Phage therapy is a potential alternative to antibiotics for the treatment of bacterial infections. Due to the good antibacterial and therapeutic effects of phages, phage therapy has received attention and the demand for clinical applications has gradually increased. Phage storage stability and shelf life are key aspects of biopharmaceutical development and registration. In this study, Aeromonas salmonicida phage was stored at different temperatures for 12 months. We found that 4 °C was the optimal storage temperature. In the case of cryopreservation, 10% dimethyl sulfoxide (DMSO) was more effective at protecting the phage at -20 °C and -80 °C than 30% glycerin. Indeed, the phage titer decreased by only one order of magnitude within one year when DMSO was added. Hydroxyapatite (HAP) reduced the inactivation of phages by six orders of magnitude during storage at 28ºC for 1 year, significantly lower than that of in SM buffer. In addition, for excipients in lyophilization, tryptic soy broth (TSB) and tryptone or skim milk powder (SMP) in combination with trehalose alleviated phage inactivation during lyophilization and subsequent storage at 28 °C. Furthermore, a model for predicting the phage shelf-life was established with the Accelerated Stability Assessment Program (ASAP) based on the Arrhenius equation. The error of the model was less than 15% by comparing the predicted value with the actual value at 28 °C, indicating high accuracy. The study demonstrated the storage stability and shelf-life model of phage for the first time, which provided a theoretical basis for the development and application of phage products.

African swine fever virus (ASFV), a highly pathogenic large DNA virus, is the cause of African swine fever worldwide. The ASFV virulence gene EP402R encodes CD2v, a structural protein that plays an important role in the ASFV infection process. In this study, a CHO-S cell line stably expressing the extracellular region of CD2v was generated and secretory CD2v(sCD2v)was purified from the cell culture supernatant. The purified glycosylated sCD2v protein possessed high immunoreactivity and immunogenicity. In addition, we found that glycosylation had a decisive effect on the immune reactivity of CD2v. Then sCD2v was used to generate five CD2v-specific monoclonal antibodies. The reactivity of all monoclonal antibodies with CD2v protein was confirmed by Western blot and indirect immunofluorescence assay (IFA). Interestingly, mAb 8D5 reactivity with sCD2v depended on sCD2v glycosylation status. Subsequent B cell epitope mapping experiments conducted using a series of overlapping synthetic peptides of the CD2v extracellular domain led to identification of mAb B cell epitopes of 128TCKKNNGTNT137 for mAb 4B11 and 148VKYTNESILE157 for mAbs 5H4 and 5F7. Due to their well-defined epitopes, these three mAbs will likely serve as valuable tools for use in ASFV CD2v structure-function studies, diagnostic assays, and prophylactic methodologies to control ASFV transmission.

Chikungunya infection leads to acute/chronic polyarthritis/polyarthralgia causing long-term morbidity among patients. Prognosis of post-chikungunya chronic arthritis (PCA) is of utmost necessity for proper disease management. Arthritic and hepatic biomarkers were evaluated among chikungunya patients without arthritis, with acute arthritis and with post-chikungunya chronic arthritis in the study. Serum levels of arthritic [CRP (C-reactive protein), anti cyclic-citrullinated-peptide (anti-CCP) antibody, soluble interleukin-2 receptor (sIL-2R), cartilage oligomeric matrix protein (COMP)] and hepatic [ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), albumin and bilirubin] biomarkers of 167 chikungunya positive patients were determined by sandwich-ELISA/immunoturbidimetry/auto-analyser. 167 chikungunya-patients and 102 healthy controls were genotyped to understand role of CRP-rs3093059/rs3091244, IL-2R-rs743777 and COMP-rs144778694 polymorphisms towards chikungunya virus (CHIKV) infectivity and arthralgic manifestation. CRP, anti-CCP antibody, IL-2R and COMP levels significantly increased among PCA patients. Concentrations of AST, ALT, AST/ALT-ratio, bilirubin and ALP increased among arthritic chikungunya patients. Principal component analysis differentiated PCA groups from acute (AA) and non-arthritic groups. Patients with IL-2R-rs743777-GA, G-allele and COMP-rs144778694-GA genotypes were susceptible to chikungunya infection. Moreover, patients with CRP-rs3093059-CT, rs3091244-TT, IL-2R-rs743777-GA and COMP-rs144778694-AA genotypes were significantly protected from arthralgia, whereas, COMP-rs144778694-GA genotype was susceptible towards it. Patients with certain genotypes of CRP, IL-2R and COMP demonstrated significantly higher biomarker serum-levels among patients suffering from AA with/without PCA. Thus, both serum biomarker levels and polymorphic genotypes of infected patients play decisive role in development of PCA.

