Laboratory Strains Of Virus Research Articles

The impact of age on vitamin D receptor expression, vitamin D metabolism and cytokine production in ex vivo Rhodococcus equi infection of equine alveolar macrophages

Rhodococcus equi (R. equi), a pneumonia-causing intracellular bacterium, results in significant morbidity and mortality in young foals, while healthy adult horses rarely develop disease. Survival and replication within alveolar macrophages (AMφ) are the hallmarks of R. equi’s pathogenicity. The vitamin D receptor (VDR) and its ligand, the active vitamin D metabolite 1,25(OH)2D, are important in immune responses to intracellular bacteria. The vitamin D/VDR pathway regulates the downstream production of cytokines in infected human AMφ. The immunomodulatory role of the vitamin D/VDR pathway in equine leukocytes is unknown. The objective of the current study was to determine the impact of R. equi infection and age on synthesis of 1,25(OH)2D, VDR expression, and cytokine production in an ex vivo model of R. equi infection in equine AMφ. AMφ were collected from ten healthy foals at 2-, 4- and 8-weeks old and from nine healthy adult horses once via bronchoalveolar lavage. AMφ were mock infected (CONTROL) or infected with a virulent laboratory strain of R. equi for 7 days (INFECTED). VDR expression was determined via RT-qPCR from cell lysates. 1,25(OH)2D and cytokines were measured in cell supernatant by immunoassays. VDR expression was impacted by age (P = 0.001) with higher expression in AMφ from 8-week-old foals than from 2-week-old foals and adults. There was no significant effect of infection in foal AMφ, but in adults, relative VDR expression was significantly lower in INFECTED AMφ compared to CONTROL AMφ (P = 0.002). There was no effect of age or infection on 1,25(OH)2D concentration (P > 0.37). Mean TNFα production was significantly higher from INFECTED compared to CONTROL AMφ from 4- and 8-week-old foals and adults (P < 0.005). Mean IFNγ production was significantly higher from AMφ from foals at 8-weeks-old compared to 2-weeks-old (P = 0.013) and higher from INFECTED AMφ than from CONTROL AMφ in foals at 4-weeks-old and in adults (P < 0.027). The proportion of samples producing IL-1β and IL-10 was also significantly higher from INFECTED compared to CONTROL AMφ isolated from 4-week-old foals (P < 0.008). Similarly, in adult samples, IL-17 was produced from a greater proportion of INFECTED compared to CONTROL samples (P = 0.031). These data document age-associated changes in VDR expression and cytokine production in equine AMφ in response to R. equi infection. This preliminary investigation supports the need for further research to fully elucidate if the vitamin D pathway has an immunomodulatory role in the horse.

Применение бисфосфонатов при экспериментальном туберкулезном остите: КТ-визуализация

Introduction The complex treatment of osteoarticular tuberculosis is based on combination of anti-tuberculosis therapy, complete (radical) removal of involved bone and restoration of the supporting and motor function of the affected musculoskeletal segment. Inhibited activity of osteoclasts involved in osteoresorption as one of the mechanisms of reparative osteoregeneration can be involved in regulation of bone formation after radical reconstructive surgery. The objective was to explore CT signs of osteoregeneration due to multimodal treatment of experimental tuberculous osteitis with use of bisphosphonates as targeted inhibitors of osteoclasts. Material and methods An experimental study was carried out on 21 mature male Chinchilla rabbits. The first stage included bone tuberculosis simulated in the medial condyle of the right femur using invasive local infection with M. tuberculosis strains H37Rv, a virulent reference laboratory strain. Pathological focus was resected and bone graft used at the second stage. Animals receiving antituberculosis therapy (ATT), ATT and bisphosphonates (BP) and BP only were divided into three groups at the third stage. Animals were sacrificed at 3 and 6 months of surgical treatment at the fourth stage. Autopsy implantation zone, bone of the contralateral condyle and intact femur were quantitatively and qualitatively assessed using micro-CT imaging. Results Positive dynamics in bone restoration was seen in the groups. ATT group showed complete lysis of the implant with bone cavities identified and no bone restoration in half of the cases seen at 6 months. Rabbits treated with BP demonstrated absence of complete lysis of the implant and CT signs of ingrowth of bone trabeculae. CT signs of maximum osteoregeneration were noted in the group of isolated BP therapy. Discussion The use of bisphosphonates can prevent lysis of grafts preserving the osteoconductive properties and facilitating formation of new bone. Conclusion Targeted osteoclast inhibitors can be safety and efficaciously used in the complex treatment of focal infectious skeletal lesions and be recommended as a potential component of pathogenetic therapy in the postoperative treatment of infectious (tuberculous) skeletal lesions.

