Viral Titers Research Articles

Effects and mechanism of Qingke Pingchuan granules against influenza virus infection.

Qingke Pingchuan granules (QPGs), which contain Houttuynia cordata Thunb, Fritillaria cirrhosa, fired licorice, and fired bitter almonds, among other components, can clear heat and ventilate the lungs, relieving cough and asthma. Clinically, QPGs are mainly used to treat cough, asthma, fever and other discomforts caused by acute or chronic bronchitis. In this study, the antiviral activity of QPGs against respiratory syncytial virus (RSV), influenza A virus A/FM/1/47 (H1N1), oseltamivir-resistant H1N1, A/Beijing/32/92 (H3N2), Sendai virus, and human adenovirus type 3 in Hep-2 or MDCK cells was evaluated using the CCK-8 method, and the cytotoxicity of QPGs to these two cell lines was tested. The effect of QPGs on mice infected with influenza A virus A/FM/1/47 (H1N1) was evaluated by measuring body weight, survival time, and survival rate, as well as virus titers and lesions in the lungs and levels of inflammatory factors in serum. In addition, the expression of TLR-7-My88-NF-κB signaling pathway-related proteins in lung tissues was analyzed by Western blotting and qRT-PCR. The results showed that QPGs had a potent inhibitory effect on the six viruses tested in vitro. Interestingly, QPGs also displayed particularly pronounced antiviral activity against H1N1-OC, similar to that of oseltamivir, a well-known antiviral drug. QPGs effectively protected mice from infection by H1N1, as indicated by significantly increased body weights, survival times, and survival rates and reduced lung virus titers of inflammatory factors and lung tissue injury. The levels of TLR-7-MyD88-NF-κB-pathway-related proteins in the lung tissue of infected mice were found to be decreased after QPG treatment, thereby alleviating lung injury caused by excessive release of inflammatory factors. Taken together, these findings indicate that QPGs have satisfactory activity against influenza virus infection.

IL-37 possesses both anti-inflammatory and antiviral effects against Middle East respiratory syndrome coronavirus infection.

The aim was to elucidate the function of IL-37 in middle east respiratory syndrome coronavirus (MERS-CoV) infection, thereby providing a novel therapeutic strategy for managing the clinical treatment of inflammatory response caused by respiratory virus infection. We investigated the development of MERS by infecting hDPP4 mice with hCoV-EMC (107 TCID50 [50% tissue culture infectious dose]) intranasally. We infected A549 cells with MERS-CoV, which concurrently interfered with IL-37, detecting the viral titer, viral load, and cytokine expression at certain points postinfection. Meanwhile, we administered IL-37 (12.5 μg/kg) intravenously to hDPP4 mice 2 h after MERS-CoV-2 infection and collected the serum and lungs 5 days after infection to investigate the efficacy of IL-37 in MERS-CoV infection. The viral titer of MERS-CoV-infected A549 cells interfering with IL-37 was significantly reduced by 4.7-fold, and the viral load of MERS-CoV-infected hDPP4 mice was decreased by 59-fold in lung tissue. Furthermore, the administration of IL-37 suppressed inflammatory cytokine and chemokine (monocyte chemoattractant protein 1, interferon-γ, and IL-17A) expression and ameliorated the infiltration of inflammatory cells in hDPP4 mice. IL-37 exhibits protective properties in severe pneumonia induced by MERS-CoV infection. This effect is achieved through attenuation of lung viral load, suppression of inflammatory cytokine secretion, reduction in inflammatory cell infiltration, and mitigation of pulmonary injury.

Japanese encephalitis virus hijacks ER-associated degradation regulators for its replication.

