Infectious Virus Research Articles

The signal peptide of BmNPV GP64 activates the ERAD pathway to regulate heterogeneous secretory protein expression

As a powerful eukaryotic expression vector, the baculovirus expression vector system (BEVS) is widely applied to the production of heterogeneous proteins for research and pharmaceutical purposes, while optimization of BEVS remains a work in progress for membrane or secreted protein expression. In this study, the impact of the signal peptide (SP) derived from Bombyx mori nucleopolyhedrovirus (BmNPV) GP64 protein on protein expression, secretion, and the endoplasmic reticulum-associated degradation (ERAD) pathway were investigated in BmN cells and BEVS. Transient expression studies in BmN cells revealed that SP alters the localization and expression levels of recombinant proteins, reducing intracellular accumulation while enhancing secretion efficiency. Quantitative analysis demonstrated that SP-mediated secretion was markedly higher compared to controls, albeit with lower total expression levels. Further exploration into SP-mediated ERAD pathway activation showed increased expression of BiP and other ERAD-associated genes (PDI, UFD1, S1P, and ASK1), correlating with higher SP-driven protein expression levels. RNA interference (RNAi) experiments elucidated that knockdown of ERAD-associated genes enhances both the secretion efficiency of SP-guided proteins and the infectivity of BmNPV. Particularly, interference with BiP demonstrated the most pronounced effect on protein secretion enhancement. Viral infection experiments further supported these findings, showing upregulated ERAD-associated genes during BmNPV infection, indicating their role in viral protein processing and infectivity. In conclusion, this study elucidates the complex interplay between SP-mediated protein secretion, ERAD pathway activation, and viral infectivity in BmNPV-infected cells. These insights suggest strategies for optimizing recombinant protein production and viral protein processing in baculovirus expression systems, with potential implications for biotechnological and biomedical applications. Further research could refine our understanding and manipulation of protein secretion pathways in insect cell-based expression systems.

Molecular characterization of reassortant infectious bursal disease virus (IBDV) strains of genogroup A3B1 detected in some areas of Britain between 2020 and 2021

Infectious bursal disease virus (IBDV) causes a major immunosuppressive disease of chickens. As part of ongoing epidemiological surveillance for IBDV, the hypervariable region (HVR) of the VP2 capsid gene encoded by segment A, and a region of the VP1 polymerase gene, encoded by segment B, were sequenced from 20 IBDV-positive bursal samples obtained in 2020 and 2021, from 16 commercial British broiler farms. Birds had received a live IBDV vaccine at 17–22 days of age, and samples were obtained at 25–55 days of age. Of the 16 farms, none contained very virulent (vv) strains, one contained a classical virulent strain, two contained vaccine strains, and five contained sequences of reassortant strains with a vv segment A and a non-vv segment B belonging to genogroup A3B1. In eight of the farms, we identified the sequences of both genogroup A3B1 reassortant strains and vaccine strains in the same samples. Therefore, the majority of the farms (13/16 (81%)) contained genogroup A3B1 reassortant viruses. Of the flocks containing reassortant strains, 5/13 (38%) had HVR mutations Q219L, G254D, D279N, and N280T, consistent with a recently described Western European clade, but the rest had other mutations or no mutations, demonstrating that multiple clades were present in the samples. Taken together, vv strains were not detected, but reassortant strains predominated in the farms, which belonged to different clades, and were frequently found together with vaccine strains.