Discrepancies in Infectivity of Flavivirus and SARS-CoV-2 Clinical Samples: An Improved Assay for Infectious Virus Shedding and Viremia Assessment.

Infectivity and neutralizing antibody titers of flavivirus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are frequently measured using the conventional plaque assay. While the assay is useful in the determination of infectivity, conventional plaque assays generally possess lower sensitivity and are time-consuming compared to nucleic acid amplification tests. In this study, a microcrystalline cellulose (MCC), Avicel, was evaluated as an alternative to the conventional virus overlay medium, methylcellulose, for a plaque assay. The plaque assay was performed using dengue and COVID-19 clinical samples and laboratory-established flavivirus and SARS-CoV-2 strains. In virus titration of clinical samples, the plaques were significantly larger, and the virus titers were higher when Avicel MCC-containing overlay medium was used than with conventional methylcellulose overlay medium. In addition, for some clinical samples and laboratory virus strains, infectious particles were detected as plaques in the Avicel MCC-containing medium, but not in the conventional methylcellulose medium. The results suggest that the viremia titer determined using the new overlay medium containing Avicel MCC may better reflect the innate infectious and plaque-forming capabilities of clinical samples and better reflect virus infectivity.

Synthesis of water-soluble prodrugs of 5-modified 2ʹ-deoxyuridines and their antibacterial activity

Recently we have synthesized a set of pyrimidine nucleoside derivatives bearing extended alkyltriazolylmethyl substituents at position 5 of the nucleic base, and showed their significant activity against Mycobacterium tuberculosis virulent laboratory strain H37Rv as well as drug-resistant MS-115 strain. The presence of a lengthy hydrophobic substituent leads to the reduction of nucleoside water solubility making their antibacterial activity troublesome to study. A series of water-soluble forms of 5-modified 2'-deoxyuridines 4a-c and 8a-c were synthesized. They appeared at least two orders more soluble compared with the parent compounds 1a and 1b. Their half-hydrolysis time was 5-12 h, which can be considered optimal for prodrugs used in clinics. Obtained compounds showed moderate activity (MIC 48-95 µg·ml-1) against some Gram-positive bacteria including resistant strains of Staphylococcus aureus and Mycobacterium smegmatis and were low cytotoxic for human cell lines (CD50 >> 100 µg·ml-1).

What a Difference a Gene Makes: Identification of Virulence Factors of Cowpox Virus.

Cowpox virus (CPXV) is a zoonotic orthopoxvirus (OPV) that causes spillover infections from its animal hosts to humans. In 2009, several human CPXV cases occurred through transmission from pet rats. An isolate from a diseased rat, RatPox09, exhibited significantly increased virulence in Wistar rats and caused high mortality compared to that caused by the mildly virulent laboratory strain Brighton Red (BR). The RatPox09 genome encodes four genes which are absent in the BR genome. We hypothesized that their gene products could be major factors influencing the high virulence of RatPox09. To address this hypothesis, we employed several BR-RatPox09 chimeric viruses. Using Red-mediated mutagenesis, we generated BR-based knock-in mutants with single or multiple insertions of the respective RatPox09 genes. High-throughput sequencing was used to verify the genomic integrity of all recombinant viruses, and transcriptomic analyses confirmed that the expression profiles of the genes that were adjacent to the modified ones were unaltered. While the in vitro growth kinetics were comparable to those of BR and RatPox09, we discovered that a knock-in BR mutant containing the four RatPox09-specific genes was as virulent as the RatPox09 isolate, causing death in over 75% of infected Wistar rats. Unexpectedly, the insertion of gCPXV0030 (g7tGP) alone into the BR genome resulted in significantly higher clinical scores and lower survival rates matching the rate for rats infected with RatPox09. The insertion of gCPXV0284, encoding the BTB (broad-complex, tramtrack, and bric-à-brac) domain protein D7L, also increased the virulence of BR, while the other two open reading frames failed to rescue virulence independently. In summary, our results confirmed our hypothesis that a relatively small set of four genes can contribute significantly to CPXV virulence in the natural rat animal model.IMPORTANCE With the cessation of vaccination against smallpox and its assumed cross-protectivity against other OPV infections, waning immunity could open up new niches for related poxviruses. Therefore, the identification of virulence mechanisms in CPXV is of general interest. Here, we aimed to identify virulence markers in an experimental rodent CPXV infection model using bacterial artificial chromosome (BAC)-based virus recombineering. We focused our work on the recent zoonotic CPXV isolate RatPox09, which is highly pathogenic in Wistar rats, unlike the avirulent BR reference strain. In several animal studies, we were able to identify a novel set of CPXV virulence genes. Two of the identified virulence genes, encoding a putative BTB/POZ protein (CPXVD7L) and a B22R-family protein (CPXV7tGP), respectively, have not yet been described to be involved in CPXV virulence. Our results also show that single genes can significantly affect virulence, thus facilitating adaptation to other hosts.

