Differences In Viral Replication Research Articles

Characterizing Infection of B Lymphocytes with Measles Virus

Abstract Wild-type (w.t.) measles virus (MeV) infection generates a state of immunosuppression, which does not occur after vaccination with live attenuated vaccine. MeV has been shown to alter the function of immune cells such as dendritic cells and lymphocytes. Though altered B cell functions contribute to immune suppression, the pathogenesis of measles infection of B cells is not understood. Here, we characterized the differences in viral replication and cytokine expression in human B cells infected with vaccine (Edmonston-Zagreb [EZ]) or w.t. MeV. To measure viral replication, human B cells were infected with an EZ virus expressing GFP and the number of GFP expressing cells and mean fluorescence intensity (MFI) were measured after 24 hours. To measure differences in cytokine responses following infection of B cells with EZ or w.t., transcription of IL-1β, IL-6, IL-10, TNFα, IFNβ, and IFNγ was measured at 24, 48, and 72 hours after infection by quantitative RT-PCR. The number of B cells expressing GFP and the MFI increased 3-fold from 0 to 24 hours after infection. Preliminary data suggested that IL-1β transcription is higher in w.t. infected B cells, while IL-6 and IL-10 transcription were higher in EZ-infected B cells. Measles virus replication was detected 24 hours after infection of isolated, human B cells. The differential expression of cytokines in B cells suggest differences in the B cell response to infection with w.t and vaccine strains of MeV. Differential cytokine expression in w.t. infected B cells could play a role in the development of immune cell dysregulation, and the mechanism could be elucidated through further functional studies of infected B cells.

The influenza NS1 protein modulates RIG-I activation via a strain-specific direct interaction with the second CARD of RIG-I

The influenza NS1 protein modulates RIG-I activation via a strain-specific direct interaction with the second CARD of RIG-I

Organ-specific small non-coding RNA responses in domestic (Sudani) ducks experimentally infected with highly pathogenic avian influenza virus (H5N1)

ABSTRACT The duck represents an important reservoir of influenza viruses for transmission to other avian and mammalian hosts, including humans. The increased pathogenicity of the recently emerging clades of highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype in ducks features systemic viral spread and organ-to-organ variation in viral transcription and tissue damage. We previously reported that experimental infection of Sudani ducks (Cairina moschata) with an Egyptian HPAI (H5N1) virus (clade 2.2.1.2) features high viral replication and severe tissue damage in lung, but lower viral replication and only mild histological changes in brain. Little is known about the involvement of miRNA in organ-specific responses to H5N1 viruses in ducks, and involvement of the other classes of small noncoding RNA (sncRNA) has not been investigated so far. Following RNA sequencing, we have annotated the duck sncRNome and compared global expression changes of the four major sncRNA classes (miRNAs, piRNAs, snoRNAs, snRNAs) between duck lung and brain during a 120 h time course of infection with this HPAI strain. We find major organ-specific differences in miRNA, piRNA and snoRNA populations even before infection and substantial reprogramming of all sncRNA classes throughout infection, which was less pronounced in brain. Pathway prediction analysis of miRNA targets revealed enrichment of inflammation-, infection- and apoptosis-related pathways in lung, but enrichment of metabolism-related pathways (including tryptophan metabolism) in brain. Thus, organ-specific differences in sncRNA responses may contribute to differences in viral replication and organ damage in ducks infected with isolates from this emerging HPAI clade, and likely other strains.

In vivo and ex vivo models of infectious bursal disease virus (IBDV) in inbred chicken lines differing in their resistance to the disease

Infectious bursal disease virus (IBDV) targets B cells in the bursa of Fabricius (BF), causing immunosuppression in chickens and mortality. Susceptibility differs between inbred chickens, with 0 % mortality in ‘resistant’ lines and up to 80 % mortality in ‘susceptible’ lines. However, the mechanism of disease resistance is not understood. In order to address this, chickens (n=18) from three ‘resistant’ lines (15, C and O) and one ‘susceptible’ line (W) were infected with the very virulent IBDV strain, UK661. Clinical scores were recorded and tissues harvested at necropsy on day one, two and three post-infection for RNA extraction and virus titration, compared to non-infected controls. Interestingly, within a given line, we observed a range of symptoms, with some individuals experiencing more severe disease than others, despite no difference in viral replication. Line 15 was the least susceptible to disease based on the average clinical scores (3.2 (15), 5.7 (C), 4.8 (O) and 4.7 (W)) and the percentage of birds with a clinical score of 2 or above (17 % (15), 100 % (C), 83 % (O) and 83 % (W)). The average peak virus replication was also significantly lower in line 15 birds (6.3 log10 fold change) compared to lines C or O (7.0 and 6.8 log10 fold change) (P<0.01). RNA-sequence analysis will be performed using BF samples to understand the biological pathways that confer IBDV resistance. Moreover, primary bursal cells harvested from resistant and susceptible lines were infected with IBDV ex vivo and ongoing work aims to quantify differentially expressed genes in these cells.

