Viruses, Volume 10, Issue 11 (November 2018) – 84 articles

To evaluate the effects of ZIKV infection on non-human primates (NHPs), as well as to investigate whether these NHPs develop sufficient viremia to infect the major urban vector mosquito, Aedes aegypti, four cynomolgus macaques (Macaca fascicularis) were subcutaneously infected with 5.0 log₁₀ focus-forming units (FFU) of DNA clone-derived ZIKV strain FSS13025 (Asian lineage, Cambodia, 2010). Following infection, the animals were sampled (blood, urine, tears, and saliva), underwent daily health monitoring, and were exposed to Ae. aegypti at specified time points. All four animals developed viremia, which peaked 3–4 days post-infection at a maximum value of 6.9 log₁₀ genome copies/mL. No virus was detected in urine, tears, or saliva. Infection by ZIKV caused minimal overt disease: serum biochemistry and CBC values largely fell within the normal ranges, and cytokine elevations were minimal. Strikingly, the minimally colonized population of Ae. aegypti exposed to viremic animals demonstrated a maximum infection rate of 26% during peak viremia, with two of the four macaques failing to infect a single mosquito at any time point. These data indicate that cynomolgus macaques may be an effective model for ZIKV infection of humans and highlights the relative refractoriness of Ae. aegypti for ZIKV infection at the levels of viremia observed. Full article

Tomato spotted wilt virus (TSWV) is one of the most destructive viral pathogens of plants. Recently, a single dominant gene conferring complete resistance to TSWV (RTSW) was identified in Nicotina alata and introgressed into cultivated tobacco (N. tabacum). However, whether the TSWV carries an avirulence (Avr) factor directed against RTSW remains obscure. In the present study, we identified the non-structural protein (NSm), the movement protein of TSWV, which is an RTSW-specific Avr factor, by using two different transient expression systems. Using amino acid (aa) substitution mutants, we demonstrated the ability to induce RTSW-mediated hypersensitive response (HR) of NSm is independent of its movement function. Moreover, key substitutions (C118Y and T120N), a 21-aa viral effector epitope, and different truncated versions of NSm, which are responsible for the recognition of the Sw-5b resistance gene of tomato, were tested for their ability to trigger HR to TSWV in tobacco. Together, our results demonstrated that RTSW-mediated resistance is triggered by NSm in the same way as by Sw-5b, however, via different elicitor active sites. Finally, an Avr gene-based diagnostic approach was established and used to determine the presence and effectiveness of resistance genes in tobacco. Full article

Despite recent advances in curative therapy, hepatitis C virus (HCV) still remains a global threat. In order to achieve global elimination, a prophylactic vaccine should be considered high priority. Previous immunogens used to induce broad neutralising antibodies (BnAbs) have been met with limited success. To improve immunogen design, factors associated with the early development of BnAbs in natural infection must first be understood. In this study, 43 subjects identified with acute HCV were analysed longitudinally using a panel of heterogeneous HCV pseudoparticles (HCVpp), to understand the emergence of BnAbs. Compared to those infected with a single genotype, early BnAb development was associated with subjects co-infected with at least 2 HCV subtypes during acute infection. In those that were mono-infected, BnAbs were seen to emerge with increasing viral persistence. If subjects acquired a secondary infection, nAb breadth was seen to boost upon viral re-exposure. Importantly, this data highlights the potential for multivalent and prime-boost vaccine strategies to induce BnAbs against HCV in humans. However, the data also indicate that the infecting genotype may influence the development of BnAbs. Therefore, the choice of antigen will need to be carefully considered in future vaccine trials. Full article