Partial cleavage of a dengue virus envelope protein, prM, by furin results in a mixture of extracellular particles with variable levels of maturation and infectivity. Partially mature particles can infect leukocytes via interaction between the prM-anti-prM antibody complex with Fcγ receptors. Known prM epitopes involved in antibody-mediated infection are localized to the pr domain. In this study, a group of murine anti-prM monoclonal antibodies with strong infection-enhancing activity was found to reduce the focus size of subsets of multiple dengue serotypes that they could enhance. By employing sets of overlapping peptides, four antibodies recognizing 2-mercaptoethanol-insensitive epitopes were mapped to a common tetrapeptide located distantly in the b-c loop and furin binding site. Substitution mutations of each, or both, of the tetrapeptides in virus-like particles, however, failed to reduce binding. Further mapping experiments were performed using immature virus-like particles with abolished furin binding site to minimize the differential influence of various pr substitutions on pr-M cleavage. Reduction of antibody binding was detected when single alanine substitutions were introduced into the 'a' strand and 'c' strand of pr domain. These findings suggest that the pr 'a and c' strands region is the major binding site of these unusual focus size-reducing anti-prM antibodies.

In the 90's, pear blister canker viroid (PBCVd), apple dimple fruit viroid (ADFVd), peach latent mosaic viroid (PLMVd) and chrysanthemum chlorotic mottle viroid (CChMVd) were identified and characterized in the Ricardo Flores' laboratory. In these studies, the autonomous replication of these infectious RNAs and their involvement in the elicitation of diseases in their natural hosts were also shown. Their discovery was achieved by classical approaches based on the physical purification of the viroid RNAs from polyacrylamide gels followed by the sequencing of their genomic RNAs and by bioassays to assess their autonomous replication and the fulfillment of Koch's postulates. The molecular characterization of these four viroids, including the study of their sequence variability, contributed to the establishment of the concept of quasispecies for viroids and to the development of reliable molecular diagnostic methods that have facilitated the control of the diseases they caused. Most importantly, some of these viroids became valuable experimental model systems that are still used nowadays to study structural-functional relationships in RNAs and to dissect evolutionary and pathogenic pathways underlying plant-viroid interaction. The differences between early viroid discovery strategies, relying on biological and pathogenic issues, and the current high-throughput sequencing-based approaches, that frequently allow the discovery of new viroids and viroid-like RNAs in symptomless hosts, is also discussed, clarifying why the traditional molecular and biological studies mentioned above are still required to conclusively define the nature of any novel viroid-like RNA.