The D253N Mutation in the Polymerase Basic 2 Gene in Avian Influenza (H9N2) Virus Contributes to the Pathogenesis of the Virus in Mammalian Hosts.

Mutations in the polymerase basic 2 (PB2) gene of avian influenza viruses are important signatures for their adaptation to mammalian hosts. Various adaptive mutations have been identified around the 627 and nuclear localization sequence (NLS) domains of PB2 protein, and these mutations contribute to the replicative ability of avian influenza viruses. However, few studies have focused on adaptive mutations in other regions of PB2. In this study, we investigated the functional roles of the D253N mutation in PB2 in an H9N2 virus. This mutation was found to affect an amino acid residue in the middle domain of the PB2 protein. The virus with the D253N mutation showed higher polymerase activity and transiently increased viral replication in human cells. However, the mutant did not show significant differences in viral replication in the respiratory tract of mice upon infection. Our results supported that the D253N mutation in the middle domain of PB2, similar to mutations at the 627 and NLS domains, specifically contributed to the replication of avian influenza viruses in human cells.

Divergent transcriptomic responses underlying the ranaviruses-amphibian interaction processes on interspecies infection of Chinese giant salamander

BackgroundRanaviruses (family Iridoviridae, nucleocytoplasmic large DNA viruses) have been reported as promiscuous pathogens of cold-blooded vertebrates. Rana grylio virus (RGV, a ranavirus), from diseased frog Rana grylio with a genome of 105.79 kb and Andrias davidianus ranavirus (ADRV), from diseased Chinese giant salamander (CGS) with a genome of 106.73 kb, contains 99% homologous genes.ResultsTo uncover the differences in virus replication and host responses under interspecies infection, we analyzed transcriptomes of CGS challenged with RGV and ADRV in different time points (1d, 7d) for the first time. A total of 128,533 unigenes were obtained from 820,858,128 clean reads. Transcriptome analysis revealed stronger gene expression of RGV than ADRV at 1 d post infection (dpi), which was supported by infection in vitro. RGV replicated faster and had higher titers than ADRV in cultured CGS cell line. RT-qPCR revealed the RGV genes including the immediate early gene (RGV-89R) had higher expression level than that of ADRV at 1 dpi. It further verified the acute infection of RGV in interspecies infection.The number of differentially expressed genes and enriched pathways from RGV were lower than that from ADRV, which reflected the variant host responses at transcriptional level. No obvious changes of key components in pathway “Antigen processing and presentation” were detected for RGV at 1 dpi. Contrarily, ADRV infection down-regulated the expression levels of MHC I and CD8. The divergent host immune responses revealed the differences between interspecies and natural infection, which may resulted in different fates of the two viruses. Altogether, these results revealed the differences in transcriptome responses among ranavirus interspecies infection of amphibian and new insights in DNA virus-host interactions in interspecies infection.ConclusionThe DNA virus (RGV) not only expressed self-genes and replicated quickly after entry into host under interspecies infection, but also avoided the over-activation of host responses. The strategy could gain time for the survival of interspecies pathogen, and may provide opportunity for its adaptive evolution and interspecies transmission.

