Golgi apparatus, which may represent a novel mechanism to regulate virus productive infection.

Prolonged existence of SARS-CoV-2 RNAs in the extracellular vesicles of respiratory specimens from patients with negative reverse transcription-polymerase chain reaction

Background and aimSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is primarily in the respiratory tract, particularly in patients with underlying comorbidities. This study aimed to investigate the presence of the virus inside the extracellular vesicles (EVs) in patients with and without chronic liver disease (CLD). MethodsEighty patients with positive SARS-CoV-2, including twenty-four patients with CLD and fifty-six patients without CLD, and five healthy controls with negative SARS-CoV-2 were enrolled. Nasal swab specimens were tested for the detection of SARS-CoV-2 using reverse transcription-polymerase chain reaction (RT-PCR). Patients with coronavirus disease 2019 (COVID-19) were followed up on days 7 and 14. Nasal swab, collected in viral transport media (VTM), and plasma samples were investigated at each time point. EVs were isolated from the nasal swabs (collected in VTM) and plasma using differential ultracentrifugation and estimated at each time point. The transmission or replication by the EVs was assessed in Vero E6 cells. ResultsIn patients with baseline RT-PCR positive, SARS-CoV-2 RNAs inside the EVs were found in 68/80 (85%) patients with higher viral load in the nasal swabs than in the EVs (cycle threshold (Ct) value, 23.4 ± 5.7 vs. 30.3 ± 5.0, P < 0.001). On follow-up at day 7, of the 32 patients negative for COVID-19, 15 (46.9%) had virus persistence in the EVs (Ct value, 30.7 ± 2.7), and on day 14, of the 56 patients with negative SARS-CoV-2, 16 patients (28.6%) had positive SARS-CoV-2 RNAs in the EVs (Ct value, 31.4 ± 3.0). The mean viral load decreased on days 7 and 14 compared to baseline in the nasal swabs (P < 0.001) but not in the EVs. Additionally, SARS-CoV-2 RNAs were undetectable in the plasma, but 12.5% of patients were positive in the plasma EVs. Significantly prolonged and high viral load was found in the EVs on day 14 in COVID-19 patients combined with CLD compared with COVID-19 patients (P = 0.0004). We found significant higher levels of EV-associated with endothelial cells and hepatocytes in the COVID-19 + CLD group than COVID-19 group (P = 0.032 and P = 0.002, respectively), suggesting more endothelial cells and hepatocytes cellular injury in liver disease patients with COVID-19. Interestingly, we also found EVs could transmit SARS-CoV-2 RNAs into Vero E6 cells at 24 h post-infection. ConclusionsThe identification of SARS-CoV-2 RNAs in the EVs in patients with negative RT-PCR indicates the persistence of infection and likely recurrence of the infection. It is suggestive of another route of transmission as EVs harbor SARS-CoV-2 RNAs. EV-associated RNAs may determine the ongoing inflammation and clinical course of subjects with undetectable SARS-CoV-2 virus and this may have relevance to better management of patients with CLD.

Assessment of thermal and temporal stability of SARS-CoV-2 samples using real-time qRT-PCR.

As per the guidelines of the Indian Council of Medical Research, nasopharyngeal and oropharyngeal swabs in viral transport medium (VTM) are to be stored at 4°C for less than 5 days and for more than 5 days at -70°C. Samples are not transported or stored as per prescribed conditions because of the limitations, resulting in an apprehensive diagnosis. The aim of the study was to test the stability of the SARS-CoV-2 sample stored in VTM at different temperatures. In this study, the stability of 21 positive and 9 negative samples for SARS-CoV-2 was evaluated in commercial VTM at different temperatures (-80°C, -20°C, 4°C, and 25 to 30°C). Stability was checked for up to 50 days in the above storage conditions at different intervals. PathoDetect™ and Hi-PCR® kits were used for the detection of the four genes of SARS-CoV-2. The Cycle Threshold (Ct) value for determining the positivity of samples for PathoDetect™ was < 40 and for Hi-PCR® was < 38. The SARS-CoV-2 confirmatory genes (RdRp and E genes) and the internal housekeeping gene remained detectable even on the 50th day of the study. The Ct of the RdRp and E genes were found to increase with storage duration, but all positive samples remained positive till the end of the study, or the Ct value remained below the cut-off level. The negative samples gave consistent results until the end of the study. When the differences in Ct values were compared between the days in a set of experiments, they were not significantly different except in a few samples. The SARS-CoV-2 genetic materials in commercial VTM were stable at room temperature to -80°C for 50 days.

