Viruses, Volume 11, Issue 8 (August 2019) – 91 articles

Innate antiviral factors in saliva play a role in protection against respiratory infections. We tested the anti-influenza virus activities of saliva samples taken from human infants, 1–12 months old, with no history of prior exposure to influenza. In contrast to the inhibitory activity we observed in mouse and ferret saliva, the activity of human infant saliva was complex, with both sialic acid-dependent and independent components, the proportion of which differed between individuals. Taken as a whole, we showed that the major anti-influenza activity of infant saliva is acquired over the first year of life and is associated with sialic acid-containing molecules. The activity of sialic acid-independent inhibitors was lower overall, more variable between individuals, and less dependent on age. The results show that the saliva of very young infants can provide a degree of protection against influenza, which may be critical in the absence of adaptive immunity. Full article

Measles elimination has been identified as a public health priority in Europe for a long time but has not yet been achieved. The World Health Organization (WHO) recommends identification of susceptible sub-populations to target supplementary immunization activities. We used three different sources of information: retrospective samples investigated for measles IgG between 1997 and 2016, vaccine coverage data from the existing electronic registry for birth cohorts 2015 to 1999, and surveillance data from 2009 until 20 July 2019. We calculated susceptibility by birth cohort using seroprevalence data, adjusting vaccine coverage data with reported effectiveness (93% for the first and 97% for the second dose, respectively), and compared it with measles incidence data, aggregated by birth cohorts and districts. Susceptibility levels for persons 10–41 years (birth cohorts 2007–1976) were 10.4% and thus far above the recommended values of WHO (5%). Older birth cohorts were sufficiently protected. Districts with the highest susceptibility estimates corresponded with districts with the highest incidence rates. Birth cohorts with susceptibility levels > 10% showed a 4.7 increased relative risk of having had more than one measles case. We conclude that retrospective serosurveys are a cheap and useful approach in identifying susceptible sub-populations, especially for older birth cohorts whose coverage data remain scarce. Full article

Dengue virus (DENV) is the most prevalent arbovirus in terms of human public health importance globally. In addition to DENV epidemiological surveillance, genomic surveillance may help investigators understand the epidemiological dynamics, geographic distribution, and temporal patterns of DENV circulation. Herein, we aimed to reconstruct the molecular epidemiology and phylogeny of DENV in Panama to connect the epidemiological history of DENV dispersal and circulation in Latin America. We retrospectively analyzed the epidemiological data obtained during 25 years of DENV surveillance in Panama. DENV was reintroduced in Panama in 1993 after a 35 year absence of autochthonous transmission. The increase in the number of total dengue cases has been accompanied by an increase in severe and fatal cases, with the highest case fatality rate recorded in 2011. All four serotypes were detected in Panama, which is characterized by serotype replacement and/or co-circulation of multiple serotypes. Phylogenetic analysis of datasets collected from envelope (E) gene sequences obtained from viruses isolated from human sera demonstrated that circulating viruses were highly diverse and clustered in distinct clades, with co-circulation of clades from the same genotype. Our analyses also suggest that Panamanian strains were related to viruses from different regions of the Americas, suggesting a continuous exchange of viruses within the Americas. Full article

The picornavirus Aichi virus (AiV) is a non-enveloped RNA virus that causes acute gastroenteritis symptoms, such as diarrhea, abdominal pain, nausea, vomiting, and fever. Antiviral host defense involves the fast response of type I interferon (IFN) and the secretion of inflammatory cytokines against pathogens. However, the intestinal inflammatory and antiviral response to AiV infection is poorly understood. This study evaluated the antiviral activity of intestinal epithelial cells (IECs), which form a single-cell layer separating the bowel wall from pathogens. Isolated primary mouse IECs were subjected to AiV infection and virion production, inducing the mRNA expression of type I/type III IFNs and inflammatory cytokines. The mechanism involved induced the expression of phospho-IFN regulatory factor 3 and mitochondrial antiviral-signaling protein of type I IFN signaling. These findings were also observed in AiV-infected human colon carcinoma cells. In summary, a viral productive and pathogenic infection of AiV in primary murine IECs is validated. Full article