Prevalence of Infection-Competent Serogroup 6 Legionella pneumophila within Premise Plumbing in Southeast Michigan.

ABSTRACTCoinciding with major changes to its municipal water system, Flint, MI, endured Legionnaires’ disease outbreaks in 2014 and 2015. By sampling premise plumbing in Flint in the fall of 2016, we found that 12% of homes harbored legionellae, a frequency similar to that in residences in neighboring areas. To evaluate the genetic diversity of Legionella pneumophila in Southeast Michigan, we determined the sequence type (ST) and serogroup (SG) of the 18 residential isolates from Flint and Detroit, MI, and the 33 clinical isolates submitted by hospitals in three area counties in 2013 to 2016. Common to one environmental and four clinical samples were strains of L. pneumophila SG1 and ST1, the most prevalent ST worldwide. Among the Flint premise plumbing isolates, 14 of 16 strains were of ST367 and ST461, two closely related SG6 strain types isolated previously from patients and corresponding environmental samples. Each of the representative SG1 clinical strains and SG6 environmental isolates from Southeast Michigan infected and survived within macrophage cultures at least as well as a virulent laboratory strain, as judged by microscopy and by enumerating CFU. Likewise, 72 h after infection, the yield of viable-cell counts increased >100-fold for each of the representative SG1 clinical isolates, Flint premise plumbing SG6 ST367 and -461 isolates, and two Detroit residential isolates. We verified by immunostaining that SG1-specific antibody does not cross-react with the SG6 L. pneumophila environmental strains. Because the widely used urinary antigen diagnostic test does not readily detect non-SG1 L. pneumophila, Legionnaires’ disease caused by SG6 L. pneumophila is likely underreported worldwide.

Isolation and Characterization of Antimycobacterial Compounds from Fruits of Aegle marmelos (L.) Correa

Root extracts of Aegle marmelos, an evergreen tree indigenous to India, is an important ingredient in Ayurvedic preparations used for the treatment of pulmonary tuberculosis. Leaves of the tree are used as a remedy for respiratory ailments in folk medicine. The fruit is extensively used in the treatment of various bacterial diseases such as dysentery, diarrhea, etc. The present study was designed to screen organic solvent extracts of fruits of A. Marmelos for their inhibitory activity on Mycobacterium tuberculosis (M. tuberculosis), and to isolate and identify the compounds responsible for antimycobacterial activity. The minimum inhibitory concentration (MIC) of the extract and purified molecules on virulent laboratory strain M. tuberculosis H37Rv was determined by resazurin microtiter assay (REMA). Structural elucidation of the compounds was carried out employing NMR. Cytotoxicity of the compounds was evaluated against human monocyte THP1-derived macrophages by MTT assay. The hexane extract of A. marmelos fruits inhibited the growth of M. tuberculosis at an MIC of 50 μg/mL. Fractionation of the hexane extract by column chromatography led to the isolation of imperatorin, β-sitosterol, plumbagin, marmesin, marmin, and stigmasterol which individually inhibited the growth of M. tuberculosis. In vitro cytotoxicity assay revealed that the compounds are not cytotoxic at the concentrations that inhibited the growth of M. tuberculosis.

