Efficiency of Commercial Air Filters Against Marek''s Disease Virus

In a series of 10 replicate trials with susceptible chickens as indicators of the presence of virus in the air, filter B(1) (Dri Pak, American Air Filter Co.), which has a dust spot rating of 93 to 97%, and filter C (Astrocel), with a dioctyl phathalate aerosol rating of over 99%, completely removed Marek's disease virus (MDV) from highly contaminated air. However, air passed through filter B(2) (Duracel), with a dust spot rating of 34 to 45%, remained infectious. Thus, an air filter with a dust spot rating of over 93% will effectively remove MDV from the air. The smallest infectious particles in the air are estimated to be greater than 0.3 mum in diameter.     

Fluorescently tagged pUL47 of Marek's disease virus reveals differential tissue expression of the tegument protein in vivo

Marek''s disease virus (MDV), a lymphotropic alphaherpesvirus, causes Marek''s disease (MD) in chickens. MD is characterized by neurological signs, chronic wasting, and T cell lymphomas that predominate in the visceral organs. MDV replicates in a highly cell-associated manner in vitro and in vivo, with infectious virus particles being released only from feather follicle epithelial (FFE) cells in the skin. Virus produced and shed from FFE cells allows transmission of MDV from infected to naïve chickens, but the mechanisms or roles of differential virus gene expression have remained elusive. Here, we generated recombinant MDV in which we fused enhanced green fluorescent protein (EGFP) to the C terminus of the tegument protein pUL47 (vUL47-EGFP) or pUL49 (vUL49-EGFP). While vUL49-EGFP was highly attenuated in vitro and in vivo, vUL47-EGFP showed unaltered pathogenic potential and stable production of pUL47-EGFP, which facilitated direct analysis of pUL47 expression in cells and tissues. Our studies revealed that pUL47-EGFP is expressed at low levels and localizes to the nucleus during lytic replication in vitro and in lymphocytes in the spleen in vivo, while it is undetectable in tumors. In contrast, pUL47-EGFP is highly abundant and localizes predominantly in the cytoplasm in FFE cells in the skin, where MDV is shed into the environment. We concluded that differential expression and localization of MDV pUL47-EGFP tegument protein is potentially important for the unique cell-associated nature of MDV in vitro and in lymphocytes in vivo, as well as production of free virus in FFE cells.     

A Virulent Bioluminescent and Fluorescent Dual-Reporter Marek's Disease Virus Unveils an Alternative Spreading Pathway in Addition to Cell-to-Cell Contact

Marek''s disease virus (MDV) is a growing threat for the poultry industry. Unfortunately, despite successful vaccination against the disease, MDV remains in circulation within vaccinated flocks, leading to the selection of increasingly virulent pathotypes. Detailed knowledge of the virus biology and the host-virus interaction is required to improve the vaccine efficiency. In the present study, I engineered an original, dual-reporter MDV to track and quantify virus replication in vitro and in vivo.     

Association between theRfp-Y haplotype and the incidence of Marek's disease in chickens

 Certain haplotypes at the major histocompatibility (B) complex (Mhc) of the chicken provide an easily demonstrated influence on tumor formation following infections with Marek’s disease virus (MDV). Recognition that there is a second histocompatibility complex of genes in the chicken, Rfp-Y, comprised of Mhc class I and class II genes, some of which are at least transcribed, evokes the question of whether this gene complex might also influence the outcome of MDV infections. To test this hypothesis, pedigree-hatched chicks in families from the original Rfp-Y-defining stock in which three Rfp-Y and two B system haplotypes are segregating were challenged with the RB1B strain of MDV. Birds with the Y ³ /Y ³ genotype were found to have 2.3 times the risk of developing a tumor compared with birds with other Rfp-Y genotypes combined (P <0.02). Additionally, birds carrying the B ^R9 /B ¹¹ genotype had 2.3 times the risk of tumor formation, relative to birds with the B ¹¹ /B ¹¹ genotype (P <0.02). We found no evidence for an interaction between genotypes within the B and Rfp-Y systems. These data provide evidence that Rfp-Y haplotypes, as well as B haplotypes, can significantly influence the outcome of infection with MDV. Received: 15 February 1996 / Revised: 23 April 1996     