For a long time, viruses have been shown to modify the clinical picture of several autoimmune diseases, including type 1 diabetes (T1D), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjögren’s syndrome (SS), herpetic stromal keratitis (HSK), celiac disease (CD), and multiple sclerosis (MS). Best examples of viral infections that have been proposed to modulate the induction and development of autoimmune diseases are the infections with enteric viruses such as Coxsackie B virus (CVB) and rotavirus, as well as influenza A viruses (IAV), and herpesviruses. Other viruses that have been studied in this context include, measles, mumps, and rubella. Epidemiological studies in humans and experimental studies in animal have shown that viral infections can induce or protect from autoimmunopathologies depending on several factors including genetic background, host-elicited immune responses, type of virus strain, viral load, and the onset time of infection. Still, data delineating the clear mechanistic interaction between the virus and the immune system to induce autoreactivity are scarce. Available data indicate that viral-induced autoimmunity can be activated through multiple mechanisms including molecular mimicry, epitope spreading, bystander activation, and immortalization of infected B cells. Contrarily, the protective effects can be achieved via regulatory immune responses which lead to the suppression of autoimmune phenomena. Therefore, a better understanding of the immune-related molecular processes in virus-induced autoimmunity is warranted. Here we provide an overview of the current understanding of viral-induced autoimmunity and the mechanisms that are associated with this phenomenon. Full article

Measles virus (MV) and canine distemper virus (CDV) are highly contagious and deadly, forming part of the morbillivirus genus. The receptor recognition by morbillivirus hemagglutinin (H) is important for determining tissue tropism and host range. Recent reports largely urge caution as regards to the potential expansion of host specificities of morbilliviruses. Nonetheless, the receptor-binding potential in different species of morbillivirus H proteins is largely unknown. Herein, we show that the CDV-H protein binds to the dog signaling lymphocyte activation molecule (SLAM), but not to the human, tamarin, or mouse SLAM. In contrast, MV-H can bind to human, tamarin and dog SLAM, but not to that of mice. Notably, MV binding to dog SLAM showed a lower affinity and faster kinetics than that of human SLAM, and MV exhibits a similar entry activity in dog SLAM- and human SLAM-expressing Vero cells. The mutagenesis study using a fusion assay, based on the MV-H–SLAM complex structure, revealed differences in tolerance for the receptor specificity between MV-H and CDV-H. These results provide insights into H-SLAM specificity related to potential host expansion. Full article

Over the past two decades, there has been tremendous progress in understanding the impact of the intestinal microbiota on mammalian metabolism, physiology, and immune development and function. There has also been substantial advancement in elucidating the interplay between commensal and pathogenic bacteria. Relatively more recently, researchers have begun to investigate the effect of the intestinal microbiota on viral pathogenesis. Indeed, a growing body of literature has reported that commensal bacteria within the mammalian intestinal tract enhance enteric virus infections through a variety of mechanisms. Commensal bacteria or bacterial glycans can increase the stability of enteric viruses, enhance virus binding to host receptors, modulate host immune responses in a proviral manner, expand the numbers of host cell targets, and facilitate viral recombination. In this review, we will summarize the current literature exploring these effects of the intestinal microbiota on enteric virus infections. Full article

Infection by diverse mycoviruses is a common phenomenon in Sclerotinia sclerotiorum. In this study, the full genome of a single-stranded RNA mycovirus, tentatively named Hubei sclerotinia RNA virus 1 (HuSRV1), was determined in the hypovirulent strain 277 of S. sclerotiorum. The HuSRV1 genome is 4492 nucleotides (nt) long and lacks a poly (A) tail at the 3ˊ- terminus. Sequence analyses showed that the HuSRV1 genome contains four putative open reading frames (ORFs). ORF1a was presumed to encode a protein with a conserved protease domain and a transmembrane domain. This protein is 27% identical to the P2a protein encoded by the subterranean clover mottle virus. ORF1b encodes a protein containing a conserved RNA-dependent RNA polymerase (RdRp) domain, which may be translated into a fusion protein by a -1 ribosome frameshift. This protein is 45.9% identical to P2b encoded by the sowbane mosaic virus. ORF2 was found to encode a putative coat protein, which shares 23% identical to the coat protein encoded by the olive mild mosaic virus. ORF3 was presumed to encode a putative protein with an unknown function. Evolutionary relation analyses indicated that HuSRV1 is related to members within Sobemovirus, but forms a unique phylogenetic branch, suggesting that HuSRV1 represents a new member within Solemoviridae. HuSRV1 virions, approximately 30 nm in diameter, were purified from strain 277. The purified virions were successfully introduced into virulent strain Ep-1PNA367, resulting in a new hypovirulent strain, which confirmed that HuSRV1 confers hypovirulence on S. sclerotiorum. Full article