Genetic characterization of H9N2 avian influenza viruses isolated from poultry in Poland during 2013/2014

The study presents molecular characterization of H9N2 avian influenza (AI) isolates from field outbreaks in turkeys that occurred in Poland in 2013–2014. Sequences of all gene segments of one isolate from 2013 (A/turkey/Poland/14/2013(H9N2)) and two isolates from 2014 (A/turkey/Poland/08/2014(H9N2), A/turkey/Poland/09/2014(H9N2)) were obtained and analyzed in search of the phylogenetic relationship and molecular markers of zoonotic potential or increased pathogenicity. All gene segments were shown to originate from the wild bird reservoir and the close relationship of the analyzed isolates proved the link between the outbreaks in 2013 and 2014. However, remarkable molecular differences between isolates from 2013 to 2014 were identified, including mutation in the HA cleavage site (CS) leading to conversion from the PAASNR*GLF to the PAASKR*GLF motif and truncation of the PB1-F2 protein. Additionally, T97I substitution in the PA protein in A/turkey/Poland/08/2014 was detected which can be responsible for enhanced activity of viral polymerase in mammalian cells. However, experimental infection of mice with both isolates from 2014 showed their low pathogenicity, and no statistically significant differences in virus replication were observed between the viruses. Nevertheless, these findings indicate the dynamic evolution of H9N2 in the field emphasizing the need for monitoring of the situation in terms of H9N2 AI in Europe.

Alternatively activated macrophages attenuate influenza-induced acute lung injury in mice heterozygous for the F508del mutation in CFTR. (INC6P.314)

Abstract Infection with influenza A virus can result in acute lung injury (ALI), characterized by pulmonary edema and hypoxemia. We found that influenza-induced ALI in C57BL/6 (WT) mice was associated with increased bronchoalveolar epithelial cell Cl- secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Influenza-induced ALI was highly attenuated in C57BL/6-congenic mice that are heterozygous for the F508del CFTR mutation (HETs), which exhibit a 50% reduction in CFTR expression and CFTR-mediated Cl- transport. This beneficial phenotype was alveolar macrophage (AM)-dependent and was not due to differences in viral replication between mouse strains. The aim of this study was to evaluate the role of HET AMs in the attenuation of influenza-induced ALI. We hypothesized that ALI attenuation in influenza-infected HET mice results from “alternative” activation (M2 polarization) of HET AMs in response to infection. Compared to WT AMs, Arginase 1 (Arg1) protein expression was higher while inducible nitric oxide synthase (iNOS) protein was lower in HET AMs following infection. Arg1 enzymatic activity was also higher in HET AM lysates. BALF from HET mice contained higher levels of urea and lower levels of nitrate/nitrite (end-products of Arg1 and iNOS, respectively) than WT. These findings indicate that reduced CFTR expression/activity promotes an anti-inflammatory, M2 AM polarization response to influenza infection, which contributes to ALI attenuation.

Genomic Characteristics and Changes of Avian Infectious Bronchitis Virus Strain CK/CH/LDL/97I after Serial Passages in Chicken Embryos

Background: We previously attenuated the infectious bronchitis virus (IBV) strain CK/CH/LDL/97I and found that it can convey protection against the homologous pathogenic virus. Objective: To compare the full-length genome sequences of the Chinese IBV strain CK/CH/LDL/97I and its embryo-passaged, attenuated level to identify sequence substitutions responsible for the attenuation and define markers of attenuation. Methods: The full-length genomes of CK/CH/LDL/97I P5 and P115 were amplified and sequenced. The sequences were assembled and compared using the MEGALIGN program (DNAStar) and a phylogenetic tree was constructed using MEGA4 software. Results: The CK/CH/LDL/97I virus population contained subpopulations with a mixture of genetic mutants. Changes were observed in nsp4, nsp9, nsp11/12, nsp14, nsp15, nsp16, and ORF3a, but these did not result in amino acid substitutions or did not show functional variations. Amino acid substitutions occurred in the remaining genes between P5 and P115; most were found in the S region, and some of the nucleotide mutations resulted in amino acid substitutions. Among the 9 nsps in the ORF1 region, nsp3 contained the most nucleotide substitutions. Conclusions: Sequence variations in different genes, especially the S gene and nsp3, in the genomes of CK/CH/LDL/97I viruses might contribute to differences in viral replication, pathogenicity, antigenicity, immunogenicity, and tissue tropism.

