Viral Pathogens Research Articles

Cocirculation of Genetically Distinct Highly Pathogenic Avian Influenza H5N5 and H5N1 Viruses in Crows, Hokkaido, Japan.

We isolated highly pathogenic avian influenza (HPAI) H5N5 and H5N1 viruses from crows in Hokkaido, Japan, during winter 2023-24. They shared genetic similarity with HPAI H5N5 viruses from northern Europe but differed from those in Asia. Continuous monitoring and rapid information sharing between countries are needed to prevent HPAI virus transmission.

Prevalence of bovine respiratory viruses in cattle calves in Kangra district of Himachal Pradesh

Respiratory diseases causing pneumonia are the 2nd leading cause of mortality in bovine calves and are primarily caused by viral pathogens. The present study was undertaken in cattle calves under twelve months of age for detecting four respiratory viruses, viz. bovine herpes virus-1 (BoHV-1), bovine viral diarrhoea virus (BVDV), bovine respiratory syncytial virus (BRSV), and bovine parainfluenza virus type 3 (BPI3), using a commercially available multiscreen antigen ELISA kit. A total of 30 cattle calves necropsied at the Department of Veterinary Pathology, DGCN COVAS, CSKHPKV, Palampur, from June 2021 to June 2022 were screened. The samples of lung tissues and nasal turbinates were collected and stored at –20°C until further processing. The collected tissue samples were homogenised and processed as per the standard protocol. Among the 30 necropsied samples, BoHV-1 and BRSV were detected in 17/30 (56.67%) samples, BPI3 was detected in 14/30 (46.67%) samples, and BVDV was detected in 8/30 (26.67%) samples. 5/30 (16.67%) samples were found positive for all four viruses. Moreover, three viruses were concurrently detected in 4/30 samples (13.33%), and two viruses were present in 10/30 samples (33.33%). Additionally, a single virus was detected in 4/30 samples (13.33%). In conclusion, the present investigation reveals the substantial presence of respiratory viruses in the respiratory tract of cattle calves. The result indicates a complex pattern of co-infections of different viruses, emphasising the need for effective surveillance and management strategies to address the diverse viral dynamics affecting bovine respiratory health.

Effect of multifunctional cationic polymer coatings on mitigation of broad microbial pathogens.

Infection control is critical for maintaining a healthy home, work, and hospital environment. We test a cationic polymer capable of capturing and eradicating viral and bacterial pathogens by applying the polymer to the air filtration material and textiles. The data suggest that the simple addition of cationic material can result in the improvement of an infectious resilient environment against viral and bacterial pathogens.

Immune status of patients with community-acquired pneumonia associated with a new coronavirus infection and other viral and bacterial pathogens