Animal cells have evolved dedicated molecular systems for sensing and delivering a coordinated response to viral threats. Our understanding of these pathways is almost entirely defined by studies in humans or model organisms like mice, fruit flies and worms. However, new genomic and functional data from organisms such as sponges, anemones and mollusks are helping redefine our understanding of these immune systems and their evolution. In this review, we will discuss our current knowledge of the innate immune pathways involved in sensing, signaling and inducing genes to counter viral infections in vertebrate animals. We will then focus on some central conserved players of this response including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and cGAS-STING, attempting to put their evolution into perspective. To conclude, we will reflect on the arms race that exists between viruses and their animal hosts, illustrated by the dynamic evolution and diversification of innate immune pathways. These concepts are not only important to understand virus-host interactions in general but may also be relevant for the development of novel curative approaches against human disease. Full article

Murid herpesvirus-4 (MuHV-4), a natural gammaherpesvirus of rodents, can infect the mouse through the nasal mucosa, where it targets sustentacular cells and olfactory neurons in the olfactory epithelium before it propagates to myeloid cells and then to B cells in lymphoid tissues. After establishment of latency in B cells, viral reactivation occurs in the genital tract in 80% of female mice, which can lead to spontaneous sexual transmission to co-housed males. Interferon-lambda (IFN-λ) is a key player of the innate immune response at mucosal surfaces and is believed to limit the transmission of numerous viruses by acting on epithelial cells. We used in vivo plasmid-mediated IFN-λ expression to assess whether IFN-λ could prophylactically limit MuHV-4 infection in the olfactory and vaginal mucosae. In vitro, IFN-λ decreased MuHV-4 infection in cells that overexpressed IFN-λ receptor 1 (IFNLR1). In vivo, prophylactic IFN-λ expression decreased infection of the olfactory epithelium but did not prevent virus propagation to downstream organs, such as the spleen where the virus establishes latency. In the olfactory epithelium, sustentacular cells readily responded to IFN-λ. In contrast, olfactory neurons did not respond to IFN-λ, thus, likely allowing viral entry. In the female genital tract, columnar epithelial cells strongly responded to IFN-λ, as did most vaginal epithelial cells, although with some variation from mouse to mouse. IFN-λ expression, however, failed to prevent virus reactivation in the vaginal mucosa. In conclusion, IFN-λ decreased MuHV-4 replication in the upper respiratory epithelium, likely by protecting the sustentacular epithelial cells, but it did not protect olfactory neurons and failed to block virus reactivation in the genital mucosa. Full article

Herpes simplex virus type 1 (HSV-1), one of the human pathogens widely epidemic and transmitted among various groups of people in the world, often causes symptoms known as oral herpes or lifelong asymptomatic infection. HSV-1 employs many sophisticated strategies to escape host antiviral immune response based on its multiple coding proteins. However, the functions involved in the immune evasion of miRNAs encoded by HSV-1 during lytic (productive) infection remain poorly studied. Dual-luciferase reporter gene assay and bioinformatics revealed that Asp-Glu-Ala-Asp (DEAD)-box helicase 41 (DDX41), a cytosolic DNA sensor of the DNA-sensing pathway, was a putative direct target gene of HSV-1-encoded miR-H2-3p. The transfection of miR-H2-3p mimics inhibited the expression of DDX41 at the level of mRNA and protein, as well as the expression of interferon beta (IFN-β) and myxoma resistance protein I (MxI) induced by HSV-1 infection in THP-1 cells, and promoted the viral replication and its gene transcription. However, the transfection of miR-H2-3p inhibitor showed opposite effects. This finding indicated that HSV-1-encoded miR-H2-3p attenuated cytosolic DNA–stimulated antiviral immune response by manipulating host DNA sensor molecular DDX41 to enhance virus replication in cultured cells. Full article