The leafhopper Recilia dorsalis (Hemiptera: Cicadellidae) is not only a significant pest in agriculture but also an important vector involved in transmitting numerous pathogens that are known to cause economic losses by affecting rice crops. Here, a new iflavirus was discovered in the leafhopper R. dorsalis by employing a transcriptomic approach. The complete viral genome was determined to be 10,711 nucleotides (nt) in length and contains a single open reading frame (ORF) encoding a putative polyprotein comprised of 3,161 amino acids (aa), which is flanked by 5' and 3' untranslated regions. The full viral genome nt and the deduced polyprotein aa sequence showed the highest similarity (71.6% and 77.8%, respectively) with Langfang leafhopper iflavirus. Phylogenetic analysis based on the RdRp domain indicated that the isolated virus, which we have tentatively named Recilia dorsalis iflavirus 2 (RdIV2), is clustered with the members of the family Iflaviridae. Moreover, the results of our surveys indicate that RdIV2 predominates in southwestern Guangdong and southeastern Guangxi, China, and was absent in the other three species of leafhoppers; Nephotettix cincticeps, N. virescens and N. nigropictus. Notably, R. dorsalis was found to be co-infected with RdIV2 and rice stripe mosaic virus (RSMV; a well-known rice-infecting virus vectored by R. dorsalis) in rice fields, although the co-infection rate is low.

Comorbidities such as diabetes worsen COVID-19 severity and recovery. Metformin, a first-line medication for type 2 diabetes, has antiviral properties and certain studies have also indicated its prognostic potential in COVID-19. Here, we report that metformin significantly inhibits SARS-CoV-2 growth in cell culture models. First, a steady increase in AMPK phosphorylation was detected as infection progressed, suggesting its important role during viral infection. Activation of AMPK in Calu3 and Caco2 cell lines using metformin revealed that metformin suppresses SARS-CoV-2 infectious titers up to 99%, in both naïve as well as infected cells. IC50 values from dose-variation studies in infected cells were found to be 0.4 and 1.43 mM in Calu3 and Caco2 cells, respectively. Role of AMPK in metformin's antiviral suppression was further confirmed using other pharmacological compounds, AICAR and Compound C. Collectively, our study demonstrates that metformin is effective in limiting the replication of SARS-CoV-2 in cell culture and thus possibly could offer double benefits as diabetic COVID-19 patients by lowering both blood glucose levels and viral load.

Since 2010, mass mortality events known as Pacific oyster mortality syndrome (POMS) have occurred in Crassostrea gigas in Australia associated with Ostreid herpesvirus 1. The virus was thought to be an OsHV-1 µVar or "microvariant", i.e. one of the dominant variants associated with POMS in Europe, but there are few data to characterize the genotype in Australia. Consequently, the genetic identity and diversity of the virus was determined to understand the epidemiology of the disease in Australia. Samples were analysed from diseased C. gigas over five summer seasons between 2011 and 2016 in POMS-affected estuaries: Georges River in New South Wales (NSW), Hawkesbury River (NSW) and Pitt Water in Tasmania. Sequencing was attempted for six genomic regions. Numerous variants were identified among these regions (n = 100 isolates) while twelve variants were identified from concatenated nucleotide sequences (n = 61 isolates). Nucleotide diversity of the seven genotypes of C region among Australian isolates (Pi 0.99 × 10-3) was the lowest globally. All Australian isolates grouped in a cluster distinct from other OsHV-1 isolates worldwide. This is the first report that Australian outbreaks of POMS were associated with OsHV-1 distinct from OsHV-1 reference genotype, µVar and other microvariants from other countries. The findings illustrate that microvariants are not the only variants of OsHV-1 associated with mass mortality events in C. gigas. In addition, there was mutually exclusive spatial clustering of viral genomic and amino acid sequence variants between estuaries, and a possible association between genotype/amino acid sequence and the prevalence and severity of POMS, as this differed between these estuaries. The sequencing findings supported prior epidemiological evidence for environmental reservoirs of OsHV-1 for POMS outbreaks in Australia.