Flaviviruses target their replication on membranous structures derived from the ER, where both viral and host proteins play crucial structural and functional roles. Here, we have characterized the involvement of the ER-associated degradation (ERAD) pathway core E3 ligase complex (SEL1L-HRD1) regulator proteins in the replication of Japanese encephalitis virus (JEV). Through high-resolution immunofluorescence imaging of JEV-infected HeLa cells, we observe that the virus replication complexes marked by NS1 strongly colocalize with the ERAD adapter SEL1L, lectin OS9, ER-membrane shuttle factor HERPUD1, E3 ubiquitin ligase HRD1 and rhomboid superfamily member DERLIN1. NS5 positive structures also show strong overlap with SEL1L. While these effectors show significant transcriptional upregulation, their protein levels remain largely stable in infected cells. siRNA mediated depletion of OS9, SEL1L, HERPUD1 and HRD1 significantly inhibit viral RNA replication and titres, with SEL1L depletion showing the maximum attenuation of replication. By performing protein translation arrest experiments, we show that SEL1L, and OS9 are stabilised upon JEV infection. Overall results from this study suggest that these ERAD effector proteins are crucial host-factors for JEV replication.

Development of a SARS-CoV-2 viral dynamic model for patients with COVID-19 based on the amount of viral RNA and viral titer.

The target-cell limited model, which is one of the mathematical modeling approaches providing a quantitative understanding of viral dynamics, has been applied to describe viral RNA profiles of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in previous studies. However, these models have been developed mainly using patient data from the early phase of the pandemic. Furthermore, no reports focused on the profiles of the viral titer. In this study, the dynamics of both viral RNA and viral titer were characterized using data reflecting the current clinical situation in which the Omicron variant has become epidemic and vaccines for SARS-CoV-2 have become available. Consecutive data for 5212 viral RNA levels and 5216 viral titers were obtained from 720 patients with coronavirus disease 2019 (COVID-19) in a phase II/III study for ensitrelvir. Our model assumed that productively infected cells would produce only infectious viruses, which could be transformed into non-infectious viruses, and has been used to describe the dynamics of both viral RNA levels and viral titer. The time from infection to symptom onset (tinf) of unvaccinated patients was estimated to be 3.0 days, which was shorter than that of the vaccinated patients. The immune-related parameter as a power function for the vaccinated patients was 1.1 times stronger than that for the unvaccinated patients. Our model allows the prediction of the viral dynamics in patients with COVID-19 from the time of infection to symptom onset. Vaccination status was identified as a factor influencing tinf and the immune function.

Need for standardization of Influenza A virus-induced cell death in vivo to improve consistency of inter-laboratory research findings.

The involvement of necroptosis in the control of influenza A virus (IAV) infection has been reported in multiple studies. Downstream of the nucleic acid sensor ZBP1, RIPK3 kinase activity is critically involved in the induction of necroptotic cell death by phosphorylating MLKL, while RIPK3 as a scaffold can induce apoptosis. Paradoxically, RIPK3-deficiency of mice may result in increased or decreased susceptibility to IAV infection. Here, we critically review the published reports on the involvement of RIPK3 in IAV infection susceptibility and try to identify differences in experimental settings that could explain seemingly conflicting outcomes. Analysis of the experimental reports revealed differences in the IAV challenge dose, the IAV inoculum preparation, IAV titer assessment, as well as the route of inoculation between studies. Furthermore, differences were noticed in the inclusion of littermate controls, which show high variance in viral sensitivity. Our evaluation argues for a standardized setup for IAV infection experiments including the preparation of the IAV virus, the use of different IAV infectious doses description and the proper experimental genetic controls of the mouse strains to increase inter-laboratory consistency in this field. Workflow for IAV infection studies in vivo: Viral preparation and titer assessment should be as standardized as possible with the use of a universal repository (such as BEI resources). Infection studies in genetically modified mice and littermate controls should include dose-response experimentation, following a defined infection route and inoculation volume. Data are generated by consistent analysis methods.

Targeting host O-linked glycan biosynthesis affects Ebola virus replication efficiency and reveals differential GalNAc-T acceptor site preferences on the Ebola virus glycoprotein.

