Viruses — Open Access Journal

Maize chlorotic mottle virus (MCMV) infection causes growth abnormalities in maize. Transcriptome sequencing was conducted to compare the global gene expression of MCMV-inoculated plants with that of mock-inoculated plants. Data analyses showed that brassinosteroid (BR)-associated genes were upregulated after MCMV infection. Exogenous 2,4-epibrassinolide (BL) or brassinazole (BRZ) applications indicated that BR pathway was involved in the susceptibility to MCMV infection. In addition, treatment of BL on maize induced the accumulation of nitric oxide (NO), and the changes of NO content played positive roles in the disease incidence of MCMV. Moreover, MCMV infection was delayed when the BL-treated plants were applied with NO scavenger, which suggested that BR induced the susceptibility of maize to MCMV infection in a NO-dependent manner. Further investigation showed the maize plants with knock-down of DWARF4 (ZmDWF4, a key gene of BR synthesis) and nitrate reductase (ZmNR, a key gene of NO synthesis) by virus-induced gene silencing displayed higher resistance to MCMV than control plants. Taken together, our results suggest that BR pathway promotes the susceptibility of maize to MCMV in a NO-dependent manner. Full article

Epizootic hemorrhagic disease virus (EHDV), an Orbivirus transmitted by Culicoides spp. vectors, is represented by seven serotypes and numerous strains worldwide. While studies comparing vector competence between serotypes exist, studies between viral strains are lacking. In this study, we examined the rates of infection, dissemination, and transmission of two strains of EHDV-2 orally fed to the known vector, Culicoides sonorensis Wirth & Jones. Culicoides sonorensis cohorts were fed an infectious blood meal containing EHDV-2 strains from either Alberta, Canada (Can-Alberta) or Florida (5.5 log₁₀ PFUe/mL) and tested for the vector’s susceptibility to infection and dissemination. In addition, transmission rates of the virus were assessed and compared using capillary tube and honey card methods. Our results show that the Florida strain had higher infection and dissemination rates than the Can-Alberta strain in spite of the Florida strain having significantly lower viral titers in C. sonorensis bodies, legs, and saliva than the Can-Alberta strain. Overall transmission rates were not significantly different between the two strains but varied significantly between the methods used. These findings suggest that the consequences of EHDV infection in C. sonorensis vary between virus strains and have huge implications in future vector competence studies involving Culicoides species and Orbiviruses. Full article

Bacteriophages (phages) or bacterial viruses have been proposed as natural antimicrobial agents to fight against antibiotic-resistant bacteria associated with human infections. Enterococcus faecalis is a gut commensal, which is occasionally found in the mouth and vaginal tract, and does not usually cause clinical problems. However, it can spread to other areas of the body and cause life-threatening infections, such as septicemia, endocarditis, or meningitis, in immunocompromised hosts. Although E. faecalis phage cocktails are not commercially available within the EU or USA, there is an accumulated evidence from in vitro and in vivo studies that have shown phage efficacy, which supports the idea of applying phage therapy to overcome infections associated with E. faecalis. In this review, we discuss the potency of bacteriophages in controlling E. faecalis, in both in vitro and in vivo scenarios. E. faecalis associated bacteriophages were compared at the genome level and an attempt was made to categorize phages with respect to their suitability for therapeutic application, using orthocluster analysis. In addition, E. faecalis phages have been examined for the presence of antibiotic-resistant genes, to ensure their safe use in clinical conditions. Finally, the domain architecture of E. faecalis phage-encoded endolysins are discussed. Full article

Zika virus (ZIKV), an emerging flavivirus that causes neurodevelopmental impairment to fetuses and has been linked to Guillain-Barré syndrome continues to threaten global health due to the absence of targeted prophylaxis or treatment. Nucleoside analogues are good examples of efficient anti-viral inhibitors, and prodrug strategies using phosphate masking groups (ProTides) have been employed to improve the bioavailability of ribonucleoside analogues. Here, we synthesized and tested a small library of 13 ProTides against ZIKV in human neural stem cells. Strong activity was observed for 2′-C-methyluridine and 2′-C-ethynyluridine ProTides with an aryloxyl phosphoramidate masking group. Substitution of a 2-(methylthio) ethyl phosphoramidate for the aryloxyl phosphoramidate ProTide group of 2′-C-methyluridine completely abolished antiviral activity of the compound. The aryloxyl phosphoramidate ProTide of 2′-C-methyluridine outperformed the hepatitis C virus (HCV) drug sofosbuvir in suppression of viral titers and protection from cytopathic effect, while the former compound’s triphosphate active metabolite was better incorporated by purified ZIKV NS5 polymerase over time. These findings suggest both a nucleobase and ProTide group bias for the anti-ZIKV activity of nucleoside analogue ProTides in a disease-relevant cell model. Full article