Foot-and-mouth disease (FMD) is a devastating livestock disease caused by foot-and-mouth disease virus (FMDV), a member of the Picornaviridae family. The 3ABC is a non-structural protein of FMDV, produced during viral replication and absent from inactivated FMD vaccines. Nucleic acid aptamers are DNA or RNA oligonucleotides capable of binding with high specificity and affinity to a molecular target. The aim of this study was to obtain DNA aptamers specific for 3ABC protein with a view of their application in the FMD diagnosis. Aptamers are usually obtained through SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure. In this study, an aptamer (termed FMDV1) was selected by a variation of this technique called Capillary Electrophoresis SELEX (CE-SELEX). The FMDV1 aptamer showed high binding affinity to the 3ABC protein with Kd value in the nano molar range: 22.69 ± 1.79 nM. The FMDV1 aptamer binding to 3ABC was significantly higher when compared with the BSA protein, used as control, demonstrating its specificity.

Ticks transmit diverse human and animal pathogens, leading to an increasing number of public health concerns. In the forest area of northeast China, the spread of tick-borne diseases (TBDs) is severe; however, little is known about the tick virome composition and evolution. Herein, we investigate the geographical distribution of tick species and related viruses in Heilongjiang and Jilin Provinces in Northeast China. To reveal the diversity of tick-borne viruses in parts of Heilongjiang and Jilin, ticks were collected at 9 collection points in these provinces in 2018. Morphology and molecular biology were used to identify tick species, and 1411 ticks from nine sampling sites were collected and analysed by next-generation sequencing (NGS). Four Ixodidae were identified, including Ixodes persulcatus, Haemaphysalis japonica, Dermacentor silvarum, and Haemaphysalis concinna. After removal of host genome sequences, 13,003 high-quality NGS reads were obtained and annotated as viruses. Further phylogenetic analysis based on amplicons revealed that these viral sequences belong to Beiji nairovirus, Alongshan virus, bovine parvovirus-2, and tick-associated circovirus; some distinct sequences are closely related to Songling virus, Changping tick virus, Norway luteo-like virus 2, and Norway partiti-like virus 1. In summary, this study describes the prevalence of local ticks and variety of tick-borne viruses in northeastern China, providing a basis for further research on tick-borne viruses in the future.

Influenza A virus (IAV) infection in pregnant women is a major public health concern. However, the effect of IAV infection on human embryogenesis is still unclear. Here we show that human induced pluripotent stem cells (hiPSCs) and hiPSC-derived ectodermal, mesodermal and endodermal cells are susceptible to IAV infection. These cell types stained positive for α2,6-linked sialic acid, the receptor for IAV infection expressed on the cell surface. While hiPSCs produced high viral titers for up to 7 days with increasing infected cell number suggesting that the viral progenies produced from hiPSCs without exogenous protease were infectious and could spread to other cells, the three germ-layer cells showed a decline in viral titers suggesting the lack of viral spreading. Amongst the three germ layers, endodermal cells were less susceptible than ectodermal and mesodermal cells. These results indicate the permissiveness of cells of early embryogenesis, and suggest a risk of detrimental effects of IAV infection in early human embryonic development.

Interleukin-33 (IL-33), which promotes M2 macrophage development, may influence the control of viruses, such as Theiler's Murine Encephalomyelitis Virus (TMEV) that infect macrophages. Because Interferon Regulatory Factor-3 (IRF3) is also critical to control of TMEV infection in macrophages, information on the relationship between IL-33 and IRF3 is important. Thus, RAW264.7 Lucia murine macrophage lineage cells with an endogenous IRF3-ISRE promoter driving secreted luciferase and IRF3KO RAW Lucia, a subline deficient in IRF3, were challenged with TMEV. After the challenge, considerable TMEV RNA detected at 18 and 24 h in RAW cells was significantly elevated in IRF3KO RAW cells. TMEV induction of ISRE-IRF3 promoter activity, IFN-β and IL-33 gene expression, and IL-6 and IL-10 protein production, which was strong in RAW cells, was less in IRF3KO RAW cells. In contrast, expression of CD206 and ARG1, classical M2 macrophage markers, was significantly elevated in IRF3KO RAW cells. Moreover, RAW and IRF3KO RAW cells produced extracellular IL-33 prior to and after infection with TMEV and antibody blockade of the IL-33 receptor, ST2, reduced CD206 and ARG1 expression, but increased IL-6 gene expression. Pre-treating both RAW and IRF3KO RAW cells with IL-33 prior to challenge significantly increased TMEV infection, but also increased IL-33, IL-10, IL-6 mRNA expression, and NO production without increasing IFN-β. Notably, IL-33 induction of IL-33, IRF3-ISRE promoter activity, and IL-10 by TMEV or poly I:C/IFN-γ was significantly dependent upon IRF3. The results show that the expression of IL-33 and the repression of M2 macrophage phenotypic markers are dependent on IRF3 and that IL-33 decreases the ability of macrophages to control infection with macrophage-tropic viruses.