Synthesis and in vitro evaluation of substituted 3-cinnamoyl-4-hydroxy-pyran-2-one (CHP) in pursuit of new potential antituberculosis agents.

Tuberculosis is an ever-evolving infectious disease that urgently needs new drugs. In the search for new antituberculosis agents, a library of 3-cinnamoyl-4-hydroxy-6-methyl-2H-pyran-2-ones (CHPs) (2a-2y) was synthesized and evaluated against a standard virulent laboratory strain of Mycobacterium tuberculosis H37Rv. Out of 25 compounds, 11, 5, 7 and 2 (2a and 2u) showed least, moderate, good and appreciable activities, respectively, based on minimum inhibitory concentrations (MICs). Both 2a and 2u exhibited an MIC value of 4 μg ml-1, which was close to those of standard antituberculosis drugs ethambutol, streptomycin and levofloxacin. Neither 2a nor 2u showed any activity against Gram-positive or Gram-negative bacteria and even against non-tuberculous mycobacterium, i.e. Mycobacterium smegmatis. Thus, like the antituberculosis drugs rifampicin, isoniazid and pretomanid, they are highly TB specific. All the pyrone-based chalcones showed no recognizable level of cytotoxicity against normal human kidney cell line (HEK-293) up to 80 μM concentration and 11 exhibited an IC50 ≤ 100 μM (highest tested concentration). On further investigation, both 2a and 2u proved to be nontoxic against four human cell lines but 2a proved to be a better choice as it did not reach IC50 even at 100 μM (highest tested concentration) while the IC50 of 2u was around 80 μM. In conclusion, our results demonstrate that 2a is specific against M. tuberculosis with no appreciable toxicity; its activity matches that of some clinically approved antituberculosis drugs and it therefore merits further evaluation.

Identification of pathogenic factors potentially involved in Staphylococcus aureus keratitis using proteomics

Staphylococcus is a leading cause of microbial keratitis, characterized by destruction of the cornea by bacterial exoproteins and host-associated factors. The aim of this study was to compare extracellular and cell-associated proteins produced by two different isolates of S. aureus, a virulent clinical isolate (Staph 38) and a laboratory strain (Staphylococcus aureus 8325-4) of weaker virulence in the mouse keratitis model. Proteins were analyzed using 2D polyacrylamide gel electrophoresis and identified by subsequent mass spectrometry. Activity of staphylococcal adhesins was assessed by allowing strains to bind to various proteins adsorbed onto polymethylmethacrylate squares. Thirteen proteins in the extracellular fraction and eight proteins in the cell-associated fractions after bacterial growth were produced in increased amounts in the clinical isolate Staph 38. Four of these proteins were S. aureus virulence factor adhesins, fibronectin binding protein A, staphopain, glyceraldehyde-3-phosphate dehydrogenase 2 and extracellular adherence protein. The clinical isolate Staph 38 adhered to a greater extent to all mammalian proteins tested, indicating the potential of the adhesins to be active on its surface. Other proteins with increased expression in Staph 38 included potential moonlighting proteins and proteins involved in transcription or translation. This is the first demonstration of the proteome of S. aureus isolates from keratitis. These results indicate that the virulent clinical isolate produces more potentially important virulence factors compared to the less virulent laboratory strain and these may be associated with the ability of a S. aureus strain to cause more severe keratitis.

Emergence of a Large-Plaque Variant in Mice Infected with Coxsackievirus B3.