We explore evolved soybean ascorbate peroxidase (APEX2) as a reporter when fused to the C-termini of llama nanobodies (single-domain antibodies, sdAb; variable domains of heavy chain-only antibodies, VHH) targeted to the E. coli periplasm. Periplasmic expression preserves authentic antibody N-termini, intra-domain disulphide bond(s), and capitalizes on efficient haem loading through the porous E. coli outer membrane. Using monomeric and dimeric anti-nucleoprotein (NP) sdAb cross-reactive within the Marburgvirus genus and cross-reactive within the Ebolavirus genus, we show that periplasmic sdAb–APEX2 fusion proteins are easily purified at multi-mg amounts. The fusions were used in Western blotting, ELISA, and microscopy to visualize NPs using colorimetric and fluorescent imaging. Dimeric sdAb–APEX2 fusions were superior at binding NPs from viruses that were evolutionarily distant to that originally used to select the sdAb. Partial conservation of the anti-Marburgvirus sdAb epitope enabled the recognition of a novel NP encoded by the recently discovered Mĕnglà virus genome. Antibody–antigen interactions were rationalized using monovalent nanoluciferase titrations and contact mapping analysis of existing crystal structures, while molecular modelling was used to reveal the potential landscape of the Mĕnglà NP C-terminal domain. The sdAb–APEX2 fusions also enabled live Marburgvirus and Ebolavirus detection 24 h post-infection of Vero E6 cells within a BSL-4 laboratory setting. The simple and inexpensive mining of large amounts of periplasmic sdAb–APEX2 fusion proteins should help advance studies of past, contemporary, and perhaps Filovirus species yet to be discovered. Full article

Gammaherpesviruses (GHVs) infect many animal species and are associated with lymphoproliferative disorders in some. Previously, we identified several novel GHVs in North American felids; however, a GHV had never been identified in Canada lynx (Lynx canadensis). We, therefore, hypothesized the existence of an unidentified GHV in lynx. Using degenerate nested and subsequently virus-specific PCR, we amplified and sequenced 3.4 kb of DNA from a novel GHV in lynx, which we named Lynx canadensis gammaherpesvirus 1 (LcaGHV1). Phylogenetic analysis determined that LcaGHV1 is a distinct GHV species belonging to the genus Percavirus. We then estimated the prevalence of LcaGHV1 in lynx by developing a PCR-based assay and detected LcaGHV1 DNA in 36% (95% CI: 22–53%) of lynx spleen DNA samples from Maine, USA and 17% (95% CI: 8–31%) from Newfoundland, Canada. The LcaGHV1 DNA sequences from Maine and Newfoundland lynx were nearly identical to each other (two nucleotide substitutions in 3.4 kb), suggesting that the unique lynx subspecies present on the island of Newfoundland (Lynx canadensis subsolanus) is infected with virus that very closely resembles virus found in mainland lynx. The potential ecologic and pathologic consequences of this novel virus for Canada lynx populations warrant further study. Full article

Parvoviruses, infecting vertebrates and invertebrates, are a family of single-stranded DNA viruses with small, non-enveloped capsids with T = 1 icosahedral symmetry. A quarter of a century after the first parvovirus capsid structure was published, approximately 100 additional structures have been analyzed. This first structure was that of Canine Parvovirus, and it initiated the practice of structure-to-function correlation for the family. Despite high diversity in the capsid viral protein (VP) sequence, the structural topologies of all parvoviral capsids are conserved. However, surface loops inserted between the core secondary structure elements vary in conformation that enables the assembly of unique capsid surface morphologies within individual genera. These variations enable each virus to establish host niches by allowing host receptor attachment, specific tissue tropism, and antigenic diversity. This review focuses on the diversity among the parvoviruses with respect to the transcriptional strategy of the encoded VPs, the advances in capsid structure-function annotation, and therapeutic developments facilitated by the available structures. Full article