Angola variant (MARV/Ang) has replaced Mt. Elgon variant Musoke isolate (MARV/MtE-Mus) as the consensus standard variant for Marburg virus research and is regarded as causing a more aggressive phenotype of disease in animal models; however, there is a dearth of published evidence supporting the higher virulence of MARV/Ang. In this retrospective study, we used data pooled from eight separate studies in nonhuman primates experimentally exposed with either 1000 pfu intramuscular (IM) MARV/Ang or MARV/MtE-Mus between 2012 and 2017 at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). Multivariable Cox proportional hazards regression was used to evaluate the association of variant type with time to death, the development of anorexia, rash, viremia, and 10 select clinical laboratory values. A total of 47 cynomolgus monkeys were included, of which 18 were exposed to MARV/Ang in three separate studies and 29 to MARV/MtE-Mus in five studies. Following universally fatal Marburg virus exposure, compared to MARV/MtE-Mus, MARV/Ang was associated with an increased risk of death (HR = 22.10; 95% CI: 7.08, 68.93), rash (HR = 5.87; 95% CI: 2.76, 12.51) and loss of appetite (HR = 35.10; 95% CI: 7.60, 162.18). Our data demonstrate an increased virulence of MARV/Ang compared to MARV/MtE-Mus variant in the 1000 pfu IM cynomolgus macaque model. Full article

Porcine reproductive and respiratory syndrome virus (PRRSV) is a continuous threat to the pork industry as it continues to cause significant economic loss worldwide. Currently, vaccination strategies provide very limited protection against PRRSV transmission. Consequently, there is an urgent need to develop new antiviral strategies. Platycodin D (PD) is one of the major bioactive triterpenoid saponins derived from Platycodon grandiflorum, a traditional Chinese medicine used as an expectorant for pulmonary diseases and a remedy for respiratory disorders. Here, we demonstrate that PD exhibits potent activity against PRRSV infection in Marc-145 cells and primary porcine alveolar macrophages. PD exhibited broad-spectrum inhibitory activities in vitro against high pathogenic type 2 PRRSV GD-HD strain and GD-XH strain as well as classical CH-1a and VR2332 strains. PD at concentrations ranging 1–4 μM significantly inhibited PRRSV RNA synthesis, viral protein expression and progeny virus production in a dose-dependent manner. EC₅₀ values of PD against four tested PRRSV strains infection in Marc-145 cells ranged from 0.74 to 1.76 μM. Mechanistically, PD inhibited PRRSV replication by directly interacting with virions therefore affecting multiple stages of the virus life cycle, including viral entry and progeny virus release. In addition, PD decreased PRRSV- and LPS-induced cytokine (IFN-α, IFN-β, IL-1α, IL-6, IL-8 and TNF-α) production in PAMs. Altogether, our findings suggested that PD is a potent inhibitor of PPRSV infection in vitro. However, further in vivo studies are necessary to confirm PD as a potential novel and effective PPRSV inhibitor in swine. Full article

Human herpesvirus-6A and -6B (HHV-6A and -6B) are two closely related betaherpesviruses that infect humans. Upon primary infection they establish a life-long infection termed latency, where the virus genome is integrated into the telomeres of latently infected cells. Intriguingly, HHV-6A/B can integrate into germ cells, leading to individuals with inherited chromosomally-integrated HHV-6 (iciHHV-6), who have the HHV-6 genome in every cell. It is known that telomeric repeats flanking the virus genome are essential for integration; however, the protein factors mediating integration remain enigmatic. We have previously shown that the putative viral integrase U94 is not essential for telomere integration; thus, we set out to assess the contribution of potential viral recombination proteins U41 and U70 towards integration. We could show that U70 enhances dsDNA break repair via a homology-directed mechanism using a reporter cell line. We then engineered cells to produce shRNAs targeting both U41 and U70 to inhibit their expression during infection. Using these cells in our HHV-6A in vitro integration assay, we could show that U41/U70 were dispensable for telomere integration. Furthermore, additional inhibition of the cellular recombinase Rad51 suggested that it was also not essential, indicating that other cellular and/or viral factors must mediate telomere integration. Full article