Chikungunya virus (CHIKV) re-emerged as a globalized health threat fifteen years ago. There are dozens of RT-PCR assays published. An inventory of the latter was made, and after in silico analysis, two assays were selected for their ability to detect strains belonging to the five CHIKV genetic lineages. They were combined in order to provide a robust assay not affected by genetic point mutations and the resulting Duo CHIKV real-time RT-PCR assay was compared to the two parental single-plex tests against five strains belonging to the five genetic lineages. The Duo CHIKV assay performed equally, or better, in terms of sensitivity, specificity, linearity and signal intensity. Dual-target assays are better suited for viruses having the propensity to evolve into new variants via point mutations or major sequence deletions/insertions. Here, we demonstrated that combining two single systems into a dual-target assay did not impair sensitivity and specificity, and proved a potent diagnostic tool to face a potential emergence of CHIKV variants by newly evolving mutations. Full article

Viruses are able to evolve in vitro by mutations after serial passages in cell cultures, which can lead to either a loss, or an increase, of virulence. Cyprinid herpesvirus 3 (CyHV-3), a 295-kb double-stranded DNA virus, is the etiological agent of the koi herpesvirus disease (KHVD). To assess the influence of serial passages, an isolate of CyHV-3 (KHV-T) was passaged 99 times onto common carp brain (CCB) cells, and virus virulence was evaluated during passages through the experimental infections of common carp. After 78 CCB passages, the isolate was much less virulent than the original form. A comparative genomic analysis of these three forms of KHV-T (P0, P78 and P99) revealed a limited number of variations. The largest one was a deletion of 1363 bp in the predicted ORF150, which was detected in P78, but not in P99. This unexpected finding was confirmed by conventional PCR and digital PCR. The results presented here primarily suggest that, CyHV-3 evolves, at least in vitro, through an assemblage of haplotypes that alternatively become dominant or under-represented. Full article

Starting in 2006, bluetongue virus serotype 8 (BTV8) was responsible for a major epizootic in Western and Northern Europe. The magnitude and spread of the disease were surprisingly high and the control of BTV improved significantly with the marketing of BTV8 inactivated vaccines in 2008. During late summer of 2011, a first cluster of reduced milk yield, fever, and diarrhoea was reported in the Netherlands. Congenital malformations appeared in March 2012 and Schmallenberg virus (SBV) was identified, becoming one of the very few orthobunyaviruses distributed in Europe. At the start of both epizootics, little was known about the pathogenesis and epidemiology of these viruses in the European context and most assumptions were extrapolated based on other related viruses and/or other regions of the World. Standardized and repeatable models potentially mimicking clinical signs observed in the field are required to study the pathogenesis of these infections, and to clarify their ability to cross the placental barrier. This review presents some of the latest experimental designs for infectious disease challenges with BTV or SBV. Infectious doses, routes of infection, inoculum preparation, and origin are discussed. Particular emphasis is given to the placental crossing associated with these two viruses. Full article

Potato virus M (PVM) is a member of the genus Carlavirus of the family Betaflexviridae and causes large economic losses of nightshade crops. Several previous studies have elucidated the population structure, evolutionary timescale and adaptive evolution of PVM. However, the synonymous codon usage pattern of PVM remains unclear. In this study, we performed comprehensive analyses of the codon usage and composition of PVM based on 152 nucleotide sequences of the coat protein (CP) gene and 125 sequences of the cysteine-rich nucleic acid binding protein (NABP) gene. We observed that the PVM CP and NABP coding sequences were GC-and AU-rich, respectively, whereas U- and G-ending codons were preferred in the PVM CP and NABP coding sequences. The lower codon usage of the PVM CP and NABP coding sequences indicated a relatively stable and conserved genomic composition. Natural selection and mutation pressure shaped the codon usage patterns of PVM, with natural selection being the most important factor. The codon adaptation index (CAI) and relative codon deoptimization index (RCDI) analysis revealed that the greatest adaption of PVM was to pepino, followed by tomato and potato. Moreover, similarity Index (SiD) analysis showed that pepino had a greater impact on PVM than tomato and potato. Our study is the first attempt to evaluate the codon usage pattern of the PVM CP and NABP genes to better understand the evolutionary changes of a carlavirus. Full article