Despite the fact that the state of the individual’s immune system plays an important role in developing community-acquired pneumonia, its clinical features are determined not only by immune response characteristics, but also by the nature of the infectious agent. The aim of the work was to assess immune status of patients with community-acquired pneumonia with lung damage ranging from 2 to 12%, in whom pathogens of a viral, bacterial and fungal nature were verified (n = 96, aged 46,3±20,5) who were admitted to the hospital 5–7 days after disease onset. Materials and methods. The selection of smears and sputum specimens was carried out on day 1 after admission. COVID-19 pathogen was analyzed in smears by PCR using the Vector-PCRR-2019-nCoV-RG kit (SSC Vector of Rospotrebnadzor, Russia). Biochemical activity, colony morphology, and pathogen concentration (in the amount of ≥ 105 CFU/ml) were also evaluated. Based on the results of preliminary studies, target patients groups were formed: with a new coronavirus infection caused by the SARS-CoV-2 gene variant B.1.1.529 (Omicron) (n = 33); with COVID-19 and bacterial pathogens (n = 17); with COVID-19 and combined bacterial and fungal infection (n = 16); patients with identified pathogens of bacterial infections (n = 13); patients with combined bacterial and fungal infection (n = 17). The relative and absolute level of CD45+CD3+ lymphocytes, CD45+CD3+CD4+ cells, CD45+CD3+CD8+ lymphocytes, CD45+CD3-CD16+CD56+ cells, CD45+CD3+CD16+CD56+ lymphocytes, CD45+CD45RO–CD45RA+ and CD45+CD45RO+CD45RA– lymphocytes, CD45+CD19+ cells, CD45+CD5+CD19–CD27– and CD45+CD19+CD5–CD27– lymphocytes, CD45+CD19+CD5–CD27+ cells was assessed in all volunteers. Results. The analysis of the immune status of patients with community-acquired pneumonia associated with Omicron gene variant new coronavirus infection revealed T-lymphocytopenia, a decreased number of CD8+ lymphocytes, an increased relative and absolute number of NK cells. In COVID-19 patients, who also had bacterial pathogens verified, there was a decrease in relative and absolute number of T-helper cells, an increased relative and absolute number of B and B2 lymphocytes. In COVID-19-negative volunteers with bacterial and bacterial-fungal community-acquired pneumonia, the relative and absolute level of B and B2 lymphocytes, as well as the general memory B cell population, was increased. In contrast to COVID-19-positive patients, in the absence of T-lymphocytopenia, a decreased number of CD4+ cells and rise in number of cytotoxic lymphocytes were recorded in patients of these groups. Conclusions. The data obtained indicate that in the immune status of patients with community-acquired pneumonia, depending on the nature of the infectious agent, changes in relative and absolute level of T-helper cells, cytotoxic lymphocytes, as well as in the qualitative and quantitative B-lymphocyte population composition are recorded.

Rapid Detection of Viral, Bacterial, Fungal, and Oomycete Pathogens on Tomatoes with Microneedles, LAMP on a Microfluidic Chip, and Smartphone Device.

Rapid detection of plant diseases before they escalate can improve disease control. Our team has developed rapid nucleic acid extraction methods with microneedles and combined these with loop-mediated amplification (LAMP) assays for pathogen detection in the field. In this work, we developed LAMP assays for early blight (Alternaria linariae, A. alternata, and A. solani) and bacterial spot of tomato (Xanthomonas perforans) and validated these LAMP assays and two previously developed LAMP assays for tomato spotted wilt virus and late blight. Tomato plants were inoculated, and disease severity was measured. Extractions were performed using microneedles, and LAMP assays were run in tubes (with hydroxynaphthol blue) on a heat block or on a newly designed microfluidic slide chip on a heat block or a slide heater. Fluorescence on the microfluidic chip slides was visualized using EvaGreen and photographed on a smartphone. Plants inoculated with X. perforans or tomato spotted wilt virus tested positive prior to visible disease symptoms, whereas Phytophthora infestans and A. linariae were detected at the time of visual disease symptoms. LAMP assays were more sensitive than PCR, and the limit of detection was 1 pg of DNA for both A. linariae and X. perforans. The LAMP assay designed for early blight detected all three species of Alternaria that infect tomato and is thus an Alternaria spp. assay. This study demonstrates the utility of rapid microneedle extraction followed by LAMP on a microfluidic chip for rapid diagnosis of four important tomato pathogens.

Protection efficacy and the safety of the synergy between modified Bazhen powder and PRRSV modified-live virus vaccine against HP-PRRSV in piglets

The highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) poses a significant threat to the global swine industry. Vaccination is a preventive measure against viral infections. However, the use of vaccines in livestock healthcare programs faces the challenge of safety and delayed immune responses. Earlier studies have shown the potential of modified Bazhen powder as an immunomodulator with significant biological properties, but its effect on vaccines against HP-PRRSV is yet to be studied. This study elucidated how modified Bazhen powder could improve the safety and efficacy of the conventional PRRSV vaccine by evaluating T-cell responses, antibody levels, clinical symptoms, levels of viremia, organ health, and cytokine production. The results revealed that the oral application of modified Bazhen powder in combination with PRRS vaccination improved both cellular and humoral immunity, accelerated viremia clearance, improved lung injury scores, and reduced viral load in the tonsils. The modified Bazhen powder also effectively reduced inflammatory responses following a PRRSV challenge. These findings further highlight the pharmacological properties of modified Bazhen powder as a potential oral immunomodulatory agent that could enhance vaccine efficacy and ensure broad-spectrum protection against HP-PRRSV in pigs.