Although Lloviu virus (LLOV) was discovered in the carcasses of insectivorous Schreiber’s Bent-winged bats in the caves of Northern Spain in 2002, its infectivity and pathogenicity remain unclear. We examined the seroprevalence of LLOV in potentially exposed Schreiber’s Bent-winged bats (n = 60), common serotine bats (n = 10) as controls, and humans (n = 22) using an immunoblot assay. We found antibodies against LLOV GP₂ in all of Schreiber’s Bent-winged bats serum pools, but not in any of the common serotine bats and human pools tested. To confirm this seroreactivity, 52 serums were individually tested using Domain Programmable Arrays (DPA), a phage display based-system serology technique for profiling filovirus epitopes. A serological signature against different LLOV proteins was obtained in 19/52 samples tested (36.5%). The immunodominant response was in the majority specific to LLOV-unique epitopes, confirming that the serological response detected was to LLOV. To our knowledge, this is the first serological evidence of LLOV exposure in live captured Schreiber’s Bent-winged bats, dissociating LLOV circulation as the cause of the previously reported die-offs. Full article

Feline foamy virus (FFV) is a retrovirus that has been detected in multiple feline species, including domestic cats (Felis catus) and pumas (Puma concolor). FFV results in persistent infection but is generally thought to be apathogenic. Sero-prevalence in domestic cat populations has been documented in several countries, but the extent of viral infections in nondomestic felids has not been reported. In this study, we screened sera from 348 individual pumas from Colorado, Southern California and Florida for FFV exposure by assessing sero-reactivity using an FFV anti-Gag ELISA. We documented a sero-prevalence of 78.6% across all sampled subpopulations, representing 69.1% in Southern California, 77.3% in Colorado, and 83.5% in Florida. Age was a significant risk factor for FFV infection when analyzing the combined populations. This high prevalence in geographically distinct populations reveals widespread exposure of puma to FFV and suggests efficient shedding and transmission in wild populations. Full article

Viral erythrocytic necrosis (VEN) affects over 20 species of marine and anadromous fishes in the North Atlantic and North Pacific Oceans. However, the distribution and strain variation of its viral causative agent, erythrocytic necrosis virus (ENV), has not been well characterized within Pacific salmon. Here, metatranscriptomic sequencing of Chinook salmon revealed that ENV infecting salmon was closely related to ENV from Pacific herring, with inferred amino-acid sequences from Chinook salmon being 99% identical to those reported for herring. Sequence analysis also revealed 89 protein-encoding sequences attributed to ENV, greatly expanding the amount of genetic information available for this virus. High-throughput PCR of over 19,000 fish showed that ENV is widely distributed in the NE Pacific Ocean and was detected in 12 of 16 tested species, including in 27% of herring, 38% of anchovy, 17% of pollock, and 13% of sand lance. Despite frequent detection in marine fish, ENV prevalence was significantly lower in fish from freshwater (0.03%), as assessed with a generalized linear mixed effects model (p = 5.5 × 10⁻⁸). Thus, marine fish are likely a reservoir for the virus. High genetic similarity between ENV obtained from salmon and herring also suggests that transmission between these hosts is likely. Full article

Composed of a few hundreds of nucleotides, viroids are infectious, circular, non-protein coding RNAs able to usurp plant cellular enzymes and molecular machineries to replicate and move in their hosts. Several secondary and tertiary RNA structural motifs have been implicated in the viroid infectious cycle, but whether modified nucleotides, such as 5C-methylcytosine (m⁵C), also play a role has not been deeply investigated so far. Here, the possible existence of m⁵C in both RNA polarity strands of potato spindle tuber viroid and avocado sunblotch viroid -which are representative members of the nucleus- and chloroplast-replicating viroids, respectively- has been assessed at single nucleotide level. We show that a standard bisulfite protocol efficiently used for identifying m⁵C in cellular RNAs may generate false positive results in the case of the highly structured viroid RNAs. Applying a bisulfite conversion protocol specifically adapted to RNAs with high secondary structure, no m⁵C was identified in both polarity strands of both viroids, indicating that this specific nucleotide modification does not likely play a role in viroid biology. Full article