Streptomyces fradiae Mitigates the Impact of Potato Virus Y by Inducing Systemic Resistance in Two Egyptian Potato (Solanum tuberosum L.) Cultivars

In this study, the impact of culture media filtrate of QD3 actinobacterial isolate on two potato cultivars, Spunta and Diamond, infected with potato virus Y (PVY) was investigated. Various parameters, including infection percentage, PVY virus infectivity, disease severity scoring, PVY optical density, photosynthetic and defense-related biochemical markers, enzymatic profiling, phenolic compounds, proline content, salicylic acid levels, and growth and yield parameters, were assessed to elucidate the potential of the QD3 actinobacterial isolate culture filtrate in mitigating PVY-induced damage. The physiological and biochemical characteristics of the QD3 actinobacterial isolate, including its salinity tolerance, pH preferences, and metabolic traits, were investigated. Molecular identification via 16S rRNA gene sequencing confirmed its classification as Streptomyces fradiae QD3, and it was deposited in GenBank with the gene accession number MN160630. Distinct responses between Spunta and Diamond cultivars, with Spunta displaying greater resistance to PVY infection. Notably, pre-infection foliar application of the QD3 filtrate significantly reduced disease symptoms and virus infection in both cultivars. For post-PVY infection, the QD3 filtrate effectively mitigated disease severity and the PVY optical density. Furthermore, the QD3 filtrate positively influenced photosynthetic pigments, enzymatic antioxidant activities, and key biochemical components associated with plant defense mechanisms. Gas chromatography‒mass spectrometry (GC‒MS) analysis revealed palmitic acid (hexadecanoic acid, methyl ester) and oleic acid (9-octadecanoic acid, methyl ester) as the most prominent compounds, with retention times of 23.23 min and 26.41 min, representing 53.27% and 23.25%, respectively, of the total peak area as primary unsaturated fatty acids and demonstrating antiviral effects against plant viruses. Cytotoxicity assays on normal human skin fibroblasts (HSFs) revealed the safety of QD3 metabolites, with low discernible toxicity at high concentrations, reinforcing their potential as safe and effective interventions. The phytotoxicity results indicate that all the seeds presented high germination rates of approximately 95–98%, suggesting that the treatment conditions had no phytotoxic effect on the Brassica oleracea (broccoli) seeds, Lactuca sativa (lettuce) seeds, and Eruca sativa (arugula or rocket) seeds. Overall, the results of this study suggest that the S. fradiae filtrate has promising anti-PVY properties, influencing various physiological, biochemical, and molecular aspects in potato cultivars. These findings provide valuable insights into potential strategies for managing PVY infections in potato crops, emphasizing the importance of Streptomyces-derived interventions in enhancing plant health and crop protection.

Differential Expression of Mitogen-Activated Protein Kinase Kinase 3 (MAP2K3) and Interleukin12B (IL12B) in the Bursa of Fabricius of chicken infected with Infectious Bursal Disease Virus

Infectious Bursal Disease (IBD) caused by Infectious Bursal Disease virus (IBDV), poses a significant threat to the poultry industry, causing immunosuppression and economic losses since its discovery in 1957. To understand the host response to IBDV infection, this study examined the modulation of MAP2K3 and IL12B in chickens infected with IBDV. A local IBDV isolate propagated in embryonated chicken eggs was inoculated into 18-day-old chicken. The mRNA transcription levels of MAP2K3 and IL12B in bursal tissue were quantified by real-time quantitative polymerase chain reaction on day two post-infection and compared with those of uninfected, age-matched control birds (sham inoculated). The study revealed increased expression of the MAP2K3 and IL12B genes, indicating their role in inflammatory response during IBDV infection. Thus, the present investigation sheds light on the involvement of MAP2K3 and IL12B in the inflammatory response to IBDV infection in chickens. Keywords: Infectious bursal disease virus, immunosuppression, MAP2K3, IL12B, pro-inflammatory cytokine gene expression

Dependence of aerosol-borne influenza A virus infectivity on relative humidity and aerosol composition.