ABSTRACTCoxsackieviruses are enteric viruses that frequently infect humans. To examine coxsackievirus pathogenesis, we orally inoculated mice with the coxsackievirus B3 (CVB3) Nancy strain. Using HeLa cell plaque assays with agar overlays, we noticed that some fecal viruses generated plaques >100 times as large as inoculum viruses. These large-plaque variants emerged following viral replication in several different tissues. We identified a single amino acid change, N63Y, in the VP3 capsid protein that was sufficient to confer the large-plaque phenotype. Wild-type CVB3 and N63Y mutant CVB3 had similar plaque sizes when agarose was used in the overlay instead of agar. We determined that sulfated glycans in agar inhibited plaque formation by wild-type CVB3 but not by N63Y mutant CVB3. Furthermore, N63Y mutant CVB3 bound heparin, a sulfated glycan, less efficiently than wild-type CVB3 did. While N63Y mutant CVB3 had a growth defect in cultured cells and reduced attachment, it had enhanced replication and pathogenesis in mice. Infection with N63Y mutant CVB3 induced more severe hepatic damage than infection with wild-type CVB3, likely because N63Y mutant CVB3 disseminates more efficiently to the liver. Our data reinforce the idea that culture-adapted laboratory virus strains can have reduced fitness in vivo. N63Y mutant CVB3 may be useful as a platform to understand viral adaptation and pathogenesis in animal studies.

Extent of Systemic Spread Determines CD8+ T Cell Immunodominance for Laboratory Strains, Smallpox Vaccines, and Zoonotic Isolates of Vaccinia Virus.

CD8(+) T cells that recognize virus-derived peptides presented on MHC class I are vital antiviral effectors. Such peptides presented by any given virus vary greatly in immunogenicity, allowing them to be ranked in an immunodominance hierarchy. However, the full range of parameters that determine immunodominance and the underlying mechanisms remain unknown. In this study, we show across a range of vaccinia virus strains, including the current clonal smallpox vaccine, that the ability of a strain to spread systemically correlated with reduced immunodominance. Reduction in immunodominance was observed both in the lymphoid system and at the primary site of infection. Mechanistically, reduced immunodominance was associated with more robust priming and especially priming in the spleen. Finally, we show this is not just a property of vaccine and laboratory strains of virus, because an association between virulence and immunodominance was also observed in isolates from an outbreak of zoonotic vaccinia virus that occurred in Brazil.

Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation

Tuberculosis, caused by Mycobacterium tuberculosis, still remains a major global health problem. The main obstacle in eradicating this disease is the ability of this pathogen to remain dormant in macrophages, and then reactivate later under immuno-compromised conditions. The physiology of hypoxic nonreplicating M. tuberculosis is well-studied using many in vitro dormancy models. However, the physiological changes that take place during the shift from dormancy to aerobic growth (reactivation) have rarely been subjected to a detailed investigation. In this study, we developed an in vitro reactivation system by re-aerating the virulent laboratory strain of M. tuberculosis that was made dormant employing Wayne's dormancy model, and compared the proteome profiles of dormant and reactivated bacteria using label-free one-dimensional LC/MS/MS analysis. The proteome of dormant bacteria was analyzed at nonreplicating persistent stage 1 (NRP1) and stage 2 (NRP2), whereas that of reactivated bacteria was analyzed at 6 and 24 h post re-aeration. Proteome of normoxially grown bacteria served as the reference. In total, 1871 proteins comprising 47% of the M. tuberculosis proteome were identified, and many of them were observed to be expressed differentially or uniquely during dormancy and reactivation. The number of proteins detected at different stages of dormancy (764 at NRP1, 691 at NRP2) and reactivation (768 at R6 and 983 at R24) was very low compared with that of the control (1663). The number of unique proteins identified during normoxia, NRP1, NRP2, R6, and R24 were 597, 66, 56, 73, and 94, respectively. We analyzed various biological functions during these conditions. Fluctuation in the relative quantities of proteins involved in energy metabolism during dormancy and reactivation was the most significant observation we made in this study. Proteins that are up-regulated or uniquely expressed during reactivation from dormancy offer to be attractive targets for therapeutic intervention to prevent reactivation of latent tuberculosis.

PATHOGENESIS AND IMMUNE RESPONSES OF FRANCISELLA TULARENSIS STRAINS IN WILD-CAUGHT COTTONTAIL RABBITS (SYLVILAGUS SPP.).