VSTAT120 REDUCES THE INNATE INFLAMMATORY RESPONSE TO INFECTION AND ENHANCES ONCOLYTIC VIRAL THERAPY FOR GLIOBLASTOMA

BACKGROUND: A major barrier to oncolytic virus (OV) efficacy for the treatment of Glioblastoma is the innate immune response; this response limits virus replication and reduces tumor cell destruction. Our lab developed a novel OV, RAMBO, which expresses the anti-angiogenic fragment Vasculostatin (Vstat120). In addition to its anti-angiogenic effects, we observe the RAMBO virus replicates more efficiently and persists longer in vivo than a control virus (rHSVQ1). The primary objective of this study is to investigate the effects RAMBO directed Vstat120 expression on microglia/macrophage activation and OV efficacy. METHODS: Mice bearing intracranial tumors were treated with RAMBO, rHSVQ1, or PBS, and flow cytometry utilized to examine monocyte/microglia activation/recruitment. Coculture of i9nfected glioma cells with microglia were utilized in in vitro experiemnts. Cytokines were analyzed via qPCR and ELISA. Virus replication was determined by plaque forming unit (PFU) assays. RESULTS: Treatment of intracranial tumors with OV resulted in significant monocyte infiltration into the tumor. Surprisingly, this infiltration was reduced 4.5 fold in tumors treated with the RAMBO virus as compared to rHSVQ1 (p < 0.05). Infiltrating monocytes from RAMBO treated tumors had significantly less MHCII, Ly6C, and CD86 than rHSVQ1 treated tumors (p < 0.05). The microglia of RAMBO treated tumors had significantly less MHCII and CD206 expression than those treated with rHSVQ1 (p< 0.05). To further examine the interaction of Vstat120 with microglia/monocytes we developed a coculture system with murine microglia and infected human glioma cells. Glioma cells were treated with PBS, rHSVQ1, or RAMBO and cocultured with microglia. We observed a 75-fold induction of TNF-α expression in microglia cultured with rHSVQ1 infected glioma cells compared to untreated microglia (p < 0.05). Interestingly, we observed a 3.4-fold reduction in microglia TNF-α expression in RAMBO treated cocultures compared to rHSVQ1 cocultures (p < 0.01). Consistent with this ELISA confirmed a 6.9 fold decrease in microglia TNF-α secretion (p < 0.01), and virus titers revealed a 2.6 fold increase in infectious virus in RAMBO Vs eHSVQ1 treated cocultures (p < 0.01). Cocultures treated with a microglia (murine) specific TNF-α blocking antibody rescued the differences in viral replication between rHSVQ1 and RAMBO virus titers. There was no difference in virus replication of glioma cells or microglia alone. These results suggest Vstat120 reduces inflammation and enhances OV replication in part through the suppression of TNF-α expression/secretion on microglia/monocytes. This transient suppression of the microglia/monocyte inflammatory response enhances OV replication and promotes anti-tumor efficacy. CONCLUSIONS: Vasculostatin affects macrophage recruitment and activity to improve viral replication and efficacy. SECONDARY CATEGORY: Immunobiology & Immunotherapy.

Integrated miRNA and mRNA transcriptomes of porcine alveolar macrophages (PAM cells) identifies strain-specific miRNA molecular signatures associated with H-PRRSV and N-PRRSV infection.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant viral diseases in swine, which causes large economic losses to the swine industry worldwide. There is considerable strain variation in PRRSV and two examples of this are the highly virulent Chinese-type PRRSV (H-PRRSV) and the classical North American type PRRSV (N-PRRSV), both with different pathogenesis. These differences may be due in part to genetic and phenotypic differences in virus replication, but also interaction with the host cell. MicroRNAs (miRNAs) are crucial regulators of gene expression and play vital roles in virus and host interactions. However, the regulation role of miRNAs during PRRSV infection has not been systematically investigated. In order to better understand the differential regulation roles of cellular miRNAs in the host response to PRRSV, miRNA expression and a global mRNA transcriptome profile was determined in primary cells infected with either H-PRRSV or N-PRRSV as multiple time points during the viral lifecycle. miRNA-mRNA interactome networks were constructed by integrating the differentially expressed miRNAs and inversely correlated target mRNAs. Using gene ontology and pathway enrichment analyses, cellular pathways associated with deregulated miRNAs were identified, including immune response, phagosome, autophagy, lysosome, autolysis, apoptosis and cell cycle regulation. To our knowledge, this is the first global analysis of strain-specific host miRNA molecular signatures associated with H- and N-PRRSV infection by integrating miRNA and mRNA transcriptomes and provides a new perspective on the contribution of miRNAs to the pathogenesis of PRRSV infection.