Ebola virus glycoprotein (EBOV GP) is one of the most heavily O-glycosylated viral glycoproteins, yet we still lack a fundamental understanding of the structure of its large O-glycosylated mucin-like domain and to what degree the host O-glycosylation capacity influences EBOV replication. Using tandem mass spectrometry, we identified 47 O-glycosites on EBOV GP and found similar glycosylation signatures on virus-like particle- and cell lysate-derived GP. Furthermore, we performed quantitative differential O-glycoproteomics on proteins produced in wild-type HEK293 cells and cell lines ablated for the three key initiators of O-linked glycosylation, GalNAc-T1, -T2, and -T3. The data show that 12 out of the 47 O-glycosylated sites were regulated, predominantly by GalNAc-T1. Using the glycoengineered cell lines for authentic EBOV propagation, we demonstrate the importance of O-linked glycan initiation and elongation for the production of viral particles and the titers of progeny virus. The mapped O-glycan positions and structures allowed to generate molecular dynamics simulations probing the largely unknown spatial arrangements of the mucin-like domain. The data highlight targeting GALNT1 or C1GALT1C1 as a possible way to modulate O-glycan density on EBOV GP for novel vaccine designs and tailored intervention approaches.IMPORTANCEEbola virus glycoprotein acquires its extensive glycan shield in the host cell, where it is decorated with N-linked glycans and mucin-type O-linked glycans. The latter is initiated by a family of polypeptide GalNAc-transferases that have different preferences for optimal peptide substrates resulting in a spectrum of both very selective and redundant substrates for each isoform. In this work, we map the exact locations of O-glycans on Ebola virus glycoprotein and identify subsets of sites preferentially initiated by one of the three key isoforms of GalNAc-Ts, demonstrating that each enzyme contributes to the glycan shield integrity. We further show that altering host O-glycosylation capacity has detrimental effects on Ebola virus replication, with both isoform-specific initiation and elongation playing a role. The combined structural and functional data highlight glycoengineered cell lines as useful tools for investigating molecular mechanisms imposed by specific glycans and for steering the immune responses in future vaccine designs.

An aggregated dataset of serial morbidity and titer measurements from influenza A virus-infected ferrets

Data from influenza A virus (IAV) infected ferrets provides invaluable information towards the study of novel and emerging viruses that pose a threat to human health. This gold standard model can recapitulate many clinical signs of infection present in IAV-infected humans, support virus replication of human, avian, swine, and other zoonotic strains without prior adaptation, and permit evaluation of virus transmissibility by multiple modes. While ferrets have been employed in risk assessment settings for >20 years, results from this work are typically reported in discrete stand-alone publications, making aggregation of raw data from this work over time nearly impossible. Here, we describe a dataset of 728 ferrets inoculated with 126 unique IAV, conducted by a single research group under a uniform experimental protocol. This collection of morbidity, mortality, and viral titer data represents the largest publicly available dataset to date of in vivo-generated IAV infection outcomes on a per-ferret level.