Use of Centrifugal Filter Devices to Concentrate Dengue Virus in Mosquito per os Infection Experiments

Dengue virus (DENV) is an arbovirus transmitted to humans by the bite of infected Aedes mosquitoes. Experimental per os infection of mosquitoes with DENV is usually a preliminary step in virus/vector studies but it requires being able to prepare artificial blood-meals with high virus titers. We report here the convenient use of centrifugal filter devices to quickly concentrate DENV particles in cell-culture supernatants. The median viral titer in concentrated-supernatants was 8.50 log₁₀ TCID₅₀/mL. By using these DENV concentrated-supernatants to prepare infectious blood-meals in Aedes aegypti per os infection experiments, we obtained a mean mosquito-infection rate of 94%. We also evaluated the use of centrifugal filter devices to recover DENV particles from non-infectious blood-meals presented to infected mosquitoes through a feeding membrane to collect their saliva.     

Some properties of grapevine Bulgarian latent virus

Grapevine Bulgarian latent virus (GBLV), obtained from symptomless vines grown in Bulgaria, was readily transmitted by inoculation of sap to a restricted range of hosts. The virus was re-inoculated into virus-free rooted cuttings and seedlings of several grapevine cultivars without inducing symptoms. Purified preparations of GBLV contained isometric particles c. 30 nm in diameter which sedimented as three components at 52 , c. 120 and 1275, respectively. The slow-sedimenting component (T) contained non-infective protein shells, whereas the two heavier classes of particles (J5_X, B₂) each contained one molecule of single-stranded ribonucleic acid (RNA) with mol. wts of 2.1 and 2.2 times 10⁶ daltons. The RNA content of B₁ and B₂ components were 39 and 41 % by particle weight respectively. The capsid was composed of one type of protein with a mol. wt of 54000. GBLV did not react with antisera to any of twenty-eight viruses with isometric particles. Its present cryptogram is R/1: 2-2/41 +2-1/39:S/S:S/*.     

Simultaneous detection of eight immunosuppressive chicken viruses using a GeXP analyser-based multiplex PCR assay

Background

Immunosuppressive viruses are frequently found as co-infections in the chicken industry, potentially causing serious economic losses. Because traditional molecular biology methods have limited detection ability, a rapid, high-throughput method for the differential diagnosis of these viruses is needed. The objective of this study is to develop a GenomeLab Gene Expression Profiler Analyser-based multiplex PCR method (GeXP-multiplex PCR) for simultaneous detection of eight immunosuppressive chicken viruses.

Results

Using chimeric primers, eight such viruses, including Marek's disease virus (MDV), three subgroups of avian leucosis virus (ALV-A/B/J), reticuloendotheliosis virus (REV), infectious bursal disease virus (IBDV), chicken infectious anaemia virus (CIAV) and avian reovirus (ARV), were amplified and identified by their respective amplicon sizes. The specificity and sensitivity of the optimised GeXP-multiplex PCR assay were evaluated, and the data demonstrated that this technique could selectively amplify these eight viruses at a sensitivity of 100 copies/20 μl when all eight viruses were present. Among 300 examined clinical specimens, 190 were found to be positive for immunosuppressive viruses according to this novel assay.

Conclusion

The GeXP-multiplex PCR assay is a high-throughput, sensitive and specific method for the detection of eight immunosuppressive viruses and can be used for differential diagnosis and molecular epidemiological surveys.

     

Avirulent Marek’s Disease Virus Type 1 Strain 814 Vectored Vaccine Expressing Avian Influenza (AI) Virus H5 Haemagglutinin Induced Better Protection Than Turkey Herpesvirus Vectored AI Vaccine

Background

Herpesvirus of turkey (HVT) as a vector to express the haemagglutinin (HA) of avian influenza virus (AIV) H5 was developed and its protection against lethal Marek’s disease virus (MDV) and highly pathogenic AIV (HPAIV) challenges was evaluated previously. It is well-known that avirulemt MDV type 1 vaccines are more effective than HVT in prevention of lethal MDV infection. To further increase protective efficacy against HPAIV and lethal MDV, a recombinant MDV type 1 strain 814 was developed to express HA gene of HPAIV H5N1.