HIV Infection: Shaping the Complex, Dynamic, and Interconnected Network of the Cytoskeleton.

HIV-1 has evolved a plethora of strategies to overcome the cytoskeletal barrier (i.e., actin and intermediate filaments (AFs and IFs) and microtubules (MTs)) to achieve the viral cycle. HIV-1 modifies cytoskeletal organization and dynamics by acting on associated adaptors and molecular motors to productively fuse, enter, and infect cells and then traffic to the cell surface, where virions assemble and are released to spread infection. The HIV-1 envelope (Env) initiates the cycle by binding to and signaling through its main cell surface receptors (CD4/CCR5/CXCR4) to shape the cytoskeleton for fusion pore formation, which permits viral core entry. Then, the HIV-1 capsid is transported to the nucleus associated with cytoskeleton tracks under the control of specific adaptors/molecular motors, as well as HIV-1 accessory proteins. Furthermore, HIV-1 drives the late stages of the viral cycle by regulating cytoskeleton dynamics to assure viral Pr55Gag expression and transport to the cell surface, where it assembles and buds to mature infectious virions. In this review, we therefore analyze how HIV-1 generates a cell-permissive state to infection by regulating the cytoskeleton and associated factors. Likewise, we discuss the relevance of this knowledge to understand HIV-1 infection and pathogenesis in patients and to develop therapeutic strategies to battle HIV-1.

Modeling the fate of viruses in aquifers: Multi-kinetics reactive transport, risk assessment, and governing parameters

The transport of viruses in groundwater is a complex process controlled by both hydrodynamic and reaction parameters. Characterizing the transport of viruses in groundwater is of crucial importance for investigating health risks associated with groundwater consumption from private individual or residential pumping wells. Setback distances between septic systems, which are the source of viruses, and pumping wells must be designed to offer sufficient groundwater travel times to allow the viral load to degrade sufficiently to be acceptable for community health needs. This study consists of developing numerical simulations for the reactive transport of viruses in the subsurface. These simulations are validated using published results of laboratory and field experiments on virus transport in the subsurface and applying previously developed analytical solutions. The numerical model is then exploited to investigate the sensitivity of the fate of viruses in saturated porous media to hydraulic parameters and the coefficients of kinetic reactions. This sensitivity analysis provides valuable insights into the prevailing factors governing health risks caused by contaminated water in private wells in rural residential contexts. The simulations of virus transport are converted into health risk predictions through dose–response relationships. Risk predictions for a wide range of input parameters are compared with the international regulatory health risk target of a maximum of 10−4 infections/person/year and a 30 m setback distance to identify critical subsurface contexts that should be the focus of regulators.

Evaluating direct amplification from viral transport medium for SARS-CoV-2 detection, strain typing, and angiotensin-converting enzyme genotyping and expression assays.

The aim of this study was to compare the performance of direct amplification of viral nucleic acid from transport medium to extracted nucleic acid for polymerase chain reaction (PCR), sequencing, and genotyping applications. XpressAmp lysate and extracted total nucleic acid from viral transport medium containing nasopharyngeal specimens were evaluated across different molecular applications to determine performance characteristics. SARS-CoV-2 quantitative PCR and angiotensin-converting enzyme (ACE) genotyping assays worked well with XpressAmp lysate, almost equal with or better than extracted nucleic acid in some specimens. However, XpressAmp completely failed to perform in next-generation sequencing for strain typing. Both protocols failed to detect ACE2 expression in viral transport medium. Direct amplification of viral nucleic acid from viral transport medium containing nasopharyngeal specimen works well for molecular assays with low thresholds of quality; however, it does have limitations with assays that require high quality nucleic acid for input. Use of the XpressAmp protocol significantly improves turnaround time and allows for easy ramp-up of PCR and genotyping assays.

A novel strategy to avoid sensitivity loss in pooled testing for SARS-CoV-2 surveillance: validation using nasopharyngeal swab and saliva samples.