Emerging evidence indicates that gut virome plays a role in human health and disease, however, much less is known about the viral communities in blood. Here we conducted a direct metatranscriptomic sequencing of virus-like-particles in blood from 1200 healthy individuals, without prior amplification to avoid potential amplification bias and with a strictly bioinformatic and manual check for candidate viral reads to reduce false-positive matches. We identified 55 different viruses from 36 viral families, including 24 human DNA, RNA and retroviruses in 70% of the studied pools. The study showed that anelloviruses are widely distributed and dominate the blood virome in healthy individuals. Human herpesviruses and pegivirus-1 are commonly prevalent in asymptomatic humans. We identified the prevalence of RNA viruses often causing acute infection, like HEV, HPIV, RSV and HCoV-HKU1, revealing of a transmissible risk of asymptomatic infection. Several viruses possible related to transfusion safety were identified, including human Merkel cell polyomavirus, papillomavirus, parvovirus B19 and herpesvirus 8 in addition to HBV. In addition, phages in Caudovirales and Microviridae, were commonly found in pools of samples with a very low abundance; a few sequences for invertebrate, plant and giant viruses were found in some of individuals; however, the remaining 31 viruses mostly reflect extensive contamination from commercial reagents and the work environments. In conclusion, this study is the first comprehensive investigation of blood virome in healthy individuals by metatranscriptomic sequencing of VLP in China. Further investigation of potential false positives representing a major challenge for the identification of novel viruses in mNGS, will offer a systemic idea and means to reveal true viral infections of human.

Diseases caused by novel duck reovirus (NDRV) have brought considerable economic losses to the poultry industry. MicroRNAs (miRNAs) have an impact on virus replication and antiviral immunity. However, the miRNA profile upon NDRV infection in duck embryo fibroblasts (DEFs) remains to be discovered. In this study, small RNA (sRNA) sequencing was performed to decipher the cellular miRNA response to NDRV infection. Based on 26 differentially expressed miRNAs (19 upregulated and 7 downregulated miRNAs) obtained from sequencing data and their target genes predicted by software, GO and KEGG analyses were performed to elucidate the functions of miRNAs in NDRV invasion, replication, and virus spread. "FoxO signaling pathway", "autophagy", and "Toll-like receptor signaling pathway" might participate in NDRV replication as revealed by KEGG enrichment analysis. The miR-155-1 sequence was found to be identical to rno-miR-155-5p and was sharply increased with the progression of NDRV infection. Moreover, NDRV-induced miR-155-1 could act as a positive factor for virus replication in DEFs, which inhibited type I interferon (IFN-I) production. Luciferase assay confirmed that miR-155-1 disturbed the abundance of suppressor of cytokine signaling (SOCS) 5 by targeting 3'-UTR. SOCS5, which is linked to increased IRF7 expression, restricts IFN expression and promotes NDRV replication in DEFs. Therefore, this study proposed that miR-155-1 was used by NDRV to restrict SOCS5 expression, attenuating the production of IFN-I and creating a favorable environment for virus replication.