Methodology/Principal Findings

A recombinant MDV-1 strain 814 expressing HA gene of HPAIV H5N1 virus A/goose/Guangdong/3/96 at the US2 site (rMDV-HA) was developed under the control of a human CMV immediate-early promoter. The HA expression in the rMDV-HA was tested by immunofluorescence and Western blot analyses, and in vitro and in vivo growth properties of rMDV-HA were also analyzed. Furthermore, we evaluated and compared the protective immunity of rMDV-HA and previously constructed rHVT-HA against HPAIV and lethal MDV. Vaccination of chickens with rMDV-HA induced 80% protection against HPAIV, which was better than the protection rate by rHVT-HA (66.7%). In the animal study with MDV challenge, chickens immunized with rMDV-HA were completely protected against virulent MDV strain J-1 whereas rHVT-HA only induced 80% protection with the same challenge dose.

Conclusions/Significance

The rMDV-HA vaccine was more effective than rHVT-HA vaccine for protection against lethal MDV and HPAIV challenges. Therefore, avirulent MDV type 1 vaccine is a better vector than HVT for development of a recombinant live virus vaccine against virulent MDV and HPAIV in poultry.     

A serum-free Vero production platform for a chimeric virus vaccine candidate

MedImmune Vaccines has engineered a live, attenuated chimeric virus that could prevent infections caused by parainfluenza virus type 3 (PIV3) and respiratory syncytial virus (RSV), causative agents of acute respiratory diseases in infants and young children. The work here details the development of a serum-free Vero cell culture production platform for this virus vaccine candidate. Efforts to identify critical process parameters and optimize culture conditions increased infectious virus titers by approximately 2 log₁₀ TCID₅₀/ml over the original serum-free process. In particular, the addition of a chemically defined lipid concentrate to the pre-infection medium along with the shift to a lower post-infection cultivation temperature increased virus titers by almost 100-fold. This improved serum-free process achieved comparable virus titers to the serum-supplemented process, and demonstrated consistent results upon scale-up: Vero cultures in roller bottles, spinner flasks and bioreactors reproducibly generated maximum infectious virus titers of 8 log₁₀ TCID₅₀/ml.     

Effects of maternal vitamin B12 deficiency from end of gestation to weaning on the growth and haematological and immunological parameters in mouse dams and offspring

Vitamin B₁₂-deficiency may induce specific symptoms as neurological alterations and unspecific symptoms such as anaemia and growth retardation. In this study, maternal vitamin B₁₂ deficiency from end of gestation to weaning was evaluated in mouse dams, which was provoked by feeding a vitamin B₁₂-deficient diet. The animals were divided into two groups (control and deficient). The control group received the vitamin B₁₂-deficient diet supplemented with commercial vitamin B₁₂. Compared to the control, the vitamin B₁₂-deficient dams and their offspring showed a significant decrease of body weight (by 20 and 39%, respectively), serum vitamin B₁₂ concentration (by 61 and 67%, respectively), haematological values as haematocrit (25 and 26%, respectively), and IgA producer cells (by 36 and 54%, respectively). In both, vitamin B₁₂-deficient mouse dams and their offspring, histological alterations of small intestine were observed, whereas growth retardation occurred in the offspring only. This experimental murine model allows assessing the incidence of maternal cobalamin deficiency in offspring and would be useful for evaluating novel adjuncts such as functional foods to prevent vitamin B₁₂ deficiency.     

Further Analysis of Marek's Disease Virus Horizontal Transmission Confirms That UL44 (gC) and UL13 Protein Kinase Activity Are Essential,while US2 Is Nonessential