We describe a novel biosafety aerosol chamber equipped with state-of-the-art instrumentation for bubble-bursting aerosol generation, size distribution measurement, and condensation-growth collection to minimize sampling artifacts when measuring virus infectivity in aerosol particles. Using this facility, we investigated the effect of relative humidity (RH) in very clean air without trace gases (except ∼400 ppm CO2) on the preservation of influenza A virus (IAV) infectivity in saline aerosol particles. We characterized infectivity in terms of 99%-inactivation time, t 99, a metric we consider most relevant to airborne virus transmission. The viruses remained infectious for a long time, namely t 99 > 5 h, if RH < 30% and the particles effloresced. Under intermediate conditions of humidity (40% < RH < 70%), the loss of infectivity was the most rapid (t 99 ≈ 15-20 min, and up to t 99 ≈ 35 min at 95% RH). This is more than an order of magnitude faster than suggested by many previous studies of aerosol-borne IAV, possibly due to the use of matrices containing organic molecules, such as proteins, with protective effects for the virus. We tested this hypothesis by adding sucrose to our aerosolization medium and, indeed, observed protection of IAV at intermediate RH (55%). Interestingly, the t 99 of our measurements are also systematically lower than those in 1-μL droplet measurements of organic-free saline solutions, which cannot be explained by particle size effects alone.

SARS-CoV-2 outbreak in lions, tigers and hyenas at Denver Zoo.

In late 2019, SARS-CoV-2 spilled-over from an animal host into humans, where it efficiently spread, resulting in the COVID-19 pandemic. Through both natural and experimental infections, we learned that many animal species are susceptible to SARS-CoV-2. Importantly, animals in close proximity to humans, including companion, farmed, and those at zoos and aquariums, became infected, and many studies demonstrated transmission to/from humans in these settings. In this study, we first review the literature of SARS-CoV-2 infections in tigers and lions, and compare species, sex, age, virus and antibody detection assay, and types, frequency and length of clinical signs, demonstrating broad heterogeneity amongst infections. We then describe a SARS-CoV-2 outbreak in lions, tigers and hyenas at Denver Zoo in late 2021. Animals were tested for viral RNA (vRNA) for four months. Lions had significantly more viral RNA in nasal swabs than both tigers and hyenas, and many individual lions experienced viral recrudescence after weeks of undetectable vRNA. Infectious virus was correlated with high levels of vRNA and was more likely to be detected earlier during infection. Four months post-infection, all tested animals generated robust neutralizing antibody titers. Animals were infected with Delta lineage AY.20 identical to a variant circulating at less than 1% in Colorado humans at that time, suggesting a single spillover event from an infected human spread within and between species housed at the zoo. Better understanding of epidemiology and susceptibility of SARS-CoV-2 infections in animals is critical to limit the current and future spread and protect animal and human health.

Co-Regulation Mechanism of Host p53 and Fos in Transcriptional Activation of ILTV Immediate-Early Gene ICP4.

Infectious laryngotracheitis virus (ILTV) exhibits a cascade expression pattern of encoded genes, and ICP4 is the only immediate-early gene of ILTV, which plays a crucial role in initiating the subsequent viral genes. Therefore, studying the transcriptional regulation mechanism of ICP4 holds promise for effectively blocking ILTV infection and spread. Host transcriptional factors p53 and Fos are proven to regulate a variety of viral infections, and our previous studies have demonstrated their synergistic effects in regulating ILTV infection. In this study, we constructed eukaryotic expression vectors for p53 and Fos as well as their specific siRNAs and transfected them into a chicken hepatoma cell line. The results showed that knocking down p53 or Fos significantly inhibited ICP4 transcription, while overexpressing p53 or Fos had an opposite effect. A further CoIP and ChIP-qPCR assay suggested p53 and Fos physically interacted with each other, and jointly bound to the upstream transcriptional regulatory region of ICP4. To elucidate the specific mechanisms of p53 and Fos in regulating ICP4 transcription, we designed p53 and Fos protein mutants by mutating their DNA binding domains, which significantly reduced their binding ability to DNA without affecting their interaction. The results showed that Fos directly bound to the promoter region of ICP4 as a binding target of p53, and the p53-Fos protein complex acted as a transcriptional co-regulator of ICP4. Studying the transcriptional process and regulatory pattern of ICP4 is of great significance for understanding the molecular mechanism of ILTV infection, and thus for finding effective methods to control and prevent it.