Coronaviruses are positive-stranded RNA viruses that infect a variety of hosts, resulting in a range of symptoms from gastrointestinal illness to respiratory distress. Bats are reservoirs for a high diversity of coronaviruses, and focused surveillance detected several strains genetically similar to MERS-coronavirus, SARS-coronavirus, and the human coronaviruses 229E and NL63. The bat fauna of central Asia, which link China to eastern Europe, are relatively less studied than other regions of the world. Kazakhstan is the world’s ninth largest country; however, little is understood about the prevalence and diversity of bat-borne viruses. In this study, bat guano was collected from bat caves in three different sites of southern Kazakhstan that tested positive for coronaviruses. Our phylogenetic reconstruction indicates these are novel bat coronaviruses that belong to the genus Alphacoronavirus. In addition, two distinct lineages of Kazakhstan bat coronaviruses were detected. Both lineages are closely related to bat coronaviruses from China, France, Spain, and South Africa, suggesting that co-circulation of coronaviruses is common in multiple bat species with overlapping geographical distributions. Our study highlights the need for collaborative efforts in understudied countries to increase integrated surveillance capabilities toward better monitoring and detection of infectious diseases. Full article

The stunting disease, incited by chrysanthemum stunt viroid (CSVd), has become a serious problem in chrysanthemum production areas worldwide. Here we identified 46 weed species from chrysanthemum fields in two producing regions of the State of São Paulo, Brazil. The mechanical inoculation of these weeds with a Brazilian CSVd isolate revealed that this viroid was able to infect 17 of these species, in addition to chrysanthemum, tomato and potato. Plants of Oxalis latifolia and chrysanthemum naturally infected with CSVd were found in chrysanthemum fields in Colombia, which is the first CSVd report in that country. Therefore, weeds have the potential to act as reservoirs of CSVd in the field. These results are the first reports of experimental CSVd infection in the following species: Amaranthus viridis, Cardamine bonariensis, Chamaesyce hirta, Conyza bonariensis, Digitaria sanguinalis, Gomphrena globosa, Helianthus annuus, Lupinus polyphyllus, Mirabilis jalapa, Oxalis latifolia, Portulaca oleracea and Catharanthus roseus. The phylogenetic analyses of the CSVd variants identified herein showed three groups with Brazilian CSVd variants distributed in them all, which suggests that Brazilian CSVd isolates may have different origins through successive introductions of infected germplasm of chrysanthemum in Brazil. Full article

Macrobrachium rosenbergii is a valuable freshwater prawn in Asian aquaculture. In recent years, a new symptom that was generally called “white head” has caused high mortality in M. rosenbergii farms in China. Samples of M. rosenbergii, M. nipponense, Procambarus clarkii, M. superbum, Penaeus vannamei, and Cladocera from a farm suffering from white head in Jiangsu Province were collected and analyzed in this study. Pathogen detection showed that all samples were positive for Decapod iridescent virus 1 (DIV1). Histopathological examination revealed dark eosinophilic inclusions and pyknosis in hematopoietic tissue, hepatopancreas, and gills of M. rosenbergii and M. nipponense. Blue signals of in situ digoxigenin-labeled loop-mediated isothermal amplification appeared in hematopoietic tissue, hemocytes, hepatopancreatic sinus, and antennal gland. Transmission electron microscopy of ultrathin sections showed a large number of DIV1 particles with a mean diameter about 157.9 nm. The virogenic stromata and budding virions were observed in hematopoietic cells. Quantitative detection with TaqMan probe based real-time PCR of different tissues in naturally infected M. rosenbergii showed that hematopoietic tissue contained the highest DIV1 load with a relative abundance of 25.4 ± 16.9%. Hepatopancreas and muscle contained the lowest DIV1 loads with relative abundances of 2.44 ± 1.24% and 2.44 ± 2.16%, respectively. The above results verified that DIV1 is the pathogen causing white head in M. rosenbergii. M. nipponense and Pr. clarkii are also species susceptible to DIV1. Full article