Lassa virus (LASV), a mammarenavirus, infects an estimated 100,000–300,000 individuals yearly in western Africa and frequently causes lethal disease. Currently, no LASV-specific antivirals or vaccines are commercially available for prevention or treatment of Lassa fever, the disease caused by LASV. The development of medical countermeasure screening platforms is a crucial step to yield licensable products. Using reverse genetics, we generated a recombinant wild-type LASV (rLASV-WT) and a modified version thereof encoding a cleavable green fluorescent protein (GFP) as a reporter for rapid and quantitative detection of infection (rLASV-GFP). Both rLASV-WT and wild-type LASV exhibited similar growth kinetics in cultured cells, whereas growth of rLASV-GFP was slightly impaired. GFP reporter expression by rLASV-GFP remained stable over several serial passages in Vero cells. Using two well-characterized broad-spectrum antivirals known to inhibit LASV infection, favipiravir and ribavirin, we demonstrate that rLASV-GFP is a suitable screening tool for the identification of LASV infection inhibitors. Building on these findings, we established a rLASV-GFP-based high-throughput drug discovery screen and an rLASV-GFP-based antibody neutralization assay. Both platforms, now available as a standard tool at the IRF-Frederick (an international resource), will accelerate anti-LASV medical countermeasure discovery and reduce costs of antiviral screens in maximum containment laboratories. Full article

The European race of the ascomycetous species Gremmeniella abietina (Lagerberg) Morelet includes causal agents of shoot blight and stem canker of several conifers in Europe and North America, which are known to host a diverse virome. GaRV6 is the latest and sixth mycovirus species reported within G. abietina. Before its description, one victorivirus and one gammapartitivirus species were described in biotype A, two mitoviruses in both biotypes A and B and a betaendornavirus in biotype B. Possible phenotypic changes produced by mycoviruses on G. abietina mycelial growth have been reported in Spanish mitovirus-free and GaRV6-hosting G. abietina isolates, which had higher growth rates at the optimal temperature of 15 °C, but no other major differences have been observed between partitivirus-like dsRNA and dsRNA-free isolates. In this review, we reappraise the diversity of viruses found in G. abietina so far, and their relevance in clarifying the taxonomy of G. abietina. We also provide evidence for the presence of two new viruses belonging to the families Fusariviridae and Endornaviridae in Spanish isolates. Full article

H9N2 avian influenza viruses are present in poultry worldwide. These viruses are considered to have pandemic potential, because recent isolates can recognize human-type receptor and several sporadic human infections have been reported. In this study, we aimed to identify mutations related to mammalian adaptation of H9N2 influenza virus. We found that mouse-adapted viruses had several mutations in hemagglutinin (HA), PB2, PA, and PB1. Among the detected mutations, PB1-K577E was a novel mutation that had not been previously reported to involve mammalian adaptation. A recombinant H9N2 virus bearing only the PB1-K577E mutation showed enhanced pathogenicity in mice, with increased virus titers in nasal turbinates compared to that in mice infected with the wild-type virus. In addition, the PB1-K577E mutation increased virus polymerase activity in human cell culture at a lower temperature. These data suggest that the PB1-K577E mutation is a novel pathogenicity determinant of H9N2 virus in mice and could be a signature for mammalian adaptation. Full article

We established a cold-adapted infectious bronchitis virus (BP-caKII) by passaging a field virus through specific pathogen-free embryonated eggs 20 times at 32 °C. We characterized its growth kinetics and pathogenicity in embryonated eggs, and its tropism and persistence in different tissues from chickens; then, we evaluated pathogenicity by using a new premature reproductive tract pathogenicity model. Furthermore, we determined the complete genomic sequence of BP-caKII to understand the genetic changes related to cold adaptation. According to our results, BP-caKII clustered with the KII genotype viruses K2 and KM91, and showed less pathogenicity than K2, a live attenuated vaccine strain. BP-caKII showed delayed viremia, resulting in its delayed dissemination to the kidneys and cecal tonsils compared to K2 and KM91, the latter of which is a pathogenic field strain. A comparative genomics study revealed similar nucleotide sequences between BP-caKII, K2 and KM91 but clearly showed different mutations among them. BP-caKII shared several mutations with K2 (nsp13, 14, 15 and 16) following embryo adaptation but acquired multiple additional mutations in nonstructural proteins (nsp3, 4 and 12), spike proteins and nucleocapsid proteins following cold adaptation. Thus, the establishment of BP-caKII and the identified mutations in this study may provide insight into the genetic background of embryo and cold adaptations, and the attenuation of coronaviruses. Full article