Optimization of SARS-CoV-2 culture from clinical samples for clinical trial applications.

RT-qPCR is commonly used for virological endpoints during clinical trials for antiviral therapy to determine the quantity and presence of virus in a sample. However, RT-qPCR identifies viral RNA and cannot determine if viable virus is present. Existing culture-based techniques for SARS-CoV-2 are insensitive and not sufficiently standardized to be employed as clinical study endpoints. The use of a culture system to monitor replicating viruses could mitigate the possibility of molecular techniques identifying viral RNA from inactive or lysed viral particles. The methodology optimized in this study for detecting infectious viruses may have application as a secondary virological endpoint in clinical trials of therapeutics for SARS-CoV-2 in addition to numerous research processes.

Characterization of the emerging recombinant infectious bronchitis virus in China.

Infectious bronchitis virus (IBV) can cause serious harm to poultry industry. It is belong to Coronaviridae which is highly variable. A kind of emerging recombinant IBV (ahysx-1) has been detected in chicken from China in 2016. To understand the epidemiology and characterization of the emerging recombinant IBV, 35,455 samples of chickens from the 15 provinces in China were collected and detected. One hundred and ninety-six out of the 537 flocks (positive rate, 36.49%), and 908 out of 35,455 samples (positive rate, 2.56%) were positive in the detection. The results showed that the emerging recombinant IBV was pandemic in China. Thirteen emerging recombinant IBV isolates were selected and continuous subcultured to the fourth generation and analyzed by Next-generation sequencing. Compared with the reported sequence of ahysx-1, the genomic analysis showed that multiple position insertions and deletions were in 1a gene, 3b gene, M gene and N gene. The identity of the S gene nucleotide sequence between all the 13 emerging recombinant IBV isolates and reference stain ahysx-1 were 98.1-99.1%, while the identity of amino acid sequence were 98.0-99.8%. To better understand the recombination mechanism of the emerging recombinant IBV, the genomic sequence of the 13 isolates were compared with turkey coronavirus or guinea fowl coronavirus. The results suggest that all the 13 emerging recombinant IBV isolates were likely to be the recombination of turkey coronavirus or guinea fowl coronavirus with IBV. Turkey coronavirus or guinea fowl coronavirus as minor parents are the donors of S gene. The major parents donors of the genome backone of these recombination events were lineages GI-19 or GVI-1 of IBV. One isolate (IBV/chicken/Henan/H1173/2021) was selected for pathogenicity analysis. The results showed that IBV/chicken/Henan/H1173/2021 was avirulent to SPF embryonated eggs, but could cause intestinal symptoms in of chicks. This study provides a foundation for understanding the epidemic situation and characterization of the emerging recombinant IBV. It is of great significance for the prevention and control of avian coronavirus infection.

Evidence of true seed transmissible nature of turnip mosaic virus in mustard species

Mustard is a commercial oilseed crop worldwide infected by a highly infectious turnip mosaic virus (TuMV). In the experimental field at ICAR-IARI, New Delhi, in 2022, a 100% incidence of TuMV infection was observed in brown, black and yellow mustard. A very low aphid population suggested the possibility of seed transmission. Earlier, the virus genome was characterized by high throughput sequencing and it was a recombinant of World-B and Asian-BR isolates. The presence of TuMV in immature seeds was confirmed in eight field-grown genotypes via RT-PCR using CP-specific primers designed from the same genome sequence. TuMV was found to be localized in embryo and cotyledon, indicating its true seed-borne nature. Presence of TuMV was also confirmed by RT-PCR in the grow out plants from seeds of field grown eight infected genotypes and 9 genotypes collected from seed stock, that were grown in an aphid-free growth chamber. Further, out of 24 seedlings of Pusa Gold (seed stock) and Pusa Karishma (seeds from field grown plants), 20 and 17 seedlings were found infected with TuMV, respectively. The internally seed-borne nature of the virus leads to its early establishment at the seedling stage, leading to stunting and leaf-puckering symptoms in the progeny plants. This study is the first evidence of seed embryo infection and seedling transmission of TuMV of all the three species of mustard plants (brown, black and yellow mustard). Seed transmission of TuMV in mustard genotypes have implications for the seed exchange programme of mustard seeds.