Within-plant genetic drift to control virus adaptation to host-resistance genes.

Manipulating evolutionary forces imposed by hosts on pathogens like genetic drift and selection could avoid the emergence of virulent pathogens. For instance, increasing genetic drift could decrease the risk of pathogen adaptation through the random fixation of deleterious mutations or the elimination of favorable ones in the pathogen population. However, no experimental proof of this approach is available for a plant-pathogen system. We studied the impact of pepper (Capsicum annuum) lines carrying the same major resistance gene but contrasted genetic backgrounds on the evolution of Potato virus Y (PVY). The pepper lines were chosen for the contrasted levels of genetic drift (inversely related to Ne, the effective population size) they exert on PVY populations, as well as for their contrasted resistance efficiency (inversely related to the initial replicative fitness, Wi, of PVY in these lines). Experimental evolution was performed by serially passaging 64 PVY populations every month on six contrasted pepper lines during seven months. These lines exhibited highly divergent evolutionary trajectories, ranging from viral extinctions to replicative fitness gains. The sequencing of the PVY VPg cistron, where adaptive mutations are likely to occur, allowed linking these replicative fitness gains to parallel adaptive nonsynonymous mutations. Evolutionary trajectories were well explained by the genetic drift imposed by the host. More specifically, Ne, Wi and their synergistic interaction played a major role in the fate of PVY populations. When Ne was low (i.e. strong genetic drift), the final PVY replicative fitness remained close to the initial replicative fitness, whereas when Ne was high (i.e. low genetic drift), the final PVY replicative fitness was high independently of the replicative fitness of the initially inoculated virus. We show that combining a high resistance efficiency (low Wi) and a strong genetic drift (low Ne) is the best solution to increase resistance durability, that is, to avoid virus adaptation on the long term.

Evolution of Phytophthora infestans on its potato host since the Irish potato famine

Phytophthora infestans is a major oomycete plant pathogen, responsible for potato late blight, which led to the Irish Potato Famine from 1845–1852. Since then, potatoes resistant to this disease have been bred and deployed worldwide. Their resistance (R) genes recognize pathogen effectors responsible for virulence and then induce a plant response stopping disease progression. However, most deployed R genes are quickly overcome by the pathogen. We use targeted sequencing of effector and R genes on herbarium specimens to examine the joint evolution in both P. infestans and potato from 1845–1954. Currently relevant effectors are historically present in P. infestans, but with alternative alleles compared to modern reference genomes. The historic FAM-1 lineage has the virulent Avr1 allele and the ability to break the R1 resistance gene before breeders deployed it in potato. The FAM-1 lineage is diploid, but later, triploid US-1 lineages appear. We show that pathogen virulence genes and host resistance genes have undergone significant changes since the Famine, from both natural and artificial selection.

Temporal Characterization of the Viral Load of Psittacine Beak and Feather Disease Virus in Rosy-Faced Lovebirds (Agapornis roseicollis)

Psittacine beak and feather disease virus (BFDV) is a widespread and highly pathogenic virus in parrots. The disease typically presents with feather and beak abnormalities, along with possible immune system suppression. No cure or commercialized vaccine is currently available. Our understanding of the Psittacine beak and feather disease often comes from infected individuals with visible symptoms. Limited knowledge exists regarding the pathology and role of asymptomatic individuals in disease transmission. Asymptomatic individuals could shed the virus in their crop secretion, feces, or feathers. In this study, we investigated the temporal change in the viral load in feather and fecal samples from 17 asymptomatic Rosy-faced Lovebirds (Agapornis roseicollis) using qPCR. Our results showed that most of the individuals had very low viral load, while three individuals with high viral load at the beginning of the experiment were observed to exhibit a decreasing trend in viral load in both fecal and feather samples. Surprisingly, the viral load in an individual can drop from a high level to an undetectable level within three months. This suggests that BFDV infection might not be lethal or highly pathogenic for some individuals. We also showed that the viral load in feathers was higher than in feces.