Proof of stability of an RSV Controlled Human Infection Model challenge agent

In 2018, SGS Belgium NV developed RSV-NICA (Respiratory Syncytial Virus-Nasobronchial Infective Challenge Agent), an RSV type A challenge agent for use in RSV Controlled Human Infection Model (CHIM) studies.It is widely recognized that the stability of RSV can be influenced by a variety of environmental parameters, such as temperature and pH. Consequently, our objective was to evaluate the stability of the viral titer of RSV-NICA following five years of controlled storage and to determine the uniformity of the viral titers across different vials of a GMP-qualified batch of RSV-NICA. In addition, we examined the capacity of RSV-NICA to infect human primary airway epithelial cells (MucilAir™), the principal target cells of RSV, and evaluated the influence of single and recurrent freeze–thaw cycles on the infectious viral titer of the challenge agent.The aliquoted RSV-NICA virus stock was subjected to standard virological and molecular methods to gather data on the titer and consistency of the viral titer contained within 24 representative vials of the stock. Our findings illustrate that over a span of five years of cryo-storage, the infectious viral titer in 75% of the tested vials exhibited a comparable average infectious viral titer (4.75 ± 0.06 vs 4.99 ± 0.11; p-value = 0.14). A considerable reduction down to an undetectable level of infectious virus was observed in the remaining vials. RSV-NICA demonstrated its capacity to effectively infect differentiated human airway epithelial cells, with active virus replication detected in these cells through increasing RSV genome copy number over time. Virus tropism for ciliated cells was suggested by the inhibition of cilia beating coupled with an increase in viral RNA titers. No discernable impact on membrane barrier function of the epithelial lung tissues nor cytotoxicity was detected. Pooling of vials with infectious titers > 4.0 log10 TCID50/ml and freeze-thawing of these combined vials showed no deterioration of the infectious titer. Furthermore, pooling and re-aliquoting of vials spanning the entire range of viral titers (including vials with undetectable infectious virus) along with subjecting the vials to three repeated freeze–thaw cycles did not result in a decrease of the infectious titers in the tested vials.Taken together, our findings indicate that long-term cryo-storage of vials containing RSV-NICA challenge agent may influence the infectious viral titer of the virus, leading to a decrease in the homogeneity of this titer throughout the challenge stock. However, our study also demonstrates that when heterogeneity of the infectious titer of an RSV stock is observed, rounds of pooling, re-aliquoting and subsequent re-titration serve as an effective method not only to restore the homogeneity of the infectious titer of an RSV-A stock, but also to optimize patient-safety, scientific and operational aspects of viral inoculation of study participants during at least the period of one RSV CHIM trial. RSV-NICA is a stable, suitable CHIM challenge agent that can be utilized in efficacy trials for RSV vaccines and antiviral entities.

Microplastics dysregulate innate immunity in the SARS-CoV-2 infected lung.

Global microplastic (MP) pollution is now well recognized, with humans and animals consuming and inhaling MPs on a daily basis, with a growing body of concern surrounding the potential impacts on human health. Using a mouse model of mild COVID-19, we describe herein the effects of azide-free 1 μm polystyrene MP beads, co-delivered into lungs with a SARS-CoV-2 omicron BA.5 inoculum. The effect of MPs on the host response to SARS-CoV-2 infection was analysed using histopathology and RNA-Seq at 2 and 6 days post-infection (dpi). Although infection reduced clearance of MPs from the lung, virus titres and viral RNA levels were not significantly affected by MPs, and overt MP-associated clinical or histopathological changes were not observed. However, RNA-Seq of infected lungs revealed that MP exposure suppressed innate immune responses at 2 dpi and increased pro-inflammatory signatures at 6 dpi. The cytokine profile at 6 dpi showed a significant correlation with the 'cytokine release syndrome' signature observed in some COVID-19 patients. The findings are consistent with the recent finding that MPs can inhibit phagocytosis of apoptotic cells via binding of Tim4. They also add to a growing body of literature suggesting that MPs can dysregulate inflammatory processes in specific disease settings.

Development and evaluation of a chicken embryo fibroblast cell culture based live attenuated Indian strain duck plague vaccine