Rift Valley fever virus (RVFV) is an emerging pathogen capable of causing severe disease in livestock and humans and can be transmitted by multiple routes including aerosol exposure. Several animal models have been developed to gain insight into the pathogenesis associated with aerosolized RVFV infection, but work with these models is restricted to high containment biosafety level (BSL) laboratories limiting their use for antiviral and vaccine development studies. Here, we report on a new RVFV inhalation infection model in STAT2 KO hamsters exposed to aerosolized MP-12 vaccine virus by nose-only inhalation that enables a more accurate delivery and measurement of exposure dose. RVFV was detected in hepatic and other tissues 4–5 days after challenge, consistent with virus-induced lesions in the liver, spleen and lung. Furthermore, assessment of blood chemistry and hematological parameters revealed alterations in several liver disease markers and white blood cell parameters. Our results indicate that STAT2 KO hamsters develop a disease course that shares features of disease observed in human cases and in other animal models of RVFV aerosol exposure, supporting the use of this BSL-2 infection model for countermeasure development efforts. Full article

Influenza still remains one of the most challenging diseases, posing a significant threat to public health. Host lipid rafts play a critical role in influenza A virus (IAV) assembly and budding, however, their role in polyvalent IAV host binding and endocytosis had remained elusive until now. In the present study, we observed co-localization of IAV with a lipid raft marker ganglioside, GM1, on the host surface. Further, we isolated the lipid raft micro-domains from IAV infected cells and detected IAV protein in the raft fraction. Finally, raft disruption using Methyl-β-Cyclodextrin revealed significant reduction in IAV host binding, suggesting utilization of host rafts for polyvalent binding on the host cell surface. In addition to this, cyclodextrin mediated inhibition of raft-dependent endocytosis showed significantly reduced IAV internalization. Interestingly, exposure of cells to cyclodextrin two hours post-IAV binding showed no such reduction in IAV entry, indicating use of raft-dependent endocytosis for host entry. In summary, this study demonstrates that host lipid rafts are selected by IAV as a host attachment factors for multivalent binding, and IAV utilizes these micro-domains to exploit raft-dependent endocytosis for host internalization, a virus entry route previously unknown for IAV. Full article

Zika virus (ZIKV) is a neuropathic virus that causes serious neurological abnormalities such as Guillain-Barre syndrome in adults and congenital Zika syndrome (CZS) in fetuses, which makes it an important concern for global human health. A catalogue of cells that support ZIKV replication, pathogenesis, and/or the persistence of the virus still remains unknown. Here, we studied the behavior of the virus in human placenta (JEG-3) and human microglia (HMC3) cell lines in order to better understand how different host tissues respond during infection. We quantified the host transcriptional response to ZIKV infection in both types of cells at 24 and 72 h post-infection. A panel of 84 genes that are involved in the innate or adaptive immune responses was used to quantify differential expression in both cell lines. HMC3 cells showed a unique set of significant differentially expressed genes (DEGs) compared with JEG-3 cells at both time points. Subsequent analysis of these data using modern pathway analysis methods revealed that the TLR7/8 pathway was strongly inhibited in HMC3 cells, while it was activated in JEG-3 cells during virus infection. The disruption of these pathways was subsequently confirmed with specific small interfering RNA (siRNA) experiments that characterize their role in the viral life cycle, and may partially explain why ZIKV infection in placental tissue contributes to extreme neurological problems in a developing fetus. Full article

One of the crucial steps during trials for Zika and other vaccines is to recruit participants and to understand how participants’ attitudes and sociodemographic characteristics affect willingness to participate (WTP). This study was conducted to assess WTP, its explanatory variables, and the impact of financial compensation on WTP in Indonesia. A health facility-based cross-sectional study was conducted in eleven regencies in the Aceh and West Sumatra provinces of Indonesia. Participants were recruited via a convenience sampling method and were interviewed. The associations between explanatory variables and WTP were assessed using a two-step logistic regression analysis. A total of 1,102 parents were approached, and of these 956 (86.8%) completed the interview and were included in analysis. Of those, 144 (15.1%) were willing to participate in a Zika vaccine trial without a financial compensation. In the multivariate analysis, WTP was tied to an age of more than 50 years old, compared to 20–29 years (odds ratio (OR): 5.0; 95% confidence interval (CI): 2.37–10.53), to being female (OR: 2.20; 95% CI: 1.11–4.37), and to having heard about Zika (OR: 2.41; 95% CI: 1.59–3.65). Participants’ WTP increased gradually with higher financial compensation. The rate of WTP increased to 62.3% at the highest offer (US$ 350.4), and those who were still unwilling to participate (37.7%) had a poorer attitude towards childhood vaccination. This study highlights that pre-existing knowledge about Zika and attitudes towards childhood vaccination are important in determining community members being willing to participate in a vaccine trial. Financial incentives are still an important factor to enhance participant recruitment during a vaccine trial. Full article