RNA viruses represent a large and important group of pathogens that infect a broad range of hosts. Segmented RNA viruses are a subclass of this group that encode their genomes in two or more molecules and package all of their RNA segments in a single virus particle. These divided genomes come in different forms, including double-stranded RNA, coding-sense single-stranded RNA, and noncoding single-stranded RNA. Genera that possess these genome types include, respectively, Orbivirus (e.g., Bluetongue virus), Dianthovirus (e.g., Red clover necrotic mosaic virus) and Alphainfluenzavirus (e.g., Influenza A virus). Despite their distinct genomic features and diverse host ranges (i.e., animals, plants, and humans, respectively) each of these viruses uses trans-acting RNA–RNA interactions (tRRIs) to facilitate co-packaging of their segmented genome. The tRRIs occur between different viral genome segments and direct the selective packaging of a complete genome complement. Here we explore the current state of understanding of tRRI-mediated co-packaging in the abovementioned viruses and examine other known and potential functions for this class of RNA–RNA interaction. Full article

A metatranscriptomic study of RNA viruses in cold-blooded vertebrates identified two related viruses from frogfish (Antennarius striatus) that represent a new genus Antennavirus in the family Arenaviridae (Order: Bunyavirales). Computational analyses were used to identify features common to class I viral fusion proteins (VFPs) in antennavirus glycoproteins, including an N-terminal fusion peptide, two extended alpha-helices, an intrahelical loop, and a carboxyl terminal transmembrane domain. Like mammarenavirus and hartmanivirus glycoproteins, the antennavirus glycoproteins have an intracellular zinc-binding domain and a long virion-associated stable signal peptide (SSP). The glycoproteins of reptarenaviruses are also class I VFPs, but do not contain zinc-binding domains nor do they encode SSPs. Divergent evolution from a common progenitor potentially explains similarities of antennavirus, mammarenavirus, and hartmanivirus glycoproteins, with an ancient recombination event resulting in a divergent reptarenavirus glycoprotein. Full article

Pseudomonas aeruginosa is an opportunistic pathogen that causes public healthcare issues. In moist environments, this Gram-negative bacterium persists through biofilm-associated contamination on surfaces. Bacteriophages are seen as a promising alternative strategy to chemical biocides. This study evaluates the potential of nine lytic bacteriophages as biocontrol treatments against nine environmental P. aerginosa isolates. The spot test method is preliminarily used to define the host range of each virus and to identify their minimum infectious titer, depending on the strain. Based on these results, newly isolated bacteriophages 14.1, LUZ7, and B1 are selected and assessed on a planktonic cell culture of the most susceptible isolates (strains MLM, D1, ST395E, and PAO1). All liquid infection assays are achieved in a mineral minimum medium that is much more representative of real moist environments than standard culture medium. Phages 14.1 and LUZ7 eliminate up to 90% of the PAO1 and D1 bacterial strains. Hence, their effectiveness is evaluated on the 24 h old biofilms of these strains, established on a stainless steel coupon that is characteristic of materials found in thermal and industrial environments. The results of quantitative PCR viability show a maximum reduction of 1.7 equivalent Log CFU/cm² in the coupon between treated and untreated surfaces and shed light on the importance of considering the entire virus/host/environment system for optimizing the treatment. Full article

Infection with Zika virus (ZIKV) came first to public attention after it was found to be associated with congenital microcephaly during the outbreak in Brazil (2015–2016). Diagnosis of ZIKV suffers from extensive cross-reactivity with other Flaviviruses, which are circulating in many ZIKV epidemic areas. Due to the fatal outcome of ZIKV infection during pregnancy, detailed knowledge about neutralizing and non-neutralizing epitopes is crucial for the development of robust detection systems of protective antibodies. Therefore, additional information about ZIKV immunogenicity and antibody response is required. In this project, we report the production, purification and characterization of six different polyclonal antibodies against ZIKV envelope (E) protein. The produced antibodies bind to isolated ZIKV E protein as well as to the surface of ZIKV particles, interestingly without being potently neutralizing. Surface plasmon resonance measurement showed that these antibodies bind with high affinity to ZIKV E protein. Epitope mapping revealed that the epitopes are distributed among the three ZIKV E domains with seven binding sites. These identified binding sites overlap only partially with the previously described epitopes recognized by neutralizing antibodies, which is in accordance with their lack of potent neutralizing activity. Additionally, these antibodies showed neither cross-reactivity nor potent neutralizing activity against West Nile virus, a related flavivirus. The gained set of data helps to extend our understanding about the distribution of neutralizing and non-/weak-neutralizing epitopes in ZIKV E protein, and provides a rationale for ZIKV vaccine design and development of robust detection assays for neutralizing antibodies. Full article