Identification of Mirror Repeats in Viral Genomes Using FPCB Analysis

The majority of living domains consist of DNA as genetic material with the minor exception of viruses. The uniquenature of every species determines by its unique pattern of genome or gene products. The genomic features become an evidentexample of evolutionary study also. Different types of repeat patterns are observed in genomes of living domains including humanbeings whose two third portion of the genome is repetitive. Among the varied type of repeat sequences Mirror Repeats (MR) playcrucial roles at the genetic level in every species. The major focus of our research is on identification &amp; to check the distributionof mirror repeats in some selected infectious viruses of three different domains (Animal, Plant &amp; Human). For this, we employeda bioinformatics-based approach refer as FASTA PARALLEL COMPLEMENT BLAST (FPCB) to identify unique mirror repeat (MR)sequences. The identified repeats vary in their length as well as found to be distributed throughout the selected viral genomes.The maximum no of MR were reported in the case of Dengue virus (229) &amp; minimum is in the case of TMV (97). In the remainingselected viruses - HCV, HPV, HTLV-1, PVY, Rabies virus 178, 156, 175, 203 &amp; 204 MR sequences were reported. These sequencescan be utilized in many ways like in molecular diagnosis, drug delivery target as well as evolutionary study, etc. The present researchalso helps in the development of novel tools of bioinformatics to study mirror repeats and their functional perspective in thecontext of their occurrence in all domains.

Effect of a loss of the mda5/ifih1 gene on the antiviral resistance in a Chinook salmon Oncorhynchus tshawytscha cell line.

Cells are equipped with intracellular RIG-like Receptors (RLRs) detecting double stranded (ds)RNA, a molecule with Pathogen-Associated Molecular Pattern (PAMPs) generated during the life cycle of many viruses. Melanoma Differentiation-Associated protein 5 (MDA5), a helicase enzyme member of the RLRs encoded by the ifih1 gene, binds to long dsRNA molecules during a viral infection and initiates production of type I interferon (IFN1) which orchestrates the antiviral response. In order to understand the contribution of MDA5 to viral resistance in fish cells, we have isolated a clonal Chinook salmon Oncorhynchus tshawytscha epithelial-like cell line invalidated for the ifih1 gene by CRISPR/Cas9 genome editing. We demonstrated that IFN1 induction is impaired in this cell line after infection with the Snakehead Rhabdovirus (SHRV), the Salmon Alphavirus (SAV) or Nervous Necrosis Virus (NNV). The cell line, however, did not show any increase in cytopathic effect when infected with SHRV or SAV. Similarly, no cytopathic effect was observed in the ifih1-/- cell line when infected with Infectious Pancreatic Necrosis Virus (IPNV), Infectious Haemorrhagic Necrotic Virus (IHNV). These results indicate the redundancy of the antiviral innate defence system in CHSE-derived cells, which helps with circumventing viral evasion strategies.