At the peak of the COVID-19 pandemic, pooled surveillance strategies were employed to alleviate the overwhelming demand for clinical testing facilities. A major drawback of most pooled-testing methods is the dilution of positive samples, which leads to a loss of detection sensitivity and the potential for false negatives. We developed a novel pooling strategy that compensates for the initial dilution with an appropriate concentration during nucleic acid extraction and real-time PCR. We demonstrated the proof of principle using laboratory-created 10-sample pools with one positive and corresponding individual positive samples by spiking a known amount of heat-inactivated SARS-CoV-2 into viral transport medium (VTM) or pooled negative saliva. No Ct difference was observed between a 10-sample pool with one positive vs. the corresponding individually analyzed positive sample by this method, suggesting that there is no detectable loss of sensitivity. We further validated this approach by using nasopharyngeal swab (NPS) specimens and showed that there is no loss of sensitivity. Serial dilutions of the virus were spiked into VTM and pooled with negative saliva in simulated 10-sample pools containing one positive to determine the LOD and process efficiency of this pooling methodology. The LOD of this approach was 10 copies/PCR, and the process efficiencies are ~95%-103% for N1 and ~87%-98% for N2 with samples in different matrices and with two different master mixes tested. Relative to TaqPath 1-step master mix, the TaqMan Fast Virus 1-Step master mix showed better sensitivity for the N2 assay, while the N1 assay showed no Ct difference. Our pooled testing strategy can facilitate large-scale, cost-effective SARS-CoV-2 surveillance screening and maintain the same level of sensitivity when analyzed individually or in a pool. This approach is highly relevant for public health surveillance efforts aimed at mitigating SARS-CoV-2 spread.

Virus Infection and mRNA Nuclear Export.

Gene expression in eukaryotes begins with transcription in the nucleus, followed by the synthesis of messenger RNA (mRNA), which is then exported to the cytoplasm for its translation into proteins. Along with transcription and translation, mRNA export through the nuclear pore complex (NPC) is an essential regulatory step in eukaryotic gene expression. Multiple factors regulate mRNA export and hence gene expression. Interestingly, proteins from certain types of viruses interact with these factors in infected cells, and such an interaction interferes with the mRNA export of the host cell in favor of viral RNA export. Thus, these viruses hijack the host mRNA nuclear export mechanism, leading to a reduction in host gene expression and the downregulation of immune/antiviral responses. On the other hand, the viral mRNAs successfully evade the host surveillance system and are efficiently exported from the nucleus to the cytoplasm for translation, which enables the continuation of the virus life cycle. Here, we present this review to summarize the mechanisms by which viruses suppress host mRNA nuclear export during infection, as well as the key strategies that viruses use to facilitate their mRNA nuclear export. These studies have revealed new potential antivirals that may be used to inhibit viral mRNA transport and enhance host mRNA nuclear export, thereby promoting host gene expression and immune responses.

An Efficient Extraction Method Allowing the Genetic Evaluation of Host DNA from Samples Collected for Virus Infection Diagnosis in Viral Transport Medium.

Introduction: During the COVID-19 pandemic, an extraordinary number of nasopharyngeal secretion samples inoculated in viral transport medium (VTM) were collected and analyzed to detect SARS-CoV-2 infection. In addition to viral detection, those samples can also be a source of host genomic material, providing excellent opportunities for biobanking and research. Objective: To describe a simple, in-house-developed DNA extraction method to obtain high yield and quality genomic DNA from VTM samples for host genetic analysis and assess its relative efficiency by comparing its yield and suitability to downstream applications to two different commercial DNA extraction kits. Methods: In this study, 13 VTM samples were processed by two commercial silica-based kits and compared with an in-House-developed protocol for host DNA extraction. An additional 452 samples were processed by the in-House method. The quantity and quality of the differentially extracted DNA samples were assessed by Qubit and spectrophotometric measurements. The suitability of extracted samples for downstream applications was tested by polymerase chain reaction (PCR) amplification followed by amplicon sequencing and allelic discrimination in real-time PCR. Results: The in-House method provided greater median DNA yield (0.81 μg), being significantly different from the PureLink® method (0.14 μg, p < 0.001), but not from the QIAamp® method (0.47 μg, p = 0.980). Overall satisfactory results in DNA concentrations and purity, in addition to cost, were observed using the in-House method, whose samples were able to produce clear amplification in PCR and sequencing reads, as well as effective allelic discrimination in real-time PCR TaqMan® assay. Conclusion: The described in-House method proved to be suitable and economically viable for genomic DNA extraction from VTM samples for biobanking purposes. These results are extremely valuable for the study of the COVID-19 pandemic and other emergent infectious diseases, allowing host genetic studies to be performed in samples initially collected for diagnosis.