Chikungunya virus (CHIKV) is a vector-borne alphavirus transmitted by the bites of mosquitoes, specifically infected, female mosquitoes of the invasive Aedes species. In nature, CHIKV can be maintained by vertical transmission, a phenomenon that relates to the transfer of CHIKV from the infected parent to their offspring within the ovary or during oviposition. In the present study, we conducted laboratory experiments to determine vertical transmission with Ae. albopictus populations from Brazil and Florida. Parental Ae. albopictus females were orally infected with the emergent Asian genotype of CHIKV in the first gonotrophic cycle (infectious blood meal) and tested for vertical transmission following the second (non-infectious blood meal) gonotrophic cycle. CHIKV infection and CHIKV viral titer in parental females were significantly related to population origin, with Brazilian Ae. albopictus showing higher viral dissemination and viral titer than the Florida population. Experimental vertical transmission of CHIKV was documented in one pool of female and four pools of male Ae. albopictus from Brazil (minimum infection rate, MIR, of 0.76% and 2.86%, respectively, for females and males). For the Florida population of Ae. albopictus, only one pool of males was positive for CHIKV infection, with an MIR of 1.06%. Our results demonstrate that Ae. albopictus populations from Brazil and Florida show heterogeneous CHIKV dissemination and vertical transmission, which may contribute to the epidemiology of CHIKV and may be particularly relevant to virus survival during inter-epidemic periods. Full article

After decades of disregard in the Western world, phage therapy is witnessing a return of interest. However, the pharmaceutical legislation that has since been implemented is basically designed for regulating industrially-made pharmaceuticals, devoid of any patient customization and intended for large-scale distribution. Accordingly, the resulting regulatory framework is hardly reconcilable with the concept of sustainable phage therapy, involving tailor-made medicinal products in the global perspective of both evolutionary and personalized medicine. The repeated appeal for a dedicated regulatory framework has not been heard by the European legislature, which, in this matter, features a strong resistance to change despite the precedent of the unhindered implementation of advanced therapy medicinal product (ATMPs) regulation. It is acknowledged that in many aspects, phage therapy medicinal products are quite unconventional pharmaceuticals and likely this lack of conformity to the canonical model hampered the development of a suitable regulatory pathway. However, the regulatory approaches of countries where phage therapy traditions and practice have never been abandoned are now being revisited by some Western countries, opening new avenues for phage therapy regulation. As a next step, supranational and international organizations are urged to take over the initiatives originally launched by national regulatory authorities. Full article

Fungi constituting the Entomophthora muscae species complex (members of subphylum Entomophthoromycotina, phylum Zoopagamycota) commonly kill their insect hosts and manipulate host behaviors in the process. In this study, we made use of public transcriptome data to identify and characterize eight new species of mitoviruses associated with several different E. muscae isolates. Mitoviruses are simple RNA viruses that replicate in host mitochondria and are frequently found in more phylogenetically apical fungi (members of subphylum Glomeromyoctina, phylum Mucoromycota, phylum Basidiomycota and phylum Ascomycota) as well as in plants. E. muscae is the first fungus from phylum Zoopagomycota, and thereby the most phylogenetically basal fungus, found to harbor mitoviruses to date. Multiple UGA (Trp) codons are found not only in each of the new mitovirus sequences from E. muscae but also in mitochondrial core-gene coding sequences newly assembled from E. muscae transcriptome data, suggesting that UGA (Trp) is not a rarely used codon in the mitochondria of this fungus. The presence of mitoviruses in these basal fungi has possible implications for the evolution of these viruses. Full article

Recent large-scale genomics studies of human papillomaviruses (HPVs) have shown a high level of genomic variability of HPV16, the most prevalent genotype in HPV-associated malignancies, and provided new insights into the biological and clinical relevance of its genetic variations in cervical cancer development. Here, we performed deep sequencing analyses of the viral genome to explore genetic variations of HPV16 that are prevalent in Japan. A total of 100 complete genome sequences of HPV16 were determined from cervical specimens collected from Japanese women with cervical intraepithelial neoplasia and invasive cervical cancer, or without cervical malignancies. Phylogenetic analyses revealed the variant distribution in the Japanese HPV16 isolates; overall, lineage A was the most prevalent (94.0%), in which sublineage A4 was dominant (52.0%), followed by sublineage A1 (21.0%). The relative risk of sublineage A4 for cervical cancer development was significantly higher compared to sublineages A1/A2/A3 (odds ratio = 6.72, 95% confidence interval = 1.78–28.9). Interestingly, a novel cluster of variants that branched from A1/A2/A3 was observed for the Japanese HPV16 isolates, indicating that unique HPV16 variants are prevalent among Japanese women. Full article