Marek''s disease virus (MDV) causes a devastating disease in chickens characterized by the development of lymphoblastoid tumors in multiple organs and is transmitted from the skin of infected chickens. We have previously reported that the U_S2, U_L44 (glycoprotein C [gC]), and U_L13 genes are essential for horizontal transmission of MDV in gain-of-function studies using an a priori spread-deficient virus that was based on an infectious clone from the highly virulent RB-1B virus (pRB-1B). To precisely determine the importance of each individual gene in the process of chicken-to-chicken transmission, we used the transmission-restored clone that readily transmits horizontally and mutated each individual gene in loss-of-function experiments. Two independent U_S2-negative mutants transmitted horizontally, eliminating U_S2 as being essential for the process. In contrast, the absence of gC expression or mutating the invariant lysine essential for U_L13 kinase activity abolished horizontal spread of MDV between chickens.Marek''s disease (MD) is caused by the oncogenic alphaherpesvirus Gallid herpesvirus 2 (GaHV-2), better known as MD virus (MDV). The most prominent sign of MD is the development of lymphoproliferative disease in chickens characterized by solid tumors in the viscera and other organs (3, 19). Natural infection begins through inhalation of virus, after which MDV is taken to the lymphoid organs and primary cytolytic infection in B and then T lymphocytes ensues. Following lytic infection, latency is established mainly in activated CD4⁺ T cells, which may be transformed with differing efficiencies, depending on the genotype of the infected chicken, and result in lymphoma formation. Irrespective of the transformation event, infection of feather follicle epithelial cells in the skin by migrating lymphocytes leads to the production of infectious particles that are shed into the environment, providing a continuous source of infectious virus. While the majority of the work on MDV has been focused on the transformation and reactivation of MDV during infection, little is known about horizontal transmission of virus from one chicken to another.We recently identified genes important for horizontal transmission of MDV. We originally used a transmission-deficient virus derived from a bacterial artificial chromosome (BAC) clone of the very virulent RB-1B strain (pRB-1B-5) (35). Following sequencing of the complete BAC (40), specific genes suspected to be important for transmission were identified. We were able to restore horizontal transmission by repair of specific genes (17). We concluded that a combination of three genes, the unique short (U_S) 2, unique long (U_L) 44 or glycoprotein (g) C, and U_L13 protein kinase genes, was essential for horizontal transmission. Repair of each gene individually did not restore spread, nor did various combinations of two genes. In this report, we further defined which genes are essential by using loss-of-function studies utilizing mutant viruses in which U_S2, U_L13, or gC was inactivated in the transmission-competent virus (17). Mutant viruses were engineered using an infectious clone and markerless Red recombination exactly as previously described (17) using primers shown in Table ​Table1.1. Following confirmation of the correct modifications by restriction fragment length polymorphism (RFLP), PCR, and sequencing analyses, mutant viruses lacking the mini-F BAC sequences after Cre-Lox excision were reconstituted in chicken embryo cell cultures and propagated in chicken kidney cell cultures as previously described (17). Groups of P2a chickens (n = 10), which are highly susceptible to the development of MD (5), were experimentally infected with 1,000 PFU of the mutant viruses intra-abdominally and placed in glove box isolators with 10 age-matched, uninfected contact chickens. All experimental procedures were conducted in compliance with approved Institutional Animal Care and Use Committee (IACUC) protocols (Cornell University protocol numbers 2002-0085 and 2008-0018).

TABLE 1.

Primers used for mutating Marek''s disease virus genes in transmission-competent pRB-1B

Marek's disease viral interleukin-8 promotes lymphoma formation through targeted recruitment of B cells and CD4+ CD25+ T cells

Marek''s disease virus (MDV) is a cell-associated and highly oncogenic alphaherpesvirus that infects chickens. During lytic and latent MDV infection, a CXC chemokine termed viral interleukin-8 (vIL-8) is expressed. Deletion of the entire vIL-8 open reading frame (ORF) was shown to severely impair disease progression and tumor development; however, it was unclear whether this phenotype was due to loss of secreted vIL-8 or of splice variants that fuse exons II and III of vIL-8 to certain upstream open reading frames, including the viral oncoprotein Meq. To specifically examine the role of secreted vIL-8 in MDV pathogenesis, we constructed a recombinant virus, vΔMetvIL-8, in which we deleted the native start codon from the signal peptide encoding exon I. This mutant lacked secreted vIL-8 but did not affect Meq–vIL-8 splice variants. Loss of secreted vIL-8 resulted in highly reduced disease and tumor incidence in animals infected with vΔMetvIL-8 by the intra-abdominal route. Although vΔMetvIL-8 was still able to spread to naïve animals by the natural route, infection and lymphomagenesis in contact animals were severely impaired. In vitro assays showed that purified recombinant vIL-8 efficiently binds to and induces chemotaxis of B cells, which are the main target for lytic MDV replication, and also interacts with CD4⁺ CD25⁺ T cells, known targets of MDV transformation. Our data provide evidence that vIL-8 attracts B and CD4⁺ CD25⁺ T cells to recruit targets for both lytic and latent infection.     