Duck plague (DP) is an acute, contagious and fatal disease, caused by duck enteritis virus (DEV), with worldwide distribution causing several outbreaks and posing severe economic losses. The present study was carried out with a goal of development of a live attenuated cell culture based DP vaccine using an Indian strain of DEV and evaluation of its safety, efficacy along with complete genome analysis. The live attenuated DP vaccine (DPvac/IVRI-19) was developed by serial propagation of a virulent isolate of DEV (DEV/India/IVRI-2016) in the chicken embryo fibroblast (CEF) primary cell culture. Adaptation of DEV in CEF cell culture was indicated by more rapid appearance of cytopathic effects (CPE) and gradual increase of virus titre, which reached up to 107.5 TCID50/mL after 41 passages. The safety, immunogenicity and efficacy of the vaccine were determined by immunization trials in ducklings. The DPvac/IVRI-19 was found to be avirulent and completely safe in the ducklings. Further, the vaccine induced both humoral and cell mediated immune responses and afforded 100% protection against the virulent DEV challenge. A comparison of the whole genome of DPvac/IVRI-19 (MZ911871) and DEV/India/IVRI-2016 (MZ824102) revealed significant number of mutations, which might be associated with viral attenuation. Phylogenetic tree of DEV/India/IVRI-2016 revealed its evolutionary relationship with other DEV isolates, but it formed a separate cluster with certain unique mutations. Thus, with the proven safety and 100% efficacy, the DPvac/IVRI-19 is suitable for large scale production with precisely pure form of vaccine and has potential utility at national and global levels.

Exploiting non-permissive CHO cells as a rapid and efficient method for recombinant HSV-1 isolation

Using herpes simplex virus type 1 (HSV-1) as a therapeutic tool has recently emerged as a promising strategy for enhancing the treatment of various cancers, particularly those associated with the nervous system, which is the virus's natural site of infection. These viruses are specifically engineered to infect and eradicate tumor cells while leaving healthy cells unharmed. To introduce targeted mutations in specific viral genes, gene-modification techniques such as shuttle vector homologous recombination are commonly employed. Plaque purification is then utilized to select and purify the recombinant virus from the parental viruses. However, plaque purification becomes problematic when the insertion of the desired gene at the target site hampers progeny virus replication, resulting in a lower titer of cell-released virus than the parental virus. This necessitates a laborious initial screening process using approximately 10–15 tissue culture dishes (10 cm), making plaque purification time-consuming and demanding. Although the recently developed CRISPR-Cas9 system significantly enhances the efficiency of homologous integration and editing precision in viral genes, the purification of recombinant variants remains a tedious task. In this study, we propose a rapid and innovative method that employs non-permissive Chinese hamster ovary (CHO) cells, representing a remarkable improvement over the aforementioned arduous process. With this approach, only 1–2 rounds of plaque purification are required. Our proposed protocol demonstrates great potential as a viable alternative to current methods for isolating and purifying recombinant HSV-1 variants expressing fluorescent reporter genes using CHO cells and plaque assays.

Ben-JNK signaling is required for host mortality during Periplaneta fuliginosa densovirus infection.

Cockroaches are widely acknowledged as significant vectors of pathogenic microorganisms. The Periplaneta fuliginosa densovirus (PfDNV) infects the smoky-brown cockroach P. fuliginosa and causes host mortality, which identifies the PfDNV as a species-specific and environmentally friendly biopesticide. However, although the biochemical characterization of PfDNV has been extensively studied, the immune response against PfDNV remains largely unclear. Here, we investigated the replication of PfDNV and its associated pathological phenotype in the foregut and hindgut. Consequently, we dissected and performed transcriptome sequencing on the foregut, midgut, and hindgut separately. We revealed the up-regulation of immune response signaling pathway c-Jun N-terminal kinase (JNK) and apoptosis in response to viral infection. Furthermore, knockdown of the JNK upstream gene Ben resulted in a decrease in virus titer and delayed host mortality. Taken together, our findings provide evidence that the Ben-JNK signaling plays a crucial role in PfDNV infection, leading to excessive apoptosis in intestinal tissues and ultimately resulting in the death of the host. Our results indicated that the host response to PfDNV fosters viral infection, thereby increasing host lethality. This underscores the potential of PfDNV as a viable, environmentally friendly biopesticide. © 2024 Society of Chemical Industry.

Duration of Highly Pathogenic Avian Influenza Virus and Newcastle Disease Virus Infectivity in Dried Ornithologic Study Skins.