Primary human airway epithelial cell (hAEC) cultures represent a universal platform to propagate respiratory viruses and characterize their host interactions in authentic target cells. To further elucidate specific interactions between human respiratory viruses and important host factors in the airway epithelium, it is important to make hAEC cultures amenable to genetic modification. However, the short and finite lifespan of primary cells in cell culture creates a bottleneck for the genetic modification of these cultures. In the current study, we show that the incorporation of the Rho-associated protein kinase (ROCK) inhibitor (Y-27632) during cell propagation extends the life span of primary human cells in vitro and thereby facilitates the incorporation of lentivirus-based expression systems. Using fluorescent reporters for fluorescence-activated cell sorting (FACS)-based sorting, we generated homogenously fluorescent hAEC cultures that differentiate normally after lentiviral transduction. As a proof-of-principle, we demonstrate that host gene expression can be modulated post-differentiation via inducible short hairpin (sh)RNA-mediated knockdown. Importantly, functional characterization of these transgenic hAEC cultures with exogenous poly (I:C), as a proxy for virus infection, demonstrates that such modifications do not influence the host innate immune response. Moreover, the propagation kinetics of both human coronavirus 229E (HCoV-229E) and human respiratory syncytial virus (hRSV) were not affected. Combined, these results validate our newly established protocol for the genetic modification of hAEC cultures, thereby unlocking a unique potential for detailed molecular characterization of virus–host interactions in human respiratory epithelium. Full article

Despite recent progress in the development of novel potent HIV-1 entry/fusion inhibitors, there are currently no licensed antiviral drugs based on inhibiting the critical interactions of the HIV-1 envelope gp120 protein with cellular receptor CD4. In this connection, studies on the design of new small-molecule compounds able to block the gp120-CD4 binding are still of great value. In this work, in silico design of drug-like compounds containing the moieties that make the ligand active towards gp120 was performed within the concept of click chemistry. Complexes of the designed molecules bound to gp120 were then generated by molecular docking and optimized using semiempirical quantum chemical method PM7. Finally, the binding affinity analysis of these ligand/gp120 complexes was performed by molecular dynamic simulations and binding free energy calculations. As a result, five top-ranking compounds that mimic the key interactions of CD4 with gp120 and show the high binding affinity were identified as the most promising CD4-mimemic candidates. Taken together, the data obtained suggest that these compounds may serve as promising scaffolds for the development of novel, highly potent and broad anti-HIV-1 therapeutics. Full article

Nucleic acid polymerases are essential enzymes that replicate the genomes of both RNA and DNA viruses. These enzymes are generally encoded by viruses themselves so as to provide biochemical functions and control elements that differ from those of the host cell polymerases. The core active site structure used by all replicative polymerases is highly conserved and composed of two key aspartate residues from the conserved motifs A and C, but beyond this there is significant divergence among structures. These differences can make it difficult to select which portions of structures to align for comparisons, yet there are extended structural similarities within different groups of viral polymerases that should clearly be considered to generate optimal alignments. This manuscript describes a comprehensive structure-based superposition of every viral polymerase structure solved thus far based on an alignment-tree approach wherein aligned regions grow in complexity as similarity among polymerases increases. The result is a set of 646 structures that have been aligned into a single common orientation. This provides a convenient resource for directly comparing viral polymerases and illustrating structural conservation among them. It also sets the stage for detailed bioinformatics analysis to further assess common structural features. The full set of protein data bank (PDB) formatted files is publicly available via the Polymerase Structures community page at the Zenodo.org open data repository. Full article

