Viruses — Open Access Journal

Feline infectious peritonitis (FIP) is a viral disease with a high morbidity and mortality by the FIP virus (FIPV, virulent feline coronavirus). Several antiviral drugs for FIP have been identified, but many of these are expensive and not available in veterinary medicine. Hydroxychloroquine (HCQ) is a drug approved by several countries to treat malaria and immune-mediated diseases in humans, and its antiviral effects on other viral infections (e.g., SARS-CoV-2, dengue virus) have been confirmed. We investigated whether HCQ in association with interferon-ω (IFN-ω) is effective for FIPV in vitro. A total of 100 μM of HCQ significantly inhibited the replication of types I and II FIPV. Interestingly, the combination of 100 μM of HCQ and 10⁴ U/mL of recombinant feline IFN-ω (rfIFN-ω, veterinary registered drug) increased its antiviral activity against type I FIPV infection. Our study suggested that HCQ and rfIFN-ω are applicable for treatment of FIP. Further clinical studies are needed to verify the combination of HCQ and rIFN-ω will be effective and safe treatment for cats with FIP. Full article

The global spread of the parasitic mite Varroa destructor has emphasized the significance of viruses as pathogens of honey bee (Apis mellifera) populations. In particular, the association of deformed wing virus (DWV) with V. destructor and its devastating effect on honey bee colonies has led to that virus now becoming one of the most well-studied insect viruses. However, there has been no opportunity to examine the effects of Varroa mites without the influence of DWV. In Papua New Guinea (PNG), the sister species, V. jacobsoni, has emerged through a host-shift to reproduce on the local A. mellifera population. After initial colony losses, beekeepers have maintained colonies without chemicals for more than a decade, suggesting that this bee population has an unknown mite tolerance mechanism. Using high throughput sequencing (HTS) and target PCR detection, we investigated whether the viral landscape of the PNG honey bee population is the underlying factor responsible for mite tolerance. We found A. mellifera and A. cerana from PNG and nearby Solomon Islands were predominantly infected by sacbrood virus (SBV), black queen cell virus (BQCV) and Lake Sinai viruses (LSV), with no evidence for any DWV strains. V. jacobsoni was infected by several viral homologs to recently discovered V. destructor viruses, but Varroa jacobsoni rhabdovirus-1 (ARV-1 homolog) was the only virus detected in both mites and honey bees. We conclude from these findings that A. mellifera in PNG may tolerate V. jacobsoni because the damage from parasitism is significantly reduced without DWV. This study also provides further evidence that DWV does not exist as a covert infection in all honey bee populations, and remaining free of this serious viral pathogen can have important implications for bee health outcomes in the face of Varroa. Full article

Mammalian Orthoreoviruses (MRV) are segmented dsRNA viruses in the family Reoviridae. MRVs infect mammals and cause asymptomatic respiratory, gastro-enteric and, rarely, encephalic infections. MRVs are divided into at least three serotypes: MRV1, MRV2 and MRV3. In Europe, swine MRV (swMRV) was first isolated in Austria in 1998 and subsequently reported more than fifteen years later in Italy. In the present study, we characterized two novel reassortant swMRVs identified in one same Italian farm over two years. The two viruses shared the same genetic backbone but showed evidence of reassortment in the S1, S4, M2 segments and were therefore classified into two serotypes: MRV3 in 2016 and MRV2 in 2018. A genetic relation to pig, bat and human MRVs and other unknown sources was identified. A considerable genetic diversity was observed in the Italian MRV3 and MRV2 compared to other available swMRVs. The S1 protein presented unique amino acid signatures in both swMRVs, with unexpected frequencies for MRV2. The remaining genes formed distinct and novel genetic groups that revealed a geographically related evolution of swMRVs in Italy. This is the first report of the complete molecular characterization of novel reassortant swMRVs in Italy and Europe, which suggests a greater genetic diversity of swMRVs never identified before. Full article

A new crAss-like genome encoding diversity-generating retroelement (DGR) was found in the fecal virome of a healthy volunteer. The genome of the phage referred to as the crAssphage LMMB, belonged to the candidate genus I of the AlphacrAssvirinae subfamily. The DGR-cassette of the crAssphage LMMB contained all the essential elements: the gene encoding reverse transcriptase (RT), the target gene (TG) encoding the tail-collar fiber protein, and variable and template repeats (VR and TR) with IMH (initiation of mutagenic homing) and IMH* sequences at the 3′-end of the VR and TR, respectively. Architecture of the DGR-cassette was TG-VR(IMH)-TR(IMH*)-RT and an accessory variable determinant (avd) was absent from the cassette. Analysis of 91 genomes and genome fragments from genus I of the AlphacrAssvirinae showed that 15 (16%) of the genomes had DGRs with the same architecture as the crAssphage LMMB, while 66 of the genomes contained incomplete DGR-cassettes or some elements of the DGR. Full article