Correlation between Marek's disease virus pathotype and replication.

Marek's disease (MD) virus (MDV) is an alphaherpesvirus that causes MD, a lymphoproliferative disease in chickens. Pathotyping has become an increasingly important assay for monitoring shifts in virulence of field strains; however, it is time-consuming and expensive, and alternatives are needed to provide fast answers in the face of current outbreaks. The purpose of this study was to determine whether differences in virus replication between pathotypes that have been reported using a small number of virulent (v) and very virulent plus (vv+) MDV strains could be confirmed with a large collection of MD viruses. Based on pilot study data, bursa, brain, and lung samples were collected at 9 and 11 days postinoculation (dpi) from birds challenged with 1 of 15 MDV strains. The correlation between virus replication and virulence was confirmed between vMDV strains and higher virulent strains, but in most cases, there was no significant difference between very virulent (vv) and vv+MDV groups. At both 9 and 11 dpi, chickens infected with vv and vv+MDV had significantly lower body weights and relative thymus and bursa weights compared with chickens challenged with vMDV. However, similar to virus quantity, there was no significant difference between weights in birds challenged with vv or vv+MDV. The significant differences observed in maternal antibody negative (ab-) chickens were not significant in maternal antibody positive (ab+) chickens, demonstrating the requirement of ab- birds for this type of comparison. These data do not support the use of virus replication or organ weights as an alternative to pathotyping for discrimination between all three virulent MDV pathotypes but may be useful for determining a virus replication threshold to choose which field strains meet a minimum virulence to be pathotyped by traditional methods.

Differences in the Pathogenicity and Inflammatory Responses Induced by Avian Influenza A/H7N9 Virus Infection in BALB/c and C57BL/6 Mouse Models

Avian influenza A/H7N9 virus infection causes pneumonia in humans with a high case fatality rate. However, virus-induced modulation of immune responses is being recognized increasingly as a factor in the pathogenesis of this disease. In this study, we compared the pathogenicity of A/H7N9 infection in BALB/c and C57BL/6 mouse models, and investigated the putative involvement of proinflammatory cytokines in lung injury and viral clearance. In both mouse strains, A/Anhui/1/2013(H7N9) infection with 106 TCID50 resulted in viral replication in lung, severe body weight loss and acute lung injury. During the early infection stage, infected C57BL/6 mice exhibited more severe lung injury, slower recovery from lung damage, less effective viral clearance, higher levels of interlukine (IL)-6, monocyte chemotactic protein (MCP)-1, and IL-1β, and lower levels of tumor necrosis factor (TNF)-α and interferon (IFN)-γ than infected BALB/c mice. These results suggest that TNF-α and IFN-γ may help suppress viral gene expression and increase viral clearance, and that IL-6 and MCP-1 may contribute to lung injury in A/H7N9-infected individuals. In addition, lung damage and the distribution of virus antigen in tissues were similar in young and middle-aged mice. These results suggest that the more serious lung injury in middle-aged or older H7N9 cases is not mainly caused by differences in viral replication in the lung but probably by a dysregulated immune response induced by underlying comorbidities. These results indicate that the extent of dysregulation of the host immune response after H7N9 virus infection most probably determines the outcome of H7N9 virus infection.

Effects of rhinovirus species on viral replication and cytokine production

Epidemiologic studies provide evidence of differential virulence of rhinovirus species (RV). We recently reported that RV-A and RV-C induced more severe illnesses than RV-B, which suggests that the biology of RV-B might be different from RV-A or RV-C. To test the hypothesis that RV-B has lower replication and induces lesser cytokine responses than RV-A or RV-C. We cloned full-length cDNA of RV-A16, A36, B52, B72, C2, C15, and C41 from clinical samples and grew clinical isolates of RV-A7 and RV-B6 in cultured cells. Sinus epithelial cells were differentiated at the air-liquid interface. We tested for differences in viral replication in epithelial cells after infection with purified viruses (10(8) RNA copies) and measured virus load by quantitative RT-PCR. We measured lactate dehydrogenase (LDH) concentration as a marker of cellular cytotoxicity, and cytokine and/or chemokine secretion by multiplex ELISA. At 24 hours after infection, the virus load of RV-B (RV-B52, RV-B72, or RV-B6) in adherent cells was lower than that of RV-A or RV-C. The growth kinetics of infection indicated that RV-B types replicate more slowly. Furthermore, RV-B released less LDH than RV-A or RV-C, and induced lower levels of cytokines and chemokines such as CXCL10, even after correction for viral replication. RV-B replicates to lower levels also in primary bronchial epithelial cells. Our results indicate that RV-B types have lower and slower replication, and lower cellular cytotoxicity and cytokine and/or chemokine production compared with RV-A or RV-C. These characteristics may contribute to reduced severityof illnesses that has been observed with RV-B infections.