Pestiviruses are distributed worldwide and are responsible for a variety of economically important diseases. They are not very host-specific, and thus sheep can be infected by well-known pestiviruses like bovine viral diarrhea virus (BVDV) and border disease virus (BDV), as well as by other recently discovered pestivirus species. The aim of this study is to describe the isolation and characterization of four pestivirus strains detected in aborted lamb fetuses from a single farm in the Brescia province (Northern Italy). A total of twelve aborted fetuses were collected and examined. After necropsy, organs were tested for the presence of infectious agents known as potential causes of abortion (Brucella spp., Listeria spp., Coxiella burnetii, Chlamydophila spp., Mycoplasma spp., Neospora caninum, and Toxoplasma gondii), and submitted to viral identification by isolation on Madin Darby bovine kidney (MDBK) cell culture and by PCR assay for Schmallenberg virus and pan-pestivirus RT-PCR real time assay. Three viral strains (Ovine/IT/1756/2017, Ovine/IT/338710-2/2017, and Ovine/IT/338710-3/2017) were isolated in the absence of cytopathic effects (CPEs) in cell cultures and identified with RT-PCR. Another pestivirus strain (Ovine/IT/16235-2/2018) was detected by PCR, but was not successfully isolated. Complete sequence genomic data of the three isolated viruses showed that they were highly similar, differed genetically from known pestivirus species, and were closely related to classical swine fever virus (CSFV). Beyond the identification of new ovine pestiviruses, this study indicates that a systematic diagnostic approach is important to identify the presence and map the distribution of both known and emerging pestiviruses. Full article

Coronaviruses (CoVs) are widespread among mammals and birds and known for their potential for cross-species transmission. In cats, infections with feline coronaviruses (FCoVs) are common. Several non-feline coronaviruses have been reported to infect feline cells as well as cats after experimental infection, supported by their ability to engage the feline receptor ortholog for cell entry. However, whether cats might become naturally infected with CoVs of other species is unknown. We analyzed coronavirus infections in cats by serological monitoring. In total 137 cat serum samples and 25 FCoV type 1 or type 2-specific antisera were screened for the presence of antibodies against the S1 receptor binding subunit of the CoV spike protein, which is immunogenic and possesses low amino acid sequence identity among coronavirus species. Seventy-eight sera were positive for antibodies that recognized one or more coronavirus S1s whereas 1 serum exclusively reacted with human coronavirus 229E (HCoV-229E) and two sera exclusively reacted with porcine delta coronavirus (PDCoV). We observed antigenic cross-reactivity between S1s of type 1 and type 2 FCoVs, and between FCoV type 1 and porcine epidemic diarrhea virus (PEDV). Domain mapping of antibody epitopes indicated the presence of conserved epitope(s) particularly in the CD domains of S1. The cross-reactivity of FCoV type 1 and PEDV was also observed at the level of virus neutralization. To conclude, we provide the first evidence of antigenic cross-reactivity among S1 proteins of coronaviruses, which should be considered in the development of serological diagnoses. In addition, the potential role of cats in cross-species transmission of coronaviruses cannot be excluded. Full article

Canine parvovirus (CPV) is a common etiological agent of acute enteritis, which occurs globally in domestic and wild carnivores. Despite the widespread use of inactivated or live attenuated vaccines, the emergence of antigenic variants and the influence of maternal antibodies have raised some concerns regarding the efficacy of commercial vaccines. While no specific antiviral therapy for CPV infection exists, the only treatment option for the infection is supportive therapy based on symptoms. Thus, there is an urgent medical need to develop antiviral therapeutic options to reduce the burden of CPV-related disease. In this study, a cytopathic effect (CPE)-based high-throughput screening assay was used to screen CPV inhibitors from a Food and Drug Administration (FDA)-approved drug library. After two rounds of screening, seven out of 1430 screened drugs were found to have >50% CPE inhibition. Three drugs—Nitazoxanide, Closantel Sodium, and Closantel—with higher anti-CPV effects were further evaluated in F81 cells by absolute PCR quantification and indirect immunofluorescence assay (IFA). The inhibitory effects of all three drugs were dose-dependent. Time of addition assay indicated that the drugs inhibited the early processes of the CPV replication cycle, and the inhibition effects were relatively high within 2 h postinfection. Western blot assay also showed that the three drugs had broad-spectrum antiviral activity against different subspecies of three CPV variants. In addition, antiapoptotic effects were observed within 12 h in Nitazoxanide-treated F81 cells regardless of CPV infection, while Closantel Sodium- or Closantel-treated cells had no pro- or antiapoptotic effects. In conclusion, Nitazoxanide, Closantel Sodium, and Closantel can effectively inhibit different subspecies of CPV. Since the safety profiles of FDA-approved drugs have already been extensively studied, these three drugs can potentially become specific and effective anti-CPV drugs. Full article