Tospovirus is a tripartite negative stranded RNA virus and is considered as one of the most devastating plant viruses. Successful virus infection in plant requires many host factors. To date, very few host factors have been identified as important in Tospovirus infection in plants. We reported earlier that NSm protein encoded by Tomato spotted wilt virus (TSWV), a type species of the genus Orthotospovirus, plays critical roles in viral cell-to-cell and long-distance movement. In this study, we determined that molecular co-chaperone NbSGT1 interacted with TSWV NSm in Nicotiana benthamiana. TSWV infection significantly upregulated the expression of NbSGT1 gene and transient overexpression of NbSGT1 in N. benthamiana leaves accelerated TSWV infection. In contrast, silencing the NbSGT1 gene expression using a virus-induced gene silencing (VIGS) approach strongly inhibited TSWV NSm cell-to-cell movement, as well as TSWV local and systemic infection in N. benthamiana plants. Furthermore, NbSGT1 was found to regulate the infection of both American and Euro/Asia type tospoviruses in N. benthamiana plant. Collectively, our findings presented in this paper and the results published previously indicated that molecular co-chaperone NbSGT1 plays important roles in modulating both positive stranded and tripartite negative stranded RNA virus infection in plants. Full article

Zika virus (ZIKV) infection and persistence during pregnancy can lead to microcephaly and other fetal neurological disorders collectively known as Congenital Zika Syndrome. The immunological and virological events that contribute to the establishment of persistent ZIKV infection in humans are unclear though. Here we show that human fetal astrocytes (HFAs), the most abundant cell type in the central nervous system, become persistently infected with ZIKV resulting in continuous viral shedding for at least one month; a process that is facilitated by TIM/TAM receptors. HFAs are relatively resistant to ZIKV-induced apoptosis, a factor that may be important for chronic infection of these cells. Once infection was established, interferon treatment did not reduce virus replication. Moreover, the fact that the innate immune system was highly activated in persistently infected HFAs indicates that the virus can thrive in the presence of a sustained antiviral response. RNAseq analyses of persistently infected cells revealed that ZIKV alters host gene expression in a manner that could affect developmental processes. Conversely, data from sequencing of ZIKV genomes in persistently infected HFAs suggest that adaptive mutations were not required for establishing chronic infection. Based on these results, we postulate that HFAs are reservoirs for ZIKV in the fetal brain and that moderate apoptosis combined with inefficient antiviral response from these cells may contribute to the establishment of chronic brain infection associated with the ZIKV neurodevelopmental abnormalities. Full article

Hepatitis C virus (HCV)-specific T cell responses are closely linked to the clinical course of infection. While T cell responses in self-limiting infection are typically broad and multi-specific, they display several distinct features of functional impairment in the chronic phase. Moreover, HCV readily adapts to immune pressure by developing escape mutations within epitopes targeted by T cells. Much of our current knowledge on HCV-specific T cell responses has been gathered under the assumption that this might eventually pave the way for a therapeutic vaccine. However, with the development of highly efficient direct acting antivirals (DAAs), there is less interest in the development of a therapeutic vaccine for HCV and the scope of T cell research has shifted. Indeed, the possibility to rapidly eradicate an antigen that has persisted over years or decades, and has led to T cell exhaustion and dysfunction, provides the unique opportunity to study potential T cell recovery after antigen cessation in a human in vivo setting. Findings from such studies not only improve our basic understanding of T cell immunity but may also advance immunotherapeutic approaches in cancer or chronic hepatitis B and D infection. Moreover, in order to edge closer to the WHO goal of HCV elimination by 2030, a prophylactic vaccine is clearly required. Thus, in this review, we will summarize our current knowledge on HCV-specific T cell responses and also provide an outlook on the open questions that require answers in this field. Full article