Ornithologic study skins are specimens of avian skins that have been preserved by drying after removing the viscera and muscle. Because of the high value of study skins for scientific studies, specimens are shared among researchers. There is concern that study skins might be contaminated with high-consequence diseases such as highly pathogenic avian influenza virus (HPAIV) or Newcastle disease virus (NDV). To mitigate risk, thermal or chemical treatment of study skins may be required before transfer; however, such treatments might damage the specimens. Therefore, a study was conducted to evaluate the duration of infectivity of HPAIV and NDV in study skins prepared from infected chickens (Gallus gallus). Study skins were prepared from 10 chickens infected with each virus. Skin and feather pulp samples were taken at the time of study skin preparation to establish starting titers. Mean starting titers in skins were 4.2 log10 and 5.1 log10 50% egg infectious doses (EID50) for HPAIV and NDV groups respectively, and were 6.7 log10 EID50 for HPAIV, and 6.4 log10 EID50 for NDV in feather pulp. Samples were collected at 2 and 4 wk of drying to quantify viable virus. At 2 wk, fewer samples had detectable virus and mean titers were 1.8 log10 (skin) and 2.1 log10 (feathers) EID50 for HPAIV, and 1.7 log10 (skin) and 3.5 log10 (feathers) EID50 for NDV. At 4 wk viable virus could not be detected in either tissue type.

Blood RNA Biomarkers Identify Bacterial and Biofilm Coinfections in COVID-19 Intensive Care Patients.

Purpose: Secondary opportunistic coinfections are a significant contributor to morbidity and mortality in intensive care unit (ICU) patients, but can be difficult to identify. Presently, new blood RNA biomarkers were tested in ICU patients to diagnose viral, bacterial, and biofilm coinfections. Methods: COVID-19 ICU patients had whole blood drawn in RNA preservative and stored at -80°C. Controls and subclinical infections were also studied. Droplet digital polymerase chain reaction (ddPCR) quantified 6 RNA biomarkers of host neutrophil activation to bacterial (DEFA1), biofilm (alkaline phosphatase [ALPL], IL8RB/CXCR2), and viral infections (IFI27, RSAD2). Viral titer in blood was measured by ddPCR for SARS-CoV2 (SCV2). Results: RNA biomarkers were elevated in ICU patients relative to controls. DEFA1 and ALPL RNA were significantly higher in severe versus incidental/moderate cases. SOFA score was correlated with white blood cell count (0.42), platelet count (-0.41), creatinine (0.38), and lactate dehydrogenase (0.31). ALPL RNA (0.59) showed the best correlation with SOFA score. IFI27 (0.52) and RSAD2 (0.38) were positively correlated with SCV2 viral titer. Overall, 57.8% of COVID-19 patients had a positive RNA biomarker for bacterial or biofilm infection. Conclusions: RNA biomarkers of host neutrophil activation indicate the presence of bacterial and biofilm coinfections in most COVID-19 patients. Recognizing coinfections may help to guide the treatment of ICU patients.

Identification of unique electromagnetic signatures from GLRaV-3 infected grapevine leaves in different stages of virus development.

Grapevine leafroll associated virus 3 (GLRaV-3) is one of the most common viruses that adversely affect grape production quality around the world. Grapevine virus is usually detected by polymerase chain reaction (PCR) tests, but the main diagnostic challenges with the PCR are uneven distribution of symptoms, low virus titers in early stage, and the tests are time consuming and expensive. In the absence of effective control and treatment for grapevine virus, the recommendation is to remove infected vines before they become a source of transmission and a rapid and early detection method for viruses is urgently needed. This study investigated how GLRaV-3 infection affects spectral behavior of grapevine leaves and the use of remote sensing tools to identify changes caused by the biotic stress. A Real-Time RT-qPCR test was performed to select representative vines with healthy and GLRaV-3 infection, and changes in electromagnetic (EM) spectrum of leaves were observed with a portable hyperspectral spectrometer and a multispectral sensor on a remotely piloted aircraft system (RPAS). The results of this study suggest that foliar EM reflectance may differ depending on the presence or absence of visible symptoms of infected leaves and different stages of virus infection progress could be indicated by changes in the relative rate of reflectance around 700 nm. This research also confirmed that the development of a narrow-band hyperspectral index for the detection of grapevine virus is feasible, further research is recommended in a much more comprehensive investigation including a combination of hyperspectral sensor and chlorophyll fluorescence sensors, interpolation of suspect wavebands into ratios or indices, larger sample sizes and a more controlled study environment.