The number and genetic relatedness of transmitted/founder virus impact clinical outcome in vaginal R5 SHIVSF162P3N infection

BackgroundSevere genetic bottleneck occurs during HIV-1 sexual transmission whereby most infections are initiated by a single transmitted/founder (T/F) virus. Similar observations had been made in nonhuman primates exposed mucosally to SIV/SHIV. We previously reported variable clinical outcome in rhesus macaques inoculated intravaginally (ivg) with a high dose of R5 SHIVSF162P3N. Given the potential contributions of viral diversity to HIV-1 persistence and AIDS pathogenesis and recombination between retroviral genomes increases the genetic diversity, we tested the hypothesis that transmission of multiple variants contributes to heightened levels of virus replication and faster disease progression in the SHIVSF162P3N ivg-infected monkeys.ResultsWe found that the differences in viral replication and disease progression between the transiently viremic (TV; n = 2), chronically-infected (CP; n = 8) and rapid progressor (RP; n = 4) ivg-infected macaques cannot be explained by which variant in the inoculum was infecting the animal. Rather, transmission of a single variant was observed in both TV rhesus, with 1–2 T/F viruses found in the CPs and 2–4 in all four RP macaques. Moreover, the genetic relatedness of the T/F viruses in the CP monkeys with multivariant transmission was greater than that seen in the RPs. Biological characterization of a subset of T/F envelopes from chronic and rapid progressors revealed differences in their ability to mediate entry into monocyte-derived macrophages, with enhanced macrophage tropism observed in the former as compared to the latter.ConclusionOur study supports the tenet that sequence diversity of the infecting virus contributes to higher steady-state levels of HIV-1 virus replication and faster disease progression and highlights the role of macrophage tropism in HIV-1 transmission and persistence.

The differential interferon responses of two strains of Stat1-deficient mice do not alter susceptibility to HSV-1 and VSV in vivo

Stat1 is a pivotal transcription factor for generation of the interferon (IFN)-dependent antiviral response. Two Stat1 knockout mouse lines have been previously generated, one deleted the N-terminal domain (ΔNTD) and one in the DNA-binding domain (ΔDBD). These widely-used strains are assumed interchangeable, and both are highly susceptible to various pathogens. In this study, primary cells derived from ΔNTD mice were shown to be significantly more responsive to IFN, and established an antiviral state with greater efficiency than cells derived from ΔDBD mice, following infection with vesicular stomatitis virus and herpes simplex virus type-1. Also, while mice from both strains succumbed rapidly and equally to virus infection, ΔDBD mice supported significantly higher replication in brains and livers than ΔNTD mice. Endpoint-type experimental comparisons of these mouse strains are therefore misleading in failing to indicate important differences in virus replication and innate response.

Human Immunodeficiency Virus Type 1 Tat-Mediated Cellular Response in Myeloid Cells

Human immunodeficiency virus type 1 (HIV-1)-infected cells respond to the infection with different outcomes depending on their cell type. The interplay of cellular and viral proteins is a key player of differences in virus replication and disease progression. Myeloid cells, including monocytes, macrophages, and myeloid dendritic cells (mDCs) play a crucial role in the transmission and pathogenesis of HIV. The viral protein Tat, which is the viral transcriptional activator, modulates the expression of both HIV and cellular genes in these myeloid cells. This review will focus on recent advances on the interplay between HIV and myeloid cells and will discuss how this interaction may contribute to HIV pathogenesis. A better understanding of the pathogenesis of HIV disease will provide us with the scientific rationale for novel approaches to prevention.