Evaluation of a Low-Temperature Immersion Immunization Strategy for the Infectious Spleen and Kidney Necrosis Virus orf037l Gene-Deleted Attenuated Vaccine

Background: Infectious spleen and kidney necrosis virus (ISKNV) poses a significant threat to aquaculture sustainability, particularly affecting mandarin fish (Siniperca chuatsi) and causing significant economic losses. Methods: To address this challenge, this study developed an ISKNV Δorf037l vaccine strain, where the orf037l gene was knocked out. Infection assays conducted at 28 °C showed that the knocking out the orf037l gene decreased the virulence of ISKNV and reduced lethality against mandarin fish by 26.7% compared to wild-type ISKNV. To further diminish residual virulence, the effect of low-temperature (22 °C) immersion immunization was evaluated. Results: The results indicate that low temperature significantly diminished the virulence of the Δorf037l vaccine strain, elevating the survival rate of mandarin fish to 90%. Furthermore, the vaccine strain effectively triggered the expression of crucial immune-related genes, such as IFN-h, IL-1, IκB, Mx, TNF-α, and Viperin, while inducing the production of specific neutralizing antibodies. Low-temperature immersion with Δorf037l achieved a high relative percentage of survival of 92.6% (n = 30) in mandarin fish, suggesting the potential of Δorf037l as a promising immersion vaccine candidate. Conclusions: These findings contribute to advancing fish immersion vaccine development and demonstrate the importance and broad applicability of temperature optimization strategies in vaccine development. Our work carries profound implications for both the theoretical understanding and practical application in aquaculture disease control.

Distinct chikungunya virus polymerase palm subdomains contribute to viral protein accumulation and virion production.

Alphaviruses encode an error-prone RNA-dependent RNA polymerase (RdRp), nsP4, required for genome synthesis, yet how the RdRp functions in the complete alphavirus life cycle is not well-defined. Previous work using chikungunya virus has established the importance of the nsP4 residue cysteine 483 in replication. Given the location of residue C483 in the nsP4 palm domain, we hypothesized that other residues within this domain and surrounding subdomains would also contribute to polymerase function. To test this hypothesis, we designed a panel of nsP4 variants via homology modeling based on the coxsackievirus B3 3D polymerase. We rescued each variant in mammalian and mosquito cells and discovered that the palm domain and ring finger subdomain contribute to host-specific replication. In C6/36 cells, we found that while the nsP4 variants had replicase function similar to that of wild-type CHIKV, many variants presented changes in protein accumulation and virion production even when viral nonstructural and structural proteins were produced. Finally, we found that WT CHIKV and nsP4 variant replication and protein production could be enhanced in mammalian cells at 28°C, yet growing virus under these conditions led to changes in virus infectivity. Taken together, these studies highlight that distinct nsP4 subdomains are required for proper RNA transcription and translation, having major effects on virion production.

Abortive infection of bat fibroblasts with SARS-CoV-2

Bats are tolerant to highly pathogenic viruses such as Marburg, Ebola, and Nipah, suggesting the presence of a unique immune tolerance toward viral infection. Here, we compared severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of human and bat (Rhinolophus ferrumequinum) pluripotent cells and fibroblasts. Since bat cells do not express an angiotensin-converting enzyme 2 (ACE2) receptor that allows virus infection, we transduced the human ACE2 (hA) receptor into the cells and found that transduced cells can be infected with SARS-CoV-2. Compared to human embryonic stem cells-hA, infected bat induced Pluripotent Stem Cells (iPSCs)-hA produced about a 100-fold lower level of infectious virus and displayed lower toxicity. In contrast, bat embryonic fibroblast-hA produced no infectious virus while being infectable and synthesizing viral RNA and proteins, suggesting abortive infection. Indeed, electron microscopy failed to detect virus-like particles in infected bat fibroblasts in contrast to bat iPSCs or human cells, consistent with the latter producing infectious viruses. This suggests that bat somatic but not pluripotent cells have an effective mechanism to control virus replication. Consistent with previous results by others, we find that bat cells have a constitutively activated innate immune system, which might limit SARS-CoV-2 infection compared to human cells.