An Elastin-like Polypeptide-fusion peptide targeting capsid-tegument interface as an antiviral against cytomegalovirus infection

The tegument protein pp150 of Human Cytomegalovirus (HCMV) is known to be essential for the final stages of virus maturation and mediates its functions by interacting with capsid proteins. Our laboratory has previously identified the critical regions in pp150 important for pp150-capsid interactions and designed peptides similar in sequence to these regions, with a goal to competitively inhibit capsid maturation. Treatment with a specific peptide (PepCR2 or P10) targeted to pp150 conserved region 2 led to a significant reduction in murine CMV (MCMV) growth in cell culture, paving the way for in vivo testing in a mouse model of CMV infection. However, the general pharmacokinetic parameters of peptides, including rapid degradation and limited tissue and cell membrane permeability, pose a challenge to their successful use in vivo. Therefore, we designed a biopolymer-stabilized elastin-like polypeptide (ELP) fusion construct (ELP-P10) to enhance the bioavailability of P10. Antiviral efficacy and cytotoxic effects of ELP-P10 were studied in cell culture, and pharmacokinetics, biodistribution, and antiviral efficacy were studied in a mouse model of CMV infection. ELP-P10 maintained significant antiviral activity in cell culture, and this conjugation significantly enhanced P10 bioavailability in mouse tissues. The fluorescently labeled ELP-P10 accumulated to higher levels in mouse liver and kidneys as compared to the unconjugated P10. Moreover, viral titers from vital organs of MCMV-infected mice indicated a significant reduction of virus load upon ELP-P10 treatment. Therefore, ELP-P10 has the potential to be developed into an effective antiviral against CMV infection.

Mayaro virus, a potential threat for Europe: vector competence of autochthonous vector species

BackgroundMayaro virus (MAYV) is an emerging alphavirus, primarily transmitted by the mosquito Haemagogus janthinomys in Central and South America. However, recent studies have shown that Aedesaegypti, Aedesalbopictus and various Anopheles mosquitoes can also transmit the virus under laboratory conditions. MAYV causes sporadic outbreaks across the South American region, particularly in areas near forests. Recently, cases have been reported in European and North American travelers returning from endemic areas, raising concerns about potential introductions into new regions. This study aims to assess the vector competence of three potential vectors for MAYV present in Europe.MethodsAedesalbopictus from Italy, Anophelesatroparvus from Spain and Culexpipiens biotype molestus from Belgium were exposed to MAYV and maintained under controlled environmental conditions. Saliva was collected through a salivation assay at 7 and 14 days post-infection (dpi), followed by vector dissection. Viral titers were determined using focus forming assays, and infection rates, dissemination rates, and transmission efficiency were calculated.ResultsResults indicate that Ae.albopictus and An.atroparvus from Italy and Spain, respectively, are competent vectors for MAYV, with transmission possible starting from 7 dpi under laboratory conditions. In contrast, Cx.pipiens bioform molestus was unable to support MAYV infection, indicating its inability to contribute to the transmission cycle.ConclusionsIn the event of accidental MAYV introduction in European territories, autochthonous outbreaks could potentially be sustained by two European species: Ae.albopictus and An.atroparvus. Entomological surveillance should also consider certain Anopheles species when monitoring MAYV transmission.Graphical abstract

Seneca valley virus 3C protease blocks EphA2-Mediated mTOR activation to facilitate viral replication