Adeno associated virus (AAV) is a versatile gene delivery tool, which has been approved as a human gene therapy vector for combating genetic diseases. AAV capsid proteins are the major components that determine the tissue specificity, immunogenicity and in vivo transduction performance of the vector. In this study, the AAV8 capsid glycosylation profile was systemically analyzed by peptide mass fingerprinting utilizing high-resolution mass spectrometry to determine the presence of capsid glycosylation. We identified N-glycosylation on the amino acid N499 of the capsid protein. We characterized the overall sugar profile for vector produced in 293 cells. Multiple N-glycosylated host-cell proteins (HCPs) copurified with AAV8 vectors and were identified by analyzing LC-MS data utilizing a human database and proteome discoverer search engine. The N-glycosylation analysis by MALDI-TOF MS, highlighted the probability of AAV8 interaction with terminal galactosylated N-glycans within the HCPs. Full article

The evolution of infectious pathogens in humans proved to be a global health problem. Technological advancements over the last 50 years have allowed better means of identifying novel therapeutics to either prevent or combat these infectious diseases. The development of humanized mouse models offers a preclinical in vivo platform for further characterization of human viral infections and human immune responses triggered by these virus particles. Multiple strains of immunocompromised mice reconstituted with a human immune system and/or human hepatocytes are susceptible to infectious pathogens as evidenced by establishment of full viral life cycles in hope of investigating viral–host interactions observed in patients and discovering potential immunotherapies. This review highlights recent progress in utilizing humanized mice to decipher human specific immune responses against viral tropism. Full article

The filoviruses Ebola virus (EBOV) and Sudan virus (SUDV) can cause severe diseases, and there are currently no licensed countermeasures available for use against them. Transmission occurs frequently via contact with bodily fluids from infected individuals. However, it can be difficult to determine when or how someone became infected, or the quantity of infectious virus to which they were exposed. Evidence suggests the infectious dose is low, but the majority of published studies use high exposure doses. This study characterized the outcome of exposure to a low dose of EBOV or SUDV, using a Macaca fascicularis model. Further, because the effect of virus passage in cell culture may be more pronounced when lower exposure doses are used, viruses that possessed either the characteristics of wild type viruses (possessing predominantly 7-uridine (7U) genotype and a high particle-to-plaque forming unit (PFU) ratio) or cell culture-passaged viruses (predominantly 8-uridine (8U) genotype, a lower particle-to-PFU ratio) were used. The time to death after a low dose exposure was delayed in comparison to higher exposure doses. These data demonstrated that an extremely low dose of EBOV or SUDV is sufficient to cause lethal disease. A low dose exposure model can help inform studies on pathogenesis, transmission, and optimization of prevention strategies. Full article

The influenza A virus (IAV) genome consists of eight single-stranded RNA segments. Each segment is associated with a protein complex, with the 3′ and 5′ ends bound to the RNA-dependent RNA polymerase (RdRp) and the remainder associated with the viral nucleoprotein. During transcription of viral mRNA, this ribonucleoprotein complex steals short, 5′-capped transcripts produced by the cellular DNA dependent RNA polymerase II (RNAPII) and uses them to prime transcription of viral mRNA. Here, we review the current knowledge on the process of IAV cap-snatching and suggest a requirement for RNAPII promoter-proximal pausing for efficient IAV mRNA transcription. Full article

The mechanisms that drive formation of the HIV capsid, first as an immature particle and then as a mature protein shell, remain incompletely understood. Recent discoveries of positively-charged rings in the immature and mature protein hexamer subunits that comprise them and their binding to the cellular metabolite inositol hexakisphosphate (IP6) have stimulated exciting new hypotheses. In this paper, we discuss how data from multiple structural and biochemical approaches are revealing potential roles for IP6 in the HIV-1 replication cycle from assembly to uncoating. Full article