Predictive signature of murine and human host response to typical and atypical pneumonia

BackgroundPneumonia due to typical bacterial, atypical bacterial and viral pathogens can be difficult to clinically differentiate. Host response-based diagnostics are emerging as a complementary diagnostic strategy to pathogen detection.MethodsWe used...

Rapid and sensitive detection of SARS-CoV-2 based on a phage-displayed scFv antibody fusion with alkaline phosphatase and NanoLuc luciferase

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the subsequent pandemic have led to devastating public health and economic losses. The development of highly sensitive, rapid and inexpensive methods to detect and monitor coronaviruses is essential for family diagnosis, preventing infections, choosing treatments and programs and laying the technical groundwork for viral diagnosis. This study established one-step immunoassays for rapid and sensitive detection of SARS-CoV-2 by using a single-chain variable fragment (scFv) fused to alkaline phosphatase (AP) or NanoLuc (NLuc) luciferase. First, a high-affinity scFv antibody specific to the SARS-CoV-2 nucleocapsid (N) protein was screened from hybridoma cells-derived and phage-displayed library. Next, prokaryotic expression of the scFv-AP and scFv-NLuc fusion proteins were induced, leading to excellent antibody binding properties and enzyme catalytic activities. The scFv-AP fusion had a detection limit of 3 pmol per assay and was used to produce eye-readable biosensor readouts. Moreover, the scFv-NLuc protein was applied a in highly sensitive luminescence immunoassay, achieving a detection limit lower than 0.1 pmol per assay. Therefore, the scFv-AP and scFv-NLuc fusion proteins can be applied for the rapid and simple diagnosis of SARS-CoV-2 to safeguard human health and provide guidance for the detection of other pathogenic viruses.

Biopharmaceutical profiling of anti-infective sanggenons from Morus alba root bark for inhalation administration

Mulberry Diels-Alder-type adducts (MDAAs), isolated from Morus alba root bark, exhibit dual activity against viral and bacterial pathogens but show sobering efficacy following oral administration. Inhalation administration may overcome issues with oral bioavailability and improve efficacy for the treatment of respiratory infections. To assess the suitability of MDAAs for inhalation administration, physicochemical (e.g. pH, pKa, logP, pH-dependent solubility) and biopharmaceutical (epithelial cytotoxicity, permeability, and uptake) properties of two bioactive MDAA stereoisomers sanggenon C (SGC) and sanggenon D (SGD) were evaluated as isolated natural compounds and within parent extracts (MA21, MA60). Despite their structural similarity, SGD exhibited a 10-fold higher solubility than SGC across pH 1.2–7.4, with slight increases at neutral pH. Both compounds were more soluble in isolated form than in the parent extracts. The more lipophilic SGC was found to be more cytotoxic when compared to SGD, indicating a better cellular penetration, which was confirmed by uptake studies. Nonetheless, SGC and SGD exhibited no measurable permeability across intact Calu-3 monolayers, highlighting their potential for increased lung retention and improved local anti-infective activity following inhalation administration. Results suggest that SGC and SGD in isolated form, rather than as extracts, are promising candidates for pulmonary drug delivery to treat lung infections.