Zika virus (ZIKV), a mosquito-borne transplacentally transmissible flavivirus, is an enveloped virus with an ~10.8 kb plus-strand RNA genome that can cause neurological disease. To facilitate the identification of potential antivirals, we developed two reporter-expressing ZIKVs, each capable of expressing an enhanced green fluorescent protein or an improved luminescent NanoLuc luciferase. First, a full-length functional ZIKV cDNA clone was engineered as a bacterial artificial chromosome, with each reporter gene under the cap-independent translational control of a cardiovirus-derived internal ribosome entry site inserted downstream of the single open reading frame of the viral genome. Two reporter-expressing ZIKVs were then generated by transfection of ZIKV-susceptible BHK-21 cells with infectious RNAs derived by in vitro run-off transcription from the respective cDNAs. As compared to the parental virus, the two reporter-expressing ZIKVs grew to lower titers with slower growth kinetics and formed smaller foci; however, they displayed a genome-wide viral protein expression profile identical to that of the parental virus, except for two previously unrecognized larger forms of the C and NS1 proteins. We then used the NanoLuc-expressing ZIKV to assess the in vitro antiviral activity of three inhibitors (T-705, NITD-008, and ribavirin). Altogether, our reporter-expressing ZIKVs represent an excellent molecular tool for the discovery of novel antivirals. Full article

Extracellular vesicles (EVs) are lipid bilayer-enclosed entities containing proteins and nucleic acids that mediate intercellular communication, in both physiological and pathological conditions. EVs resemble enveloped viruses in both structural and functional aspects. In full analogy with viral biogenesis, some of these vesicles are generated inside cells and, once released into the extracellular milieu, are called “exosomes”. Others bud from the plasma membrane and are generally referred to as “microvesicles”. In this review, we will discuss the state of the art of the current studies on the relationship between EVs and viruses and their involvement in three important viral infections caused by HIV, HCV and Severe Acute Respiratory Syndrome (SARS) viruses. HIV and HCV are two well-known pathogens that hijack EVs content and release to create a suitable environment for viral infection. SARS viruses are a new entry in the world of EVs studies, but are equally important in this historical framework. A thorough knowledge of the involvement of the EVs in viral infections could be helpful for the development of new therapeutic strategies to counteract different pathogens. Full article

Approximately 250 million people are living with chronic hepatitis B virus (HBV) infections, which claim nearly a million lives annually. The target of all current HBV drug therapies (except interferon) is the viral polymerase; specifically, the reverse transcriptase domain. Although no high-resolution structure exists for the HBV polymerase, several recent advances have helped to map its functions to specific domains. The terminal protein (TP) domain, unique to hepadnaviruses such as HBV, has been implicated in the binding and packaging of the viral RNA, as well as the initial priming of and downstream synthesis of viral DNA—all of which make the TP domain an attractive novel drug target. This review encompasses three types of analysis: sequence conservation analysis, secondary structure prediction, and the results from mutational studies. It is concluded that the TP domain of HBV polymerase is comprised of seven subdomains (three unstructured loops and four helical regions) and that all three loop subdomains and Helix 5 are the major determinants of HBV function within the TP domain. Further studies, such as modeling inhibitors of these critical TP subdomains, will advance the TP domain of HBV polymerase as a therapeutic drug target in the progression towards a cure. Full article

White adipose tissue (WAT) produces interleukin-10 and other immune suppressors in response to pathogen-associated molecular patterns (PAMPs). It also homes a subset of B-cells specialized in the production of IL-10, referred to as regulatory B-cells. We investigated whether viral stimuli, polyinosinic: polycytidylic acid (poly(I:C)) or whole replicative murine cytomegalovirus (MCMV), could stimulate the expression of IL-10 in murine WAT using in vivo and ex vivo approaches. Our results showed that in vivo responses to systemic administration of poly(I:C) resulted in high levels of endogenously-produced IL-10 and IL-21 in WAT. In ex vivo WAT explants, a subset of B-cells increased their endogenous IL-10 expression in response to poly(I:C). Finally, MCMV replication in WAT explants resulted in decreased IL-10 levels, opposite to the effect seen with poly(I:C). Moreover, downregulation of IL-10 correlated with relatively lower number of Bregs. To our knowledge, this is the first report of IL-10 expression by WAT and WAT-associated B-cells in response to viral stimuli. Full article