Machine Learning Early Detection of SARS-CoV-2 High-Risk Variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved many high-risk variants, resulting in repeated COVID-19 waves overthe past years. Therefore, accurate early warning of high-risk variants is vital for epidemic prevention and control. However, detecting high-risk variants through experimental and epidemiological research is time-consuming and often lags behind the emergence and spread of these variants. In this study, HiRisk-Detector a machine learning algorithm based on haplotype network, is developed for computationally early detecting high-risk SARS-CoV-2 variants. Leveraging over 7.6 million high-quality and complete SARS-CoV-2 genomes and metadata, the effectiveness, robustness, and generalizability of HiRisk-Detector are validated. First, HiRisk-Detector is evaluated on actual empirical data, successfully detecting all 13 high-risk variants, preceding World Health Organization announcements by 27 days on average. Second, its robustness is tested by reducing sequencing intensity to one-fourth, noting only a minimal delay of 3.8 days, demonstrating its effectiveness. Third, HiRisk-Detector is applied to detect risks among SARS-CoV-2 Omicron variant sub-lineages, confirming its broad applicability and high ROC-AUC and PR-AUC performance. Overall, HiRisk-Detector features powerful capacity for early detection of high-risk variants, bearing great utility for any public emergency caused by infectious diseases or viruses.

Cross-Species Susceptibility of Emerging Variants of SARS-CoV-2 Spike.

The continuous evolution of SARS-CoV-2 and the emergence of novel variants with numerous mutations have heightened concerns surrounding the possibility of cross-species transmission and the establishment of natural animal reservoirs for the virus, but the host range of emerging SARS-CoV-2 variants has not been fully explored yet. We employed an in vitro model comprising VSV∆G* pseudotyped viruses bearing SARS-CoV-2 spike proteins to explore the plausible host range of SARS-CoV-2 emerging variants. The overall host tropism of emerging SARS-CoV-2 variants are consistent with that of the SARS-CoV-2 wuhan-hu-1 strain with minor difference. Pseudotyped viruses bearing spike protein from RaTG13 and RmYN02 can enter cell cultures from a broad range of mammalian species, revealing that mink and hamsters may act as potential intermediate hosts. We further investigated 95 potential site-specific mutations in the SARS-CoV-2 spike protein that could impact viral infectivity across different species. The results showed that 13 of these mutations notably increased the transduction rates by more than two-fold when compared to the wild-type spike protein. Further examination of these 13 mutations within cell cultures from 31 different species revealed heightened sensitivity in cells derived from palm civets, minks, and Chinese horseshoe bats to the VSV∆G*-SARS2-S mutants. Specific mutations, such as L24F, R158G, and L212I, were seen to significantly enhance the capacity for SARS-CoV-2 of cross-species transmission. This study offers critical insights for the ongoing surveillance and monitoring efforts of SARS-CoV-2 evolution, emphasizing the need for the vigilant monitoring of specific mutations in both human and animal populations.

TMT-based quantitative proteomic analysis of IBDV-infected CEF cells reveals a favorable role of chicken IRF10 in IBDV replication via suppressing type-I interferon expression

Infectious bursal disease (IBD) is an acute, highly contagious disease caused by infectious bursal disease virus (IBDV), causing huge economic losses to the poultry industry worldwide. Currently, the emerging variant strains of IBDV and new recombinants in the field are circulating in many countries and poses severe threats to the development of poultry industry. Elucidation of the pathogenesis of IBDV infection will be of great help to the development of vaccines for control of IBDV infection. In this study, liquid chromatography tandem-mass spectrometry (LC–MS/MS) combined with tandem mass tag (TMT) labeling was performed to determine the expressions of nucleus proteins in IBDV-infected chicken embryonic fibroblast (CEF) cells 24 h post-infection (hpi). Our data show that a total of 236 nucleus proteins were differentially expressed in IBDV-infected cells vs mock-infected controls, and that among those proteins, 171 were significantly upregulated while 65 downregulated. Bioinformatics analysis reveals that the differentially expressed proteins (DEPs) were mainly involved in immune response, DNA replication, mismatch repair, and RIG-I-like receptor (RLR) signaling. Consistently, the expression of ten selected upregulated genes (IRF10, IRF7, IRF1, STAT1, ATF3, GTF3A, CSRP3, RARB, BASP1, and NF-κB1) markedly increased as examined by quantitative real‐time PCR (qRT-PCR). Furthermore, the expression of IRF10 was upregulated both in the cytoplasm and nucleus of DF-1 cells as examined by Western Blot. Moreover, knockdown of IRF10 remarkably inhibited IBDV replication via promoting IFN-I response, and overexpression of IRF10 significantly suppressed type I interferon and ISGs expression in both mock and IBDV-infected cells, suggesting that IRF10 serve as a negative regulator for host antiviral response. These results provide clues to further investigation into host–IBDV interactions and the underlying mechanisms of IBDV infection.