The pattern of exposure to static magnetic field of nurses involved in activities related to contrast administration into patients diagnosed in 1.5 T MRI scanners

Static magnetic fields (SMFs) and time-varying electromagnetic fields exposure is necessary to obtain the diagnostic information regarding the structure of patient's tissues, by the use of magnetic resonance imaging (MRI) scanners. A diagnostic procedure may also include the administration of pharmaceuticals called contrast, which are to be applied to a patient during the examination. The nurses involved in administering contrast into a patient during the pause in examination are approaching permanently active magnets of MRI scanners and are exposed to SMF. There were performed measurements of spatial distribution of SMF in the vicinity of MRI magnets and parameters of personal exposure of nurses (i.e. individual exposimetric profiles of variability in time of SMF affecting the nurse who is performing tasks in the vicinity of magnet, characterized by statistical parameters of recorded magnetic flux density affecting the nurse). The SMF exposure in the vicinity of various MRI magnets depends on both magnetic flux density of the main field B ₀ (applicable to a patient) and the construction of the scanner, but the most important factor determining the workers' exposure is the work practice. In the course of a patient's routine examination in scanners of B ₀ = 1.5 T, the nurses are present over ～0.4–2.9 min in SMF exceeding 0.03% of B ₀ (i.e. 0.5 mT), but only sometimes they are present in SMF exceeding 5% of B ₀ (i.e. 75 mT). When patients need more attention because of their health status/condition, the nurses' exposure may be significantly longer – it may even exceed 10 min and 30% of B ₀ (i.e. 500 mT). We have found that the level of exposure of nurses to SMF may vary from < 5% of the main field (a median value: 0.5–1.5%; inter-quartile range: 0.04–8.8%; max value: 1.3–12% of B ₀) when a patient is moved from the magnets bore before contrast administration, up to the main field level (B ₀) when a patient stays in the magnets bore and nurse is crawling into the bore.     

Hydrocellulase of Aspergillus aculeatus

Conditions for extracellular production of vitamin B₆ compounds (B₆), especially pyridoxal 5'-phosphate (PLP) by Schizosaccharomyces pombe leu1 strain were examined. The productivity was dependent on concentration of L-leucine in the culture medium: 30 mg/l gave the highest concentrations of total B₆ and PLP. The viable cells harvested at different growth phases showed different productivity: middle and late exponential phase cells showed the highest productivity of total B₆ and PLP, respectively. D-Glucose (1%, w/v) among other sugars gave the best productivity. Supplementation of air and ammonium sulfate significantly increased extracellular production of PLP. Superoxide anion producers, menadione and plumbagin, and H₂O₂ increased the productivity of PLP. Cycloheximide inhibited the increase of PLP by the oxidative stress and in contrast, increased pyridoxine.     

Effects on Myzus persicae (Sulz.) and transmission of beet yellows virus of applying certain trace elements to sugar beet

Applications of lithium chloride (LiCl), zinc sulphate (ZnSO₄) or nickel sulphate (NiSO₄) to the roots of sugar-beet plants in the glasshouse encouraged settling on the leaves of adult apterae from a clone of Myzus persicae (Sulz.); conversely, treatment with boric acid (H₂B₂O₇) inhibited aphid settling. Larviposition of M. persicae was increased by NiSO₄ and tin chloride (SnCl₂). Viruliferous M. persicae transmitted beet yellows virus (BYV) more efficiently to plants treated with LiCl or H₂B₂O₇ than to those treated with copper sulphate (CuSO₄), ZnSO₄ or SnCl₂. The sulphate and chloride anions of the applied chemicals appeared to have little effect on M. persicae and virus transmission. It is suggested that applications of trace elements to sugar beet affected M. persicae and virus transmission by changing the concentrations of trace elements in the aphids' diet and by altering the metabolism of the leaf tissues in the host plant.