Retroviruses package their full-length, dimeric genomic RNA (gRNA) via specific interactions between the Gag polyprotein and a “Ψ” packaging signal located in the gRNA 5′-UTR. Rous sarcoma virus (RSV) gRNA has a contiguous, well-defined Ψ element, that directs the packaging of heterologous RNAs efficiently. The simplicity of RSV Ψ makes it an informative model to examine the mechanism of retroviral gRNA packaging, which is incompletely understood. Little is known about the structure of dimerization initiation sites or specific Gag interaction sites of RSV gRNA. Using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), we probed the secondary structure of the entire RSV 5′-leader RNA for the first time. We identified a putative bipartite dimerization initiation signal (DIS), and mutation of both sites was required to significantly reduce dimerization in vitro. These mutations failed to reduce viral replication, suggesting that in vitro dimerization results do not strictly correlate with in vivo infectivity, possibly due to additional RNA interactions that maintain the dimers in cells. UV crosslinking-coupled SHAPE (XL-SHAPE) was next used to determine Gag-induced RNA conformational changes, revealing G218 as a critical Gag contact site. Overall, our results suggest that disruption of either of the DIS sequences does not reduce virus replication and reveal specific sites of Gag–RNA interactions. Full article

We identified and isolated a novel Tembusu virus (TMUV) strain TP1906 (TMUV-TP1906) from a Culex annulus mosquito pool collected from the northern part of Taiwan in 2019. The TMUV-TP1906 genome is a 10,990-nucleotide-long, positive-sense, single-stranded RNA, consisting of a single open reading frame (ORF) encoding a polyprotein of 3425 amino acids, with 5′ and 3′ untranslated regions (UTRs) of 94 and 618 nucleotides, respectively. The nucleotide sequence of the TMUV-TP1906 of ORF exhibited 93.71% and 91.27% similarity with Sitiawan virus (STWV) and the TMUV prototype strain MM1775, respectively. The 3′-UTR variable region of TMUV-TP1906 showed nucleotide sequence divergence with other TMUV strains. Phylogenetic analysis of the complete ORF and polyprotein sequences revealed that TMUV-TP1906 is most closely related to STWV which causes encephalitis and retarded growth in chickens. We found that the TMUV-TP1906 caused a cytopathic effect (CPE) in the DF-1 chicken fibroblast cell line, while no apparent CPE was observed in Vero and C6/36 cells. In this study, we first identified and isolated a novel TMUV strain in Taiwan. In addition, to our knowledge, it is the first time that the TMUV strain was isolated from the Cx. annulus mosquitoes. Further study is warranted to investigate the host range and virulence of TMUV-TP1906. Full article

Honey bees are key agricultural pollinators, but beekeepers continually suffer high annual colony losses owing to a number of environmental stressors, including inadequate nutrition, pressures from parasites and pathogens, and exposure to a wide variety of pesticides. In this review, we examine how two such stressors, pesticides and viruses, may interact in additive or synergistic ways to affect honey bee health. Despite what appears to be a straightforward comparison, there is a dearth of studies examining this issue likely owing to the complexity of such interactions. Such complexities include the wide array of pesticide chemical classes with different modes of actions, the coupling of many bee viruses with ectoparasitic Varroa mites, and the intricate social structure of honey bee colonies. Together, these issues pose a challenge to researchers examining the effects pesticide-virus interactions at both the individual and colony level. Full article

Selenium binding protein 1 (SELENBP1) has been known to be reduced in various types cancer, and epigenetic change is shown to be likely to account for the reduction of SELNEBP1 expression. With cDNA microarray comparative analysis, we found that SELENBP1 is markedly decreased in hepatitis B virus-X (HBx)-expressing cells. To clarify the effect of HBx on SELENBP1 expression, we compared the expression levels of SELENBP1 mRNA and protein by semi-quantitative RT-PCR, Northern blot, and Western blot. As expected, SELENBP1 expression was shown to be reduced in cells expressing HBx, and reporter gene analysis showed that the SELENBP1 promoter is repressed by HBx. In addition, the stepwise deletion of 5' flanking promoter sequences resulted in a gradual decrease in basal promoter activity and inhibition of SELENBP1 expression by HBx. Moreover, immunohistochemistry on tissue microarrays containing 60 pairs of human liver tissue showed decreased intensity of SELENBP1 in tumor tissues as compared with their matched non-tumor liver tissues. Taken together, our findings suggest that inhibition of SELENBP1 expression by HBx might act as one of the causes in the development of hepatocellular carcinoma caused by HBV infection. Full article