Human bocaviruses (HBoVs) 1–4 belong to the Parvoviridae family, and they infect the respiratory or gastrointestinal tracts in children. We investigated the prevalence of HBoV1–4 DNAs in the blood and stool samples, and of HBoV1–4 IgG and IgM in the plasma samples, of children presenting with acute gastroenteritis (AGE). In addition, we identified HBoV co-infections with the five most frequent gastrointestinal pathogens. A total of 83 paired blood and stool samples were collected from children aged five years or less. Infection markers of HBoV1, 2, or 3 (viral DNA in blood and/or stool and/or antibodies) were detected in 61 out of 83 (73.5%) patients. HBoV1, 2, or 3 DNA as a monoinfection was revealed in 18.1%, 2.4%, and 1.2%, respectively, and 21.7% in total. In 56.1% of the HBoV DNA-positive patients, the presence in stool of another virus—most frequently norovirus or rotavirus—was observed. In conclusion, this study, for the first time, illustrates the prevalence and genetic diversity of HBoVs in Latvian children with gastroenteritis, and shows a widespread distribution of these viruses in the community. HBoV1 and 2 are commonly found as single infectious agents in children with AGE, suggesting that the viruses can be as pathogenic by themselves as other enteric agents are. Full article

Bacteriophage-derived endolysins have gained increasing attention as potent antimicrobial agents and numerous publications document the in vivo efficacy of these enzymes in various rodent models. However, little has been documented about their safety and toxicity profiles. Here, we present preclinical safety and toxicity data for two pneumococcal endolysins, Pal and Cpl-1. Microarray, and gene profiling was performed on human macrophages and pharyngeal cells exposed to 0.5 µM of each endolysin for six hours and no change in gene expression was noted. Likewise, in mice injected with 15 mg/kg of each endolysin, no physical or behavioral changes were noted, pro-inflammatory cytokine levels remained constant, and there were no significant changes in the fecal microbiome. Neither endolysin caused complement activation via the classic pathway, the alternative pathway, or the mannose-binding lectin pathway. In cellular response assays, IgG levels in mice exposed to Pal or Cpl-1 gradually increased for the first 30 days post exposure, but IgE levels never rose above baseline, suggesting that hypersensitivity or allergic reaction is unlikely. Collectively, the safety and toxicity profiles of Pal and Cpl-1 support further preclinical studies. Full article

Base-By-Base is a comprehensive tool for the creation and editing of multiple sequence alignments that is coded in Java and runs on multiple platforms. It can be used with gene and protein sequences as well as with large viral genomes, which themselves can contain gene annotations. This report describes new features added to Base-By-Base over the last 7 years. The two most significant additions are: (1) The recoding and inclusion of “consensus-degenerate hybrid oligonucleotide primers” (CODEHOP), a popular tool for the design of degenerate primers from a multiple sequence alignment of proteins; and (2) the ability to perform fuzzy searches within the columns of sequence data in multiple sequence alignments to determine the distribution of sequence variants among the sequences. The intuitive interface focuses on the presentation of results in easily understood visualizations and providing the ability to annotate the sequences in a multiple alignment with analytic and user data. Full article

Glycans are, with nucleic acids, proteins and lipids, one of the four founding structures of cellular life. Due to their non-template synthesis, they are inherently heterogeneous and difficult to study with regards to their structure and function. Since 2016, the research group ViroCarb, funded by the German Research Foundation, has investigated the role of glycans in non-enveloped virus infections with a highly interdisciplinary approach. The core idea was to bring together scientists and students from various disciplines such as structural biology, cell biology, virology and chemistry to advance research by an interdisciplinary means. In 2018, ViroCarb hosted the 1st International Symposium on Glycovirology in Schöntal, Germany, with a similar aim. Scientists from various disciplines gathered to discuss their area of study, present recent findings, establish or strengthen collaborations, and mentor the next generation of glycovirologists through formal presentations and informal discussions. The secluded meeting at the monastery of Schöntal gave ample time for in-depth discussions. On behalf of ViroCarb, this report summarizes the reports and highlights advances in the field. Full article