Since 2015, severe outbreaks of hepatitis-hydropericardium syndrome (HHS), caused by hypervirulent fowl adenovirus serotype 4 (FAdV-4), have emerged in several provinces in China, posing a great threat to poultry industry. So far, factors contributing to the pathogenesis of hypervirulent FAdV-4 have not been fully uncovered. Elucidation of the pathogenesis of FAdV-4 will facilitate the development of effective FAdV-4 vaccine candidates for the control of HHS and vaccine vector. The interaction between pathogen and host defense system determines the pathogenicity of the pathogen. Therefore, the present review highlights the knowledge of both viral and host factors contributing to the pathogenesis of hypervirulent FAdV-4 strains to facilitate the related further studies. Full article

NS3 protein is a member of the non-structural protein of duck Tembusu virus (DTMUV), which contains three domains, each of which has serine protease, nucleotide triphosphatase, and RNA helicase activities, respectively. It performs a variety of biological functions that are involved in the regulation of the viral life cycle and host immune response. Based on the yeast two-hybrid system, we successfully transformed pGBKT7-NS3 bait plasmid into Y2H Gold, tested it to prove that it has no self-activation and toxicity, and then hybridized it with the prey yeast strain of the duck embryo fibroblast cDNA library for screening. After high-stringency selection, positive alignment with the National Center for Biotechnology Information database revealed nine potential interactive proteins: MGST1, ERCC4, WIF1, WDR75, ACBD3, PRDX1, RPS7, ND5, and LDHA. The most interesting one (PRDX1) was selected to be verified with full-length NS3 protein and its three domains S7/DEXDc/HELICc using yeast regressive verification and GST Pull-Down assay. It denoted that PRDX1 does indeed interact with HELICc domains of NS3. NS3 is involved in the RNA uncoiling process of viral replication, which may cause mitochondrial overload to create oxidative stress (OS) during DTMUV attack. We deduced that the HELICc domain binding partner PRDX1, which regulates the p38/mitogen-activated protein kinase pathway (p38/MAPK) to avert OS, causing apoptosis, making it possible for viruses to escape host immune responses. Full article

Treatment drugs, besides their specific activity, often have multiple effects on the body. The undesired effect of the drug may be repurposed as therapeutics, saving significant investigative time and effort. Minocycline has anti-cancer, anti-oxidant, anti-inflammatory, and anti-apoptotic properties. Presently, minocycline is also known to show anti-viral activity against Influenza virus, Japanese encephalitis virus, Simian immunodeficiency virus, Human immunodeficiency virus and West Nile virus. Here, we investigate the effect of minocycline on Respiratory syncytial virus (RSV), a common respiratory virus that causes severe mortality and morbidity in infants, children, and older adult populations. Currently, there is no effective vaccine or treatment for RSV infection; hence, there is a critical need for alternative and effective drug choices. Our study shows that minocycline reduces the RSV-mediated cytopathic effect and prevents RSV infection. This is the first study demonstrating the anti-viral activity of minocycline against RSV. Full article

Double-stranded RNA (dsRNA) molecules of viral origin trigger a post-transcriptional gene-silencing mechanism called RNA interference (RNAi). Specifically, virally derived dsRNA is recognized and cleaved by the enzyme Dicer2 into short interfering RNAs (siRNAs), which further direct sequence-specific RNA silencing, ultimately silencing replication of the virus. Notably, RNAi can also be artificially triggered by the delivery of gene-specific dsRNA, thereby leading to endogenous gene silencing. This is a widely used technology that holds great potential to contribute to novel pest control strategies. In this regard, research efforts have been set to find methods to efficiently trigger RNAi in the field. In this article, we demonstrate the generation of dsRNA- and/or virus-derived siRNAs—the main RNAi effectors—in six insect species belonging to five economically important orders (Lepidoptera, Orthoptera, Hymenoptera, Coleoptera, and Diptera). In addition, we describe that the siRNA length distribution is species-dependent. Taken together, our results reveal interspecies variability in the (antiviral) RNAi mechanism in insects and show promise to contribute to future research on (viral-based) RNAi-triggering mechanisms in this class of animals. Full article

Phages have shown a high biotechnological potential with numerous applications. The advent of high-resolution microscopy techniques aligned with omic and molecular tools are revealing innovative phage features and enabling new processes that can be further exploited for biotechnological applications in a wide variety of fields. This special issue is a collection of original and review articles focusing on the most recent advances in phage-based biotechnology with applications for human benefit. Full article