Even with an efficient combination of antiretroviral therapy (ART), which significantly decreases viral load in human immunodeficiency virus type 1 (HIV-1)-positive individuals, the occurrence of HIV-1-associated neurocognitive disorders (HAND) still exists. Microglia have been shown to have a significant role in HIV-1 replication in the brain and in subsequent HAND pathogenesis. However, due to the limited ability of ART drugs to cross the blood–brain barrier (BBB) after systemic administration, in addition to efflux transporter expression on microglia, the efficacy of ART drugs for viral suppression in microglia is suboptimal. Previously, we developed novel poly (lactic-co-glycolic acid) (PLGA)-based elvitegravir nanoparticles (PLGA-EVG NPs), which showed improved BBB penetration in vitro and improved viral suppression in HIV-1-infected primary macrophages, after crossing an in vitro BBB model. Our objective in the current study was to evaluate the efficacy of our PLGA-EVG NPs in an important central nervous system (CNS) HIV-1 reservoir, i.e., microglia. In this study, we evaluated the cyto-compatibility of the PLGA-EVG NPs in microglia, using an XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay and cellular morphology observation. We also studied the endocytosis pathway and the subcellular localization of PLGA NPs in microglia, using various endocytosis inhibitors and subcellular localization markers. We determined the ability of PLGA-EVG NPs to suppress HIV-1 replication in microglia, after crossing an in vitro BBB model. We also studied the drug levels in mouse plasma and brain tissue, using immunodeficient NOD scid gamma (NSG) mice, and performed a pilot study, to evaluate the efficacy of PLGA-EVG NPs on viral suppression in the CNS, using an HIV-1 encephalitic (HIVE) mouse model. From our results, the PLGA-EVG NPs showed ~100% biocompatibility with microglia, as compared to control cells. The internalization of PLGA NPs in microglia occurred through caveolae-/clathrin-mediated endocytosis. PLGA NPs can also escape from endo-lysosomal compartments and deliver the therapeutics to cells efficiently. More importantly, the PLGA-EVG NPs were able to show ~25% more viral suppression in HIV-1-infected human-monocyte-derived microglia-like cells after crossing the in vitro BBB compared to the EVG native drug, without altering BBB integrity. PLGA-EVG NPs also showed a ~two-fold higher level in mouse brain and a trend of decreasing CNS HIV-1 viral load in HIV-1-infected mice. Overall, these results help us to create a safe and efficient drug delivery method to target HIV-1 reservoirs in the CNS, for potential clinical use. Full article

Bacteriophage 80α is a representative of a class of temperate phages that infect Staphylococcus aureus and other Gram-positive bacteria. Many of these phages carry genes encoding toxins and other virulence factors. This phage, 80α, is also involved in high-frequency mobilization of S. aureus pathogenicity islands (SaPIs), mobile genetic elements that carry virulence factor genes. Bacteriophage 80α encodes a minor capsid protein, gp44, between the genes for the portal protein and major capsid protein. Gp44 is essential for a productive infection by 80α but not for transduction of SaPIs or plasmids. We previously demonstrated that gp44 is an ejection protein that acts to promote progression to the lytic cycle upon infection and suggested that the protein might act as an anti-repressor of CI in the lytic–lysogenic switch. However, an 80α Δ44 mutant also exhibited a reduced rate of lysogeny. Here, we show that gp44 is a non-specific DNA binding protein with affinity for the blunt ends of linear DNA. Our data suggest a model in which gp44 promotes circularization of the genome after injection into the host cell, a key initial step both for lytic growth and for the establishment of lysogeny. Full article

Identification and characterization of viral genomes in vectors including ticks and mosquitoes positive for pathogens of great public health concern using metagenomic next generation sequencing (mNGS) has challenges. One such challenge is the ability to efficiently recover viral RNA which is typically dependent on sample processing. We evaluated the quantitative effect of six different extraction methods in recovering viral RNA in vectors using negative tick homogenates spiked with serial dilutions of tick-borne encephalitis virus (TBEV) and surrogate Langat virus (LGTV). Evaluation was performed using qPCR and mNGS. Sensitivity and proof of concept of optimal method was tested using naturally positive TBEV tick homogenates and positive dengue, chikungunya, and Zika virus mosquito homogenates. The amount of observed viral genome copies, percentage of mapped reads, and genome coverage varied among different extractions methods. The developed Method 5 gave a 120.8-, 46-, 2.5-, 22.4-, and 9.9-fold increase in the number of viral reads mapping to the expected pathogen in comparison to Method 1, 2, 3, 4, and 6, respectively. Our developed Method 5 termed ROVIV (Recovery of Viruses in Vectors) greatly improved viral RNA recovery and identification in vectors using mNGS. Therefore, it may be a more sensitive method for use in arbovirus surveillance. Full article