Francisella tularensis is a highly virulent, zoonotic bacterium that causes significant natural disease and is of concern as an organism for bioterrorism. Serologic testing of wildlife is frequently used to monitor spatial patterns of infection and to quantify exposure. Cottontail rabbits (Sylvilagus spp.) are a natural reservoir for F. tularensis in the US, although very little work has been done experimentally to determine how these animals respond to infection; thus, information gathered from field samples can be difficult to interpret. We characterized clinical disease, bacteremia, pathology, and antibody kinetics of North American cottontail rabbits experimentally infected with five strains of F. tularensis. Rabbits were infected with four field strains, including MA00-2987 (type A1b), WY96-3418 (type A2), KY99-3387, and OR96-0246 (type B), and with SchuS4 (type A1a), a widely used, virulent laboratory strain. Infection with the different strains of the bacterium resulted in varied patterns of clinical disease, gross pathology, and histopathology. Each of the type A strains were highly virulent, with rabbits succumbing to infection 3-13 d after infection. At necropsy, numerous microabscesses were observed in the livers and spleens of most rabbits, associated with high bacterial organ burdens. In contrast, most rabbits infected with type B strains developed mild fever and became lethargic, but the disease was infrequently lethal. Those rabbits infected with type B strains that survived past 14 d developed a robust humoral immune response, and F. tularensis was not isolated from liver, spleen, or lung of those animals. Understanding F. tularensis infection in a natural reservoir species can guide serosurveillance and generate new insights into environmental maintenance of this pathogen.

A conservative amino acid mutation in the master regulator FleQ renders Pseudomonas aeruginosa aflagellate.

Flagellar-based motility plays a critical role in Pseudomonas aeruginosa pathogenesis, influencing both the establishment of bacterial infection and the host's response to the pathogen. Nonetheless, aflagellate clinical strains are often isolated from acutely and chronically infected patients and include the virulent laboratory strain PA103. We determined that PA103's aflagellate phenotype is the result of a single amino acid change (G240V) in the master flagellar regulator, FleQ. This mutation, which lies just outside the Walker B box of FleQ, abrogates the ability of FleQ to positively regulate flagellar gene expression. Reversal of this seemingly conservative amino acid substitution is sufficient to restore swimming motility to PA103, despite the presence of mutations in other flagellar genes of PA103. We also investigated the consequences of restoring flagellar assembly on PA103 virulence. Although a negative correlation between flagellar assembly and Type 3 secretion system (T3SS) expression has been reported previously, we did not observe downregulation of T3SS expression or function in Fla+ PA103. Restoration of flagellar assembly did, however, amplify IL-1 signals measured during murine pulmonary infection and was associated with increased bacterial clearance. These experiments suggest that loss of flagellar motility may primarily benefit PA103 by attenuating pathogen recognition and clearance during acute infection.

Low Levels of Antibody-Dependent Enhancement in Vitro Using Viruses and Plasma from Dengue Patients

BackgroundThe majority of dengue patients infected with any serotype of dengue virus (DENV) are asymptomatic, but the remainder may develop a wide spectrum of clinical symptoms, ranging from mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF). Severe cases occur more often in patients who experience a secondary infection with a different virus serotype. A phenomenon called antibody-dependent enhancement (ADE) has been proposed to explain the onset of these severe cases, but the exact mechanism of ADE remains unclear.Methodology/Principal FindingVirus neutralization and ADE assays were performed using ultracentrifugation supernatants of acute-phase sera from patients with secondary infections or human monoclonal antibodies (HuMAbs) as anti-DENV antibodies. Virus sources included infectious serum-derived viruses from the ultracentrifugation precipitates, laboratory-culture adapted DENV, or recombinant DENVs derived from patient sera. In contrast to the high levels of ADE observed with laboratory virus strains, low ADE was observed with autologous patient-derived viruses, when patient sera were used to provide the antibody component in the ADE assays. Similar results were obtained using samples from DF and DHF patients. Recombinant-viruses derived from DHF patients showed only minor differences in neutralization and ADE activity in the presence of HuMAbs or plasma derived from the same DHF patient.Conclusion/SignificanceSerum or plasma taken from patients during the acute phase of a secondary infection showed high levels of ADE, but no neutralization activity, when assayed in the presence of laboratory-adapted virus strains. By contrast, serum or plasma from the same patient showed high levels of neutralization activity but failed to induce significant ADE when the assays were performed with autologous virus. These results demonstrate the significance of the virus source when measuring ADE. They also suggest that repeated passage of DENV in cell culture has endowed it with the capacity to induce high levels of ADE.