Gene editing of pigs to control influenza A virus infections

Proteolytic activation of the hemagglutinin (HA) glycoprotein by host cellular proteases is pivotal for influenza A virus (IAV) infectivity. Highly pathogenic avian influenza viruses possess the multibasic cleavage site of the HA which is cleaved by ubiquitous proteases, such as furin; in contrast, the monobasic HA motif is recognized and activated by trypsin-like proteases, such as the transmembrane serine protease 2 (TMPRSS2). Here, we aimed to determine the effects of TMPRSS2 on the replication of pandemic H1N1 and H3N2 subtype IAVs in the natural host, the pig. The use of the CRISPR/Cas 9 system led to the establishment of homozygous gene edited (GE) TMPRSS2 knockout (KO) pigs. Delayed IAV replication was demonstrated in primary respiratory cells of KO pigs in vitro. IAV infection in vivo resulted in significant reduction of virus shedding in the upper respiratory tract, and lower virus titers and pathological lesions in the lower respiratory tract of TMPRSS2 KO pigs as compared to wild-type pigs. Our findings support the commercial use of GE pigs to mitigate influenza A virus infection in pigs, as an alternative approach to prevent zoonotic influenza A transmissions from pigs to human.

Machine learning approaches for influenza A virus risk assessment identifies predictive correlates using ferret model in vivo data

In vivo assessments of influenza A virus (IAV) pathogenicity and transmissibility in ferrets represent a crucial component of many pandemic risk assessment rubrics, but few systematic efforts to identify which data from in vivo experimentation are most useful for predicting pathogenesis and transmission outcomes have been conducted. To this aim, we aggregated viral and molecular data from 125 contemporary IAV (H1, H2, H3, H5, H7, and H9 subtypes) evaluated in ferrets under a consistent protocol. Three overarching predictive classification outcomes (lethality, morbidity, transmissibility) were constructed using machine learning (ML) techniques, employing datasets emphasizing virological and clinical parameters from inoculated ferrets, limited to viral sequence-based information, or combining both data types. Among 11 different ML algorithms tested and assessed, gradient boosting machines and random forest algorithms yielded the highest performance, with models for lethality and transmission consistently better performing than models predicting morbidity. Comparisons of feature selection among models was performed, and highest performing models were validated with results from external risk assessment studies. Our findings show that ML algorithms can be used to summarize complex in vivo experimental work into succinct summaries that inform and enhance risk assessment criteria for pandemic preparedness that take in vivo data into account.

Andrographolide Attenuates RSV-induced Inflammation by Suppressing Apoptosis and Promoting Pyroptosis after Respiratory Syncytial Virus Infection In Vitro.

Respiratory syncytial virus (RSV), which is the predominant viral pathogen responsible for causing acute lower respiratory tract infections in children, currently lacks specific therapeutic drugs. Despite andrographolide's demonstrated effectiveness against various viral infections, its effects on RSV infection remain unclear. In this study, RSV infection and andrographolide-intervened A549 cell lines were used. The virus load of RSV and the levels of IL-6 and IL-8 in the cell supernatant were quantified. The potential targets of andrographolide in the treatment of RSV-infected airway epithelial cells were analyzed using the Gene Expression Omnibus (GEO) database and the PharmMapper Database, and the changes in mRNA expression of these target genes were measured. To further illustrate the effect of andrographolide on the death pattern of RSV-infected airway epithelial cells, Annexin V-FITC/PI apoptosis assays and Western blotting were conducted. Andrographolide decreased the viral load and attenuated IL-6 and IL-8 levels in cell supernatant post-RSV infection. A total of 25 potential targets of andrographolide in the treatment of RSV-infected airway epithelial cells were discovered, and CASP1, CCL5, JAK2, and STAT1 were identified as significant players. Andrographolide noticeably suppressed the increased mRNA expressions of these genes post-RSV infection as well as IL-1β. The flow cytometry analysis demonstrated that andrographolide alleviated apoptosis in RSV-infected cells. Additionally, RSV infection decreased the protein levels of caspase-1, cleaved caspase-1, cleaved IL-1β, N-terminal of GSDMD, and Bcl-2. Conversely, andrographolide increased their levels. These results suggest that andrographolide may reduce RSV-induced inflammation by suppressing apoptosis and promoting pyroptosis in epithelial cells, leading to effective viral clearance.