The Seneca Valley virus (SVV) is a recently discovered porcine pathogen that causes vesicular diseases and poses a significant threat to the pig industry worldwide. Erythropoietin-producing hepatoma receptor A2 (EphA2) is involved in the activation of the AKT/mTOR signaling pathway, which is involved in autophagy. However, the regulatory relationship between SVV and EphA2 remains unclear. In this study, we demonstrated that EphA2 is proteolysed in SVV-infected BHK-21 and PK-15 cells. Overexpression of EphA2 significantly inhibited SVV replication, as evidenced by decreased viral protein expression, viral titers, and viral load, suggesting an antiviral function of EphA2. Subsequently, viral proteins involved in the proteolysis of EphA2 were screened, and the SVV 3C protease (3Cpro) was found to be responsible for this cleavage, depending on its protease activity. However, the protease activity sites of 3Cpro did not affect the interactions between 3Cpro and EphA2. We further determined that EphA2 overexpression inhibited autophagy by activating the mTOR pathway and suppressing SVV replication. Taken together, these results indicate that SVV 3Cpro targets EphA2 for cleavage to impair its EphA2-mediated antiviral activity and emphasize the potential of the molecular interactions involved in developing antiviral strategies against SVV infection.

Further preclinical characterization of molnupiravir against SARS-CoV-2: Antiviral activity determinants and viral genome alteration patterns

The SARS-CoV-2 pandemic has highlighted the need for broad-spectrum antiviral drugs to respond promptly to viral emergence. We conducted a preclinical study of molnupiravir (MOV) against SARS-CoV-2 to fully characterise its antiviral properties and mode of action. The antiviral activity of different concentrations of MOV was evaluated ex vivo on human airway epithelium (HAE) and in vivo in a hamster model at three escalating doses (150, 300 and 400 mg/kg/day) according to three different regimens (preventive, pre-emptive and curative). We assessed viral loads and infectious titres at the apical pole of HAE and in hamster lungs, and MOV trough concentration in plasma and lungs. To explore the mode of action of the MOV, the entire genomes of the collected viruses were deep-sequenced. MOV effectively reduced viral titres in HAE and in the lungs of treated animals. Early treatment after infection was a key factor in efficacy, probably associated with high lung concentrations of MOV, suggesting good accumulation in the lung. MOV induced genomic alteration in viral genomes with an increase in the number of minority variants, and predominant G to A transitions. The observed reduction in viral replication and its mechanism of action leading to lethal mutagenesis, supported by clinical trials showing antiviral action in humans, provide a convincing basis for further research as an additional means in the fight against COVID-19 and other RNA viruses.

Growth and replication of infectious bursal disease virus in the fish cell line as an experimental vaccine

Recently, several attempts have been made to replace egg-based with cell-based vaccines to prevent and control Infectious Bursal Disease Virus (IBDV). This study aimed to evaluate a new fish cell line (M99) for culturing and replicating IBDV. After observing complete cytopathic effects (CPE) on the M99 cell line, virus titers were determined using the TCID50 test, and the presence of the virus was confirmed using an RT-PCR test. Subsequently, 135 broiler chickens (14 days old) were randomly divided into three equal groups for immune response measurements: G1: immunized with a commercial vaccine, G2: immunized with an experimental vaccine, and G3: control. Antibody responses, bursal index, and histopathological evaluations were examined on different days after immunization. Based on the results, CPE of the virus was noticeable from the first passage, becoming complete by the third passage. The infectious titer of the virus was log106.9. Antibody titer measured 21 days after immunization in both vaccinated groups were significantly differed from the control group (p < 0.05). The results obtained from examining the bursal index and histopathological evaluations showed no significant difference between the studied groups at different times. Overall, this research is the first report on the successful cultivation of infectious bursal virus on a permanent cell line of fish origin, with the advantages of tolerance to a wide temperature range (26–40 degrees Celsius). Therefore, this cell line has potential for use to attenuate, cultivate, and adapt other pathogens to cold temperatures in future studies.