Strikingly poor CD8+ T‐cell immunogenicity of vaccinia virus strain MVA in BALB/c mice

Vaccinia virus (VACV) strain MVA is a highly attenuated vector for vaccines that is being explored in clinical trials. We compared the CD8+ T cell immunogenicity of MVA with that of a virulent laboratory strain of VACV (strain WR) in BALB/c mice by examining epitope-specific responses as well as estimating the total number of activated CD8+ T cells, irrespective of specificity. We found that MVA elicited total CD8+ T cell responses that were reduced by at least 20-fold compared with strain WR in BALB/c mice. In C57Bl/6 mice we also found a substantial difference in immunogenicity between these VACV strains, but it was more modest at around 5-fold. Of note, the size of responses to the virulent WR virus were similar in both strains of mice suggesting that BALB/c mice can mount robust CD8+ T cell responses to VACV. While the data for total responses clearly showed that MVA overall is poorly immunogenic in BALB/c mice, we found one epitope for which strong responses were made irrespective of virus strain. Therefore in the context of a vaccine, some recombinant epitopes may have similar immunogenicity when expressed from MVA and other strains of VACV, but we would expect these to be exceptions. These data show clearly the substantial difference in immunogenicity between MVA and virulent VACV strains and suggest that the impact of host genetics on responses to attenuated vaccine vectors like MVA requires more consideration.

Protection by a broadly neutralizing monoclonal antibody to pandemic Influenza requires selective Fc receptor engagement (P4216)

Abstract Neutralizing Abs against influenza viruses have traditionally been thought to provide protection by interfering with virus entry and/or fusion with host cells, events that rely solely on Ab variable region binding to viral spike proteins. However, the contributions of in vivo mechanisms conferred by Fc-Fc receptor (FcγR) interactions during protection by Ab remain unclear. We assessed the role of FcγRs during influenza virus neutralization in mice using mAbs directed to the HA antigen. We found that a broadly neutralizing mAb (bNAb) targeting the conserved stem region of HA confers protection to mice from lethal viral challenge, and required Fc-FcγR interactions for in vivo activity against both laboratory virus strains as well as the 2009 pandemic H1N1 virus. By contrast, a strain-specific mAb to the variable head domain of HA was equally protective both in the presence or absence of FcγR interactions. We then generated a chimeric human IgG1 Fc version of the anti-stem bNAb, modified to selectively enhance human activating FcγR engagement. We utilized FcγR-humanized mice, which express all human FcγRs on a genetic background lacking mouse FcγRs, to demonstrate augmented protection by the modified bNAb compared to wild type bNAb. Thus, Fc-FcγR interactions are required for bNAb-mediated protection and can be manipulated for optimal protection in vivo. These findings represent a general approach towards enhancing bNAb-mediated protection and warrant clinical investigation.

Mice lacking tissue factor in myeloid cells are susceptible to Mycobacterium tuberculosis infection (P1243)

Abstract Tissue Factor (TF) is a trans membrane glycoprotein important for initiation of the coagulation cascade. Recent observations indicate that TF is expressed in cancer, metabolic diseases and infections. In the current study, we determined the role of TF in M. tuberculosis (M. tb) infection using mice lacking the TF gene in myeloid cells (Cre+). One month after infection with the M. tb virulent laboratory strain, H37Rv, the lung bacterial burden was higher in Cre+ than Cre- mice (3.7 ± 0.2 X 106 vs 1.9 ± 0.1 X 106 CFU, p=0.01). M.tb infected Cre+ mice had more CD4+ and CD8+ lymphocytes and expressed 33 and 38 fold less of IFN-γ and IL-17 mRNA in the lungs, compared to Cre- mice, as measured by real time PCR. In contrast, lung IL-10 mRNA levels were higher in M. tb-infected Cre+ mice than Cre- mice (16.04 ± 3.3 vs 4.4 ± 1 fold, p=0.03). Immunohistochemical analysis indicated that M. tb-infected Cre+ mice had more foamy macrophages in the lungs than Cre- mice. M. tbH37Rv grew more rapidly in peritoneal exudate macrophages of Cre+ mice, compared to Cre- macrophages (6.1 ± 1 X 106 vs 1.5 ± 0.6 X 106 CFU, p=0.02). Further studies indicated that M. tb-infected Cre+ macrophages undergo less mitochondrial apoptosis than Cre- macrophages (16.2 ± 2.1vs 27.9 ± 1.4 %, p=0.01). Studies are underway to determine the mechanism/s involved in increased IL-10 production, reduced mitochondrial apoptosis and increased bacterial burden of M tb infected Cre+ mice.