TBC1D15-regulated mitochondria–lysosome membrane contact exerts neuroprotective effects by alleviating mitochondrial calcium overload in seizure

Mitochondrial calcium overload plays an important role in the neurological insults in seizure. The Rab7 GTPase-activating protein, Tre-2/Bub2/Cdc16 domain family member 15 (TBC1D15), is involved in the regulation of mitochondrial calcium dynamics by mediating mitochondria–lysosome membrane contact. However, whether TBC1D15-regulated mitochondria–lysosome membrane contact and mitochondrial calcium participate in neuronal injury in seizure is unclear. We aimed to investigate the effect of TBC1D15-regulated mitochondria–lysosome membrane contact on epileptiform discharge-induced neuronal damage and further explore the underlying mechanism. Lentiviral vectors (Lv) infection and stereotaxic adeno-associated virus (AAV) injection were used to regulate TBC1D15 expression before establishing in vitro epileptiform discharge and in vivo status epilepticus (SE) models. TBC1D15’s effect on inter-organellar interactions, mitochondrial calcium levels and neuronal injury in seizure was evaluated. The results showed that abnormalities in mitochondria–lysosome membrane contact, mitochondrial calcium overload, mitochondrial dysfunction, increased levels of reactive oxygen species, and prominent neuronal damage were partly relieved by TBC1D15 overexpression, whereas TBC1D15 knockdown markedly deteriorated these phenomena. Further examination revealed that epileptiform discharge-induced mitochondrial calcium overload in primary hippocampal neurons was closely associated with abnormal mitochondria–lysosome membrane contact. This study highlights the crucial role played by TBC1D15-regulated mitochondria–lysosome membrane contact in epileptiform discharge-induced neuronal injury by alleviating mitochondrial calcium overload.

Synthesis of novel silica-encapsulated core-satellite nanotags for enhanced SERS detection

Developing a SERS nanotag with high signal intensity, excellent signal stability, and biocompatibility is of significant importance in the fields of food safety, drug testing, and virus detection. In this work, silica-encapsulated, indocyanine green (ICG)-modified Au octahedral core-small Au spherical nanoparticles (NPs) satellite nanostructures (Au/Au@ICG@SiO2) are uniformly synthesized as surface-enhanced Raman scattering (SERS) tags, which provide abundant “hot spots” and also increase the stability of Raman reporter molecule ICG to greatly facilitate subsequent functionalization and detection. Both experimental and numerical results demonstrate that this Au/Au@ICG nanostructure achieve a detection limit of 10⁻⁷ M for ICG in aqueous solution ascribed to the increased number of hot spots compared with Au octahedral NPs. As-prepared Au/Au@ICG@SiO2 SERS nanotags are further integrated with a lateral flow immunoassay (LFIA) for the detection of the SARS-CoV-2 nucleocapsid protein. Compared to the traditional bio-recognition method on LFIA strips with the detection limit of 8 pg/mL, the detection capability for the SARS-CoV-2 nucleocapsid protein is improved approximately 16 times (500 fg/mL) based on this novel SERS tag and custom-built adaptive Raman mapping spectrometer. This Au/Au@ICG@SiO2 SERS tag is expected to play a significant role in early detection, prevention, and control of similar infectious viruses in the future.