West Nile virus (WNV) is a widespread zoonotic arbovirus and a threat to public health in Germany since its first emergence in 2018. It has become of particular relevance in Germany in 2019 due to its rapid geographical spread and the detection of the first human clinical cases. The susceptibility of indigenous Culex pipiens (biotypes pipiens and molestus) for a German WNV lineage 2 strain was experimentally compared to that of Serbian Cx. pipiens biotype molestus and invasive German Aedes albopictus. All tested populations proved to be competent laboratory vectors of WNV. Culex pipiens biotype pipiens displayed the highest transmission efficiencies (40.0%–52.9%) at 25 °C. This biotype was also able to transmit WNV at 18 °C (transmission efficiencies of 4.4%–8.3%), proving that temperate climates in Central and Northern Europe may support WNV circulation. Furthermore, due to their feeding behaviors, Cx. pipiens biotype molestus and Ae. albopictus can act as “bridge vectors”, leading to human WNV infections. Full article

The fast replication rate and lack of repair mechanisms of human immunodeficiency virus (HIV) contribute to its high mutation frequency, with some mutations resulting in the evolution of resistance to antiretroviral therapies (ART). As such, studying HIV drug resistance allows for real-time evaluation of evolutionary mechanisms. Characterizing the biological process of drug resistance is also critically important for sustained effectiveness of ART. Investigating the link between “black box” deep learning methods applied to this problem and evolutionary principles governing drug resistance has been overlooked to date. Here, we utilized publicly available HIV-1 sequence data and drug resistance assay results for 18 ART drugs to evaluate the performance of three architectures (multilayer perceptron, bidirectional recurrent neural network, and convolutional neural network) for drug resistance prediction, jointly with biological analysis. We identified convolutional neural networks as the best performing architecture and displayed a correspondence between the importance of biologically relevant features in the classifier and overall performance. Our results suggest that the high classification performance of deep learning models is indeed dependent on drug resistance mutations (DRMs). These models heavily weighted several features that are not known DRM locations, indicating the utility of model interpretability to address causal relationships in viral genotype-phenotype data. Full article

The efficacy of phages in multispecies infections has been poorly examined. The in vitro lytic efficacies of phage cocktails AB-SA01, AB-PA01, which target Staphylococcus aureus and Pseudomonas aeruginosa, respectively, and their combination against their hosts were evaluated in S. aureus and P. aeruginosa mixed-species planktonic and biofilm cultures. Green fluorescent protein (GFP)-labelled P. aeruginosa PAO1 and mCherry-labelled S. aureus KUB7 laboratory strains and clinical isolates were used as target bacteria. During real-time monitoring using fluorescence spectrophotometry, the density of mCherry S. aureus KUB7 and GFP P. aeruginosa PAO1 significantly decreased when treated by their respective phage cocktail, a mixture of phage cocktails, and gentamicin. The decrease in bacterial density measured by relative fluorescence strongly associated with the decline in bacterial cell counts. This microplate-based mixed-species culture treatment monitoring through spectrophotometry combine reproducibility, rapidity, and ease of management. It is amenable to high-throughput screening for phage cocktail efficacy evaluation. Each phage cocktail, the combination of the two phage cocktails, and tetracycline produced significant biofilm biomass reduction in mixed-species biofilms. This study result shows that these phage cocktails lyse their hosts in the presence of non-susceptible bacteria. These data support the use of phage cocktails therapy in infections with multiple bacterial species. Full article

The host-vector shuttle and the bottleneck in dengue transmission is a significant aspect with regard to the study of dengue outbreaks. As mosquitoes require 100–1000 times more virus to become infected than human, the transmission of dengue virus from human to mosquito is a vulnerability that can be targeted to improve disease control. In order to capture the heterogeneity in the infectiousness of an infected patient population towards the mosquito population, we calibrate a population of host-to-vector virus transmission models based on an experimentally quantified infected fraction of a mosquito population. Once the population of models is well-calibrated, we deploy a population of controls that helps to inhibit the human-to-mosquito transmission of the dengue virus indirectly by reducing the viral load in the patient body fluid. We use an optimal bang-bang control on the administration of the defective virus (transmissible interfering particles (TIPs)) to symptomatic patients in the course of their febrile period and observe the dynamics in successful reduction of dengue spread into mosquitoes. Full article