The antigenic characterization of IBDV, a virus that causes an immunosuppressive disease in young chickens, has been historically addressed using cross virus neutralization (VN) assay and antigen-capture enzyme-linked immunosorbent (AC-ELISA). However, VN assay has been usually carried out either in specific antibody negative embryonated eggs, for non-cell culture adapted strains, which is tedious, or on chicken embryo fibroblasts (CEF), which requires virus adaptation to cell culture. AC-ELISA has provided crucial information about IBDV antigenicity, but this information is limited to the epitopes included in the tested panel with a lack of information of overall antigenic view. The present work aimed at overcoming those technical limitations and providing an extensive antigenic landscape based on original cross VN assays employing primary chicken B cells, where no previous IBDV adaptation is required. Sixteen serotype 1 IBDV viruses, comprising both reference strains and documented antigenic variants were tested against eleven chicken post-infectious sera. The VN data were analysed by antigenic cartography, a method which enables reliable high-resolution quantitative and visual interpretation of large binding assay datasets. The resulting antigenic cartography revealed i) the existence of several antigenic clusters of IBDV, ii) high antigenic relatedness between some genetically unrelated viruses, iii) a highly variable contribution to global antigenicity of previously identified individual epitopes and iv) broad reactivity of chicken sera raised against antigenic variants. This study provides an overall view of IBDV antigenic diversity. Implementing this approach will be instrumental to follow the evolution of IBDV antigenicity and control the disease.

Human rhinovirus (HRV), the main etiologic agent of the common cold, is responsible for significant morbidity, medical costs, and the loss of productivity in the workplace and school. To prevent the spread of HRV, accurate, low-cost and rapid diagnostics of HRV is crucial for identifying those at-risk for the illness associated with HRV, with the most frequently detected species, including HRV species A (HRV-A) and C (HRV-C). Here, a novel HRV-A and/or HRV-C molecular diagnostic assay that integrates reverse-transcription recombinase polymerase amplification assay (RT-RPA) amplification with CRISPR/Cas12a detection, with the result readout using a fluorescence detector or lateral flow strip (LFS). The established assay could be completed within 50 min without complex instruments and skilled technicians. The limit of detection of the RT-RPA-Cas12a-mediated real-time fluorescence or LFS assay could reach 0.1 copy/μl, and 0.5 copy/μl for the end-point fluorescence assay with a UV light illuminator readout, respectively. Meanwhile, the assay demonstrates excellent specificity without cross-reactivity to non-target viruses. Furthermore, they were appraised using 80 clinical samples, and RT-RPA-Cas12a-mediated fluorescence or LFS assay displayed high-accuracy with positive and negative predictive agreement of 96.7%, 95% and 100%, respectively. Taken together, the RT-RPA-Cas12a-mediated assay is a rapid, sensitive, and specific detection tool for routine and on-site detection method for HRV-A and/or HRV-C infections, and shows great promise for use in resource-poor or constrained settings.

Mayaro virus (MAYV) is mainly found in Central and South America and causes a febrile illness followed by debilitating arthritis and arthralgia similar to chikungunya virus (CHIKV). Infection leads to long-term sequelae with a direct impact on the patient's productive capacity, resulting in economic losses. Mayaro fever is a neglected disease due to the limited epidemiological data. In Brazil, it is considered a potential public health risk with the number of cases increasing every year. Most of our knowledge about MAYV biology is inferred from data obtained from other alphaviruses as well as more recent studies on MAYV. Here, we analyzed the kinetics of viral replication through standard growth curves, quantification of intracellular and extracellular particles, and RNA quantification. We compared transmission electron microscopy data during different stages of infection. This approach allowed us to establish a chronological order of events during MAYV replication and its respective timepoints including cell entry through clathrin-mediated endocytosis occurring at 15-30 min, genome replication at 2-3 h, morphogenesis at 4 hpi, and release at 4-6 hpi. We also present evidence of uncharacterized events such as ribosome reorganization as well as clusters of early viral precursors and release through exocytosis in giant forms. Our work sheds new and specific light on the MAYV replication cycle and may contribute to future studies on the field.