Pathogenic and Harmful Bacteria in Dairy Technology: Genomic Characterization and Its Correlation with Physicochemical Parameters of Sheep’s Cheese Sold in Southern Brazil

The distinct microbiological and physicochemical characteristics of sheep milk enable the production of cheeses with unique organoleptic properties. The scenario of sheep cheeses in Brazil is still uncertain, lacking data and regulations. This study aims to characterize the diversity of bacterial groups with potential pathogenic and/or harmful properties to milk technology and correlate the presence of these bacteria with the physicochemical properties of sheep cheese. Additionally, it investigates the presence of virulence genes, resistance genes, and toxins in reference genomes. The main steps were (a) sample preparation and fractionation; (b) physicochemical analysis; (c) analysis of the free fatty acids profile by GC-FID; (d) analysis of the volatile compounds profile by GC-MS; (e) molecular analysis—Next Generation Sequencing of the 16S rRNA gene (V3-V4 region); and (f) in silico analysis—Genomics. A total of 1,061,392 sequences were identified, corresponding to 45 genera and 57 species. Of the total abundance, 95.7% were considered beneficial, while 3.9% were classified as bacteria with pathogenic potential (BPP) and/or bacteria harmful to milk technology (BHMT). Five phyla were identified: Firmicutes, Proteobacteria, Actinobacteriota, Bacteroidota, and Fusobacteriota. The genera Acinetobacter, Pseudomonas, and Staphylococcus stood out in the BPP and BHMT groups, showing higher abundance. Alpha diversity of the cheese samples showed that the cheese origin significantly influences the richness, evenness, and abundance of bacterial species. Some physicochemical parameters, fatty acids, and volatile organic compounds showed a negative correlation with Staphylococcus. Reference genomes of two species exhibited a higher number of resistance and virulence genes. This reinforces the need to monitor bacteria considered of lesser relevance to prevent the transmission, through food, of potentially resistant and virulent pathogens.

Molecular Characterization and Pathogenicity of Groundnut Bud Necrosis Virus (Orthotospovirus arachinecrosis) Inducing Necrosis in Black Gram

Orthotospovirus arachinecrosis commonly known as Groundnut bud necrosis virus (GBNV), is the causal agent for necrosis disease in black gram. GBNV is the second most important challenge to black gram production in major growing areas following yellow mosaic disease. Symptoms of necrosis disease of black gram include severe downward curling and twisting of leaf lamina, veinal necrosis, petiole necrosis, bud necrosis, and necrotic streaks on the stem. Infection at early growth stages of black gram leads to plant death, while surviving plants exhibited stunted growth with a significant reduction in pod yield. The GBNV-BG isolate was maintained in pure form on cowpea cv. C-152, resulted in chlorotic and necrotic local lesions on primary leaves at seven days post-inoculation. The pathogenicity of GBNV on black gram (LBG-645) was further confirmed by artificial sap inoculation. Amplification of the coat protein gene of GBNV was confirmed through RT-PCR using gene-specific primers, producing an amplicon of 800 bp. Sequence analysis of the coat protein gene revealed that the present study isolates OQ683310 and OR295403 shared sequence identity of 98.0 % and 99.3 % respectively at the nucleotide level, with other isolates in the NCBI database. The phylogenetic analysis of the coat protein gene revealed that the isolates under this study were closely linked with the GBNV tomato isolate from Tamil Nadu (MN718651 and MZ505083).

Genotype classification and pathogenicity of infectious bursal disease virus circulating in vaccinated broiler chicken farms.