The in ovo delivery of cytosine-guanosine (CpG) oligodeoxynucleotides (ODNs) protects chickens against many bacterial and viral infections, by activating the toll-like receptor (TLR)21 signaling pathway. Although the delivery of CpG ODNs in ovo at embryo day (ED) 18 has been shown to reduce infectious bronchitis virus (IBV) loads in embryonic chicken lungs pre-hatch, whether in ovo delivered CpG ODNs are capable of protecting chickens against a post-hatch challenge is unknown. Thus, our objectives were to determine the protective effect of the in ovo delivery of CpG ODNs at ED 18 against IBV infection encountered post-hatch and, then, to investigate the mechanisms of protection. We found significantly higher survival rates and reduced IBV infection in the chickens following the pre-treatment of the ED 18 eggs with CpG ODNs. At 3 days post infection (dpi), we found an increased recruitment of macrophages, cluster of differentiation (CD)8α+ and CD4+ T lymphocytes, and an up-regulation of interferon (IFN)-γ mRNA in the respiratory tract of the chickens. Overall, it may be inferred that CpG ODNs, when delivered in ovo, provide protection against IBV infection induced morbidity and mortality with an enhanced immune response. Full article

Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that can cause severe disease, including congenital birth defect and Guillain−Barré syndrome during pregnancy. Although, several molecular diagnostic methods have been developed to detect the ZIKV, these methods pose challenges as they cannot detect early viral infection. Furthermore, these methods require the extraction of RNA, which is easy to contaminate. Nonstructural protein 1 (NS1) is an important biomarker for early diagnosis of the virus, and the detection methods associated with the NS1 protein have recently been reported. The aim of this study was to develop a rapid and sensitive detection method for the detection of the ZIKV based on the NS1 protein. The sensitivity of this method is 120 ng mL⁻¹ and it detected the ZIKV in the supernatant and lysates of Vero and BHK cells, as well as the sera of tree shrews infected with the ZIKV. Without the isolation of the virus and the extraction of the RNA, our method can be used as a primary screening test as opposed to other diagnosis methods that detect the ZIKV. Full article

Over recent years, there has been increasing interest in the use of the anelloviruses, the major component of the human virome, for the prediction of post-transplant complications such as severe infections. Due to an important diversity, the comprehensive characterization of this viral family over time has been poorly studied. To overcome this challenge, we used a metagenomic next-generation sequencing (mNGS) approach with the aim of determining the individual anellovirus profile of autologous stem cell transplant (ASCT) patients. We conducted a prospective pilot study on a homogeneous patient cohort regarding the chemotherapy regimens that included 10 ASCT recipients. A validated viral mNGS workflow was used on 108 plasma samples collected at 11 time points from diagnosis to 90 days post-transplantation. A complex interindividual variability in terms of abundance and composition was noticed. In particular, a strong sex effect was found and confirmed using quantitative PCR targeting torque teno virus, the most abundant anellovirus. Interestingly, an important turnover in the anellovirus composition was observed during the course of the disease revealing a strong intra-individual variability. Although more studies are needed to better understand anellovirus dynamics, these findings are of prime importance for their future use as biomarkers of immune competence. Full article

The cell entry mechanism of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is not fully understood. Previous studies showed that AcMNPV entered host cells primarily through clathrin-mediated endocytosis, and could efficiently infect cells via fusion with the plasma membrane after a low-pH trigger. However, whether AcMNPV enters cells via these two pathways simultaneously, and the exact manner in which AcMNPV particles are internalized into cells remains unclear. In this study, using single-virus tracking, we observed that AcMNPV particles were first captured by pre-existing clathrin-coated pits (CCP), and were then delivered to early endosomes. Population-based analysis of single-virus tracking and quantitative electron microscopy demonstrated that the majority of particles were captured by CCPs and internalized via invagination. In contrast, a minority of virus particles were not delivered to CCPs, and were internalized through direct fusion with the plasma membrane without invagination. Quantitative electron microscopy also showed that, while inhibition of CCP assembly significantly impaired viral internalization, inhibition of endosomal acidification blocked virus particles out of vesicles. Collectively, these findings demonstrated that approximately 90% of AcMNPV particles entered cells through clathrin-mediated endocytosis and 10% entered via direct fusion with the plasma membrane. This study will lead toward a better understanding of AcMNPV infection. Full article