The impact of COVID-19 pandemic on surveillance of influenza and influenza-like viruses: a single center experience

Aims: Influenza and the agents responsible for influenza-like illness (ILI) are a significant cause of upper and lower respiratory tract infections, with a notable pattern of seasonal incidence. The surveillance of influenza and agents causing ILI is important for the developmentof a prophylaxis approach and infection control. In the present study, we utilize the Reverse transcription-polymerase chain reaction (RT-PCR) method to identify the presence of influenza virus and other agents associated with ILI in nasopharyngeal smears obtained from patients exhibiting symptoms of influenza and ILI. The study sample included patients who were admitted to multiple clinics and intensive care units (ICU) of the Health Sciences University Ankara Training and Research Hospital in the period preceding, and in the initial months and first year following the coronavirus disease 2019 (COVID-19) outbreak. Throughthe comparisons made in the study, the intention is to gain a better understanding of the influence of the COVID-19 pandemic on other viral infectious agents transmitted via the respiratory tract.&#x0D; Methods: Included in the study were 257 admitted to different wards and ICUs of our hospital due to symptoms of upper or lower respiratory tract infection and ILI between 2015 and 2021 (excluding data from 2017–2018). The study was conducted prospectively within the scope of the Global Influenza Hospital Surveillance Network project. Using sterile swabs, nasopharyngeal swab samples were collected from inpatients who provided informed consent for their participation in the study, and the samples were placed in a viral transport medium. The presence of influenza viruses (Influenza A, subtypes of Influenza A and Influenza B) and agents causing ILI (i.e. respiratory syncytial virus, coronavirus) was investigated with an RT-PCR approach.&#x0D; Results: The most common symptoms among the 257 patients included in the study with pre-diagnoses of influenza and ILI were cough (82.2%), fever (67.7%), shortness of breath (66.1%) and myalgia (40%). The RT-PCR detected a viral agent in 60 (23.3%) of the 257 patients, whereas no agent could be detected in 197 (76.6%) patients. Furthermore, 51 (18.5%) tested positive for influenza virus, five (1.9%) for respiratory syncytial virus (RSV), and four (1.5%) for SARS-CoV-2. An analysis of the results within two distinct time frames, namely prior to the COVID-19 pandemic (2016–2017, 2019–prior to December 10, 2020) and during the COVID-19 pandemic (between December 11, 2020 and 2021), influenza viruses (influenza A H1N1 and influenza A H3N2), RSV and influenza type B were identified as the dominant viruses before the COVID-19 pandemic, while the predominant viruses were a single influenza strain and four SARS-CoV-2 variants during the COVID-19 pandemic.A significant difference was noted in the distribution of viruses between the two time frames – prior to the pandemic and during the pandemic. Of the patients, 199 (77.4%) were discharged with full recovery while 58 patients died (22.6%). Of the 58 patients that died, 25 were female (25/131 females) and 33 were male (33/126 males), 11 had tested positive for influenza virus andone for RSV, while no infectious agent could be identified in 46 patients. &#x0D; Conclusion: The implementation of molecular testing methods for the identification of viral infectious agents among inpatients during influenza and ILI outbreaks, administering antiviral and prophylactic treatments targeting influenza, RSV and SARS-CoV-2 infections, and adopting infection control measures, could significantly decrease mortality and morbidity rates while mitigating the complications associated with these infectious agents.

KRT10 plays an important role in the release of viral genome from endosomes during H9N2 subtype AIV replication in HeLa cells

The infection and replication of avian influenza virus (AIV) in host cells is a complex biological process that involves the transport of viral genes through the host cell's transport systems. Actin, microtubules and vimentin are known to facilitate transport of endosomes to the perinuclear region, but the biological role of Keratin, another intermediate filament, in viral transport during AIV replication is not well understood. In this study, the viral NS2 protein was used as the target protein to identify the potential interacting proteins following GST-Pulldown method and protein mass spectrometry. It was discovered that Keratin10 interacted with NS2. Subsequently, it was found AIV infection did not affect the gene level or protein level of keratin10 in HeLa cells, but when Keratin10 was knocked down, the expressions of viral NP mRNA and protein were reduced, and the generation of offspring virus also was also decreased. Furthermore, in early viral infection, Keratin10 could aggregate and co-localize with NP proteins, suggesting that Keratin10 might be connected to early viral transport. Additionally, it was demonstrated that Keratin10 co-localized with Lamp1 and that AIV particles were trapped in late endosomes/Lysosomes after Keratin10 was knocked down. Finally, it was discovered that the knocking down Keratin10 in HeLa cells led to an increase in the acidic pH of endosomes and lysosomes, which prevented AIV from undergoing fusion and uncoating, and then inhibited the process of the viral infection. Overall, the results suggested that Keratin10 might play the critical role in the release of vRNPs from LEs/Ls and can affect the generation of offspring virus. The study provides the novel insights into the role of Keratin10 in the process of AIV infection and transmission, which may have implications for developing new strategies to against AIV infections.