High-content screening to distinguish between attachment and post-attachment steps of human cytomegalovirus entry into fibroblasts and epithelial cells

Human cytomegalovirus (HCMV) enters cells through a complex pathway involving the interaction of multiple viral glycoproteins and cellular receptors. While HCMV clinical isolates enter a wide range of cell types, entry has historically been studied using a laboratory strain of virus that can only infect fibroblasts. Herein, we have constructed a HCMV reporter strain that contains GFP fused to the abundant tegument protein pp65 to allow for the direct visualization of virus attachment and entry. Furthermore, the UL131 gene of this strain was restored to clinical isolate sequence to expand our studies of entry into physiologically relevant epithelial cell types. Using the HCMV-GFP reporter virus, we developed an image-based assay and screened a library containing 65,000 compounds for the inhibition of virus entry into fibroblasts. In addition to assessing the effect on virus entry, automated image analysis provided information on compound toxicity and whether the compounds acted as attachment or post-attachment inhibitors. To identify therapeutically viable inhibitors capable of blocking entry in multiple cell types, the inhibitors were screened further for their ability to inhibit virus entry into epithelial cells. Compounds were identified that were able to inhibit virus entry into both cell types at either attachment or post-attachment steps.

The current status and future directions of myxoma virus, a master in immune evasion

Myxoma virus (MYXV) gained importance throughout the twentieth century because of the use of the highly virulent Standard Laboratory Strain (SLS) by the Australian government in the attempt to control the feral Australian population of Oryctolagus cuniculus (European rabbit) and the subsequent illegal release of MYXV in Europe. In the European rabbit, MYXV causes a disease with an exceedingly high mortality rate, named myxomatosis, which is passively transmitted by biting arthropod vectors. MYXV still has a great impact on European rabbit populations around the world. In contrast, only a single cutaneous lesion, restricted to the point of inoculation, is seen in its natural long-term host, the South-American Sylvilagus brasiliensis and the North-American S. Bachmani. Apart from being detrimental for European rabbits, however, MYXV has also become of interest in human medicine in the last two decades for two reasons. Firstly, due to the strong immune suppressing effects of certain MYXV proteins, several secreted virus-encoded immunomodulators (e.g. Serp-1) are being developed to treat systemic inflammatory syndromes such as cardiovascular disease in humans. Secondly, due to the inherent ability of MYXV to infect a broad spectrum of human cancer cells, the live virus is also being developed as an oncolytic virotherapeutic to treat human cancer. In this review, an update will be given on the current status of MYXV in rabbits as well as its potential in human medicine in the twenty-first century.Table of contents1. The virus2. History3. Pathogenesis and disease symptoms4. Immunomodulatory proteins of MYXV4.1. MYXV proteins with anti-apoptotic functions4.1.1. Inhibition of pro-apoptotic molecules4.1.2. Inhibition by protein-protein interactions by ankyrin repeat viral proteins4.1.3. Inhibition of apoptosis by enhancing the degradation of cellular proteins4.1.4. Inhibition of apoptosis by blocking host Protein Kinase R (PKR)4.2. MYXV proteins interfering with leukocyte chemotaxis4.3. MYXV serpins that inhibit cellular pro-inflammatory or pro-apoptotic proteases4.4. MYXV proteins that interfere with leukocyte activation4.5. MYXV proteins with sequence similarity to HIV proteins4.6. MYXV proteins with unknown immune function5. Vaccination strategies against myxomatosis5.1. Current MYXV vaccines5.2. Vaccination campaigns to protect European rabbits in the wild6. Applications of myxoma virus for human medicine6.1. MYXV proteins as therapeutics for allograft vasculopathy and atherosclerosis6.2. Applications for MYXV as a live oncolytic virus to treat cancer7. Discussion and Conclusions8. List of AbbreviationsReferencesAuthor DetailsAuthors' contributionsCompeting interestsFigure LegendsAcknowledgements