Future Risk Assessments for the Impact of Climate Change on Vector-Borne Viruses of Livestock: Potential of Genomics, Transcriptomics and Proteomics Approaches – A Review

Climate and habitat change, together with globalisation, are important factors in the emergence of arthropodborne viruses (arboviruses). Changes in the range and abundance of the virus, vectors and hosts may create new combinations of host-pathogen-vector interactions for which data are not available. This paper reviews the genomics data currently available for arboviruses, their vectors and hosts, and assesses the level of understanding of the genetic factors affecting their adaptation to climate. It is anticipated here that genomics, transcriptomics and proteomics approaches may enable the breakdown of the traditional risk assessment pathways into the individual biochemical steps for each of the three interfaces between the virus, vector and host such that future risk assessments could be based on looking for certain gene combinations and their resulting expression profiles. Differences in the virus interaction with the arthropod midgut have been implicated in specifically affecting the extrinsic incubation period for some arboviruses, while differences in viral replication and dissemination through the arthropod may affect vector competence for other arboviruses. Genomics approaches to identify the proteins involved will enhance our understanding of vector competence and may explain why some arbovirus genotypes are more efficiently replicated in the vector at elevated temperatures. Such studies may also reflect the diversity present in real-world systems to a greater extent than experimental systems involving a single combination of vector and virus genotypes. Understanding the genetic basis for tissue tropism will facilitate prediction of new routes of transmission.

Vaccinia Virus-mediated Expression of Human Erythropoietin in Tumors Enhances Virotherapy and Alleviates Cancer-related Anemia in Mice

Recombinant human erythropoietin (rhEPO), a glycoprotein hormone regulating red blood cell (RBC) formation, is used for the treatment of cancer-related anemia. The effect of rhEPO on tumor growth, however, remains controversial. Here, we report the construction and characterization of the recombinant vaccinia virus (VACV) GLV-1h210, expressing hEPO. GLV-1h210 was shown to replicate in and kill A549 lung cancer cells in culture efficiently. In mice bearing A549 lung cancer xenografts, treatment with a single intravenous dose of GLV-1h210 resulted in tumor-specific production and secretion of functional hEPO, which exerted an effect on RBC progenitors and precursors in the mouse bone marrow, leading to a significant increase in the number of RBCs and in the level of hemoglobin. Furthermore, virally expressed hEPO, but not exogenously added rhEPO, enhanced virus-mediated green fluorescent protein (GFP) expression in tumors and subsequently accelerated tumor regression when compared with the treatment with the parental virus GLV-1h68 or GLV-1h209 that expressed a nonfunctional hEPO protein. Moreover, intratumorally expressed hEPO caused enlarged tumoral microvessels, likely facilitating virus spreading. Taken together, VACV-mediated intratumorally expressed hEPO not only enhanced oncolytic virotherapy but also simultaneously alleviated cancer-related anemia.

Foot-and-Mouth Disease Virus, Epithelial Cell Death and PDE Models.

Foot-and-mouth disease virus (FMDV) is a highly infectious anima lvirus that affects cloven-hoofed animals (including cattle, sheep and pigs).Because FMDV is a serious global socio-economic threat, it has been studied extensively for many decades. However, there are still several significant knowledge gaps in the pathogenesis of the disease. In particular, the predilection for certain epithelial tissues to develop vesicular lesions is currently unexplained. For example, epithelial cell lysis is extensive in the epithelial tissue of the bovine tongue, which results in the development of vesicular lesions. Nevertheless, the epithelium of the dorsal soft palate(DSP) does not show similar signs, even though it is a primary infectionsite of FMDV. The factors which inuence epithelial cell death and the development of lesions are the focus of this work, which is one of the few modelling studies on the within-host dynamics of FMDV.With the aim of identifying potential determinants of FMDV-induced epithelial cell lysis in cattle, a spatially explicit 1D PDE model was de-veloped to investigate the roles played by bovine epithelial thickness and cell layer structures. Numerical investigations demonstrated that these two factors alone do not explain the formation of lesions and, consequently,additional biological complexity is essential to explain the bifurcation inepithelial cell behaviour. Detailed exploration of parameter space offered an insight on the importance of potential differences in viral replication and receptor distribution between epithelia and between epithelial cell layers.The 1D epithelial tissue model has been subsequently expanded to a 3D structure, to facilitate the study of the size, as well as the occurrence of lesions, while greater biological realism has been added to the model byincorporating the antiviral activity of interferon to allow its eects on theFMDV dynamics in epithelium to be explored.