This study investigated the genotype classification and pathogenicity of infectious bursal disease virus (IBDV) circulating in vaccinated broiler chicken farms in Egypt. A total of 150 samples were collected from 30 vaccinated commercial broiler chicken farms and pooled into 30 working samples. IBDV was tested using reverse transcriptase polymerase chain reaction (RT-PCR) amplification of the hypervariable region of the viral protein 2 (hvVP2) and the VP1 gene 5' extremity. Both RT-PCR fragments were sequenced from six samples, and then the obtained nucleotide sequences were analyzed. The IBDV genotypes were identified using nucleotide sequences. Five sequences of the six strains examined were classified as genotype A3B2 for the highly virulent segments A and B (vv-A/vv-B IBDV). Interestingly, this study identified and classified a novelsegment-reassortant strainas the A1B2 genotype. Specifically, it involved the segment reassortment of classical virulent segment A (cv-A) with vv-B producing cv-A/vv-B reassortant IBDV. Subsequently, we compared the pathogenicity of reassortant (cv-A/vv-B) IBDV and vvIBDV strains identified in this study. Both strains developed typical IBD clinical signs, postmortem lesions, histopathology, immunohistochemistry, and lesion scores, which were more severe in vvIBDV than reassortant IBDV. In conclusion, this is the first report of the genotype classification based on both genome segments (hvVP2 and VP1) with pathogenicity of IBDV circulating in vaccinated broiler chicken farms and this pathogenicity is more severe in vvIBDV strain than a novel reassortant IBDV strain.

Establishment of an Infection System for Gentian (Gentiana spp.) Sclerotial Flower Blight Disease.

Gentians (Gentiana spp.) as floriculture crops are constantly exposed to several fungal and viral pathogens in the field. Among the fungal diseases afflicting gentian production, gentian sclerotial flower blight caused by Ciborinia gentianae incurs economic losses, as it affects flowers before and after harvest. Currently, preventive measures for this disease are limited, and no resistant cultivars have been reported. This is partly because of the lack of a reliable infection system that could promote research on this plant-fungus interaction. In this study, Gentiana plant tissue culture material was inoculated with C. gentianae culture filtrate. We successfully demonstrated non-ascospore-mediated infection of C. gentianae. Inoculation of individual hyphal structures present in the culture filtrate suggested that sclerotial primordia are the main agents of this infection. Interestingly, our results indicated that primary infection of C. gentianae occurs in petals rather than leaves, which enables systemic infection and therefore mirrors the fungus's infection strategy observed in the field. Moreover, we showed that (i) non-ascospore hyphal structures can also cause disease in flowers grown in the field, and (ii) ascosporic infection can also be observed using the in vitro system, opening possibilities for both practical and basic research aimed to combat gentian sclerotial flower blight disease.

Adaptive truncation of the S gene in IBV during chicken embryo passaging plays a crucial role in its attenuation.

Like all coronaviruses, infectious bronchitis virus, the causative agent of infectious bronchitis in chickens, exhibits a high mutation rate. Adaptive mutations that arise during the production of live attenuated vaccines against IBV often decrease virulence. The specific impact of these mutations on viral pathogenicity, however, has not been fully elucidated. In this study, we identified a mutation at the 3' end of the S gene in an IBV strain that was serially passaged in chicken embryos, and showed that this mutation resulted in a 9-aa truncation of the cytoplasmic tail (CT) of the S protein. This phenomenon of CT truncation has previously been observed in the production of attenuated vaccines against other coronaviruses such as the porcine epidemic diarrhea virus. We next discovered that the 9-aa truncation in the S protein CT resulted in the loss of the endoplasmic-reticulum-retention signal (KKSV). Rescue experiments with recombinant viruses confirmed that the deletion of the KKSV motif impaired the localization of the S protein to the endoplasmic-reticulum-Golgi intermediate compartment (ERGIC) and increased its expression on the cell surface. This significantly reduced the incorporation of the S protein into viral particles, impaired early subgenomic RNA and protein synthesis, and ultimately reduced viral invasion efficiency in CEK cells. In vivo experiments in chickens confirmed the reduced pathogenicity of the mutant IBV strains. Additionally, we showed that the adaptive mutation altered the TRS-B of ORF3 and impacted the transcriptional regulation of this gene. Our findings underscore the significance of this adaptive mutation in the attenuation of IBV infection and provide a novel strategy for the development of live attenuated IBV vaccines.