Computer Assisted Planning for Curved Laser Interstitial Thermal Therapy.

Laser interstitial thermal therapy (LiTT) isa minimally invasive alternative to conventional open surgery for drug-resistant focal mesial temporal lobe epilepsy (MTLE). Recent studies suggest that higher seizure freedom rates are correlated with maximal ablation of the mesialhippocampal head, whilst sparing of the parahippocampal gyrus (PHG) may reduce neuropsychological sequelae. Current commercially available laser catheters are inserted following manually planned straight-line trajectories, which cannot conform to curved brain structures, such as the hippocampus, without causing collateral damage or requiring multiple insertions. OBJECTIVES: The clinical feasibility and potential of curved LiTT trajectories through steerable needles has yet to be investigated. This is the focus of our work. METHODS: We propose a GPU-accelerated computer-assisted planning (CAP) algorithm for steerable needle insertions that generates optimized curved 3D trajectories with maximal ablation of the amygdalohippocampal complex and minimal collateral damage to nearby structures, while accounting for a variable ablation diameter ( 5-15 mm). RESULTS: Simulated trajectories and ablations were performed on 5 patients with mesial temporal sclerosis (MTS), which were identified from a prospectively managed database. The algorithm generated obstacle-free paths with significantly greater target area ablation coverage and lower PHG ablation variance compared to straight line trajectories. CONCLUSIONS: The presented CAP algorithm returns increased ablation of the amygdalohippocampal complex, with lower patient risk scores compared to straight-line trajectories. SIGNIFICANCE: This is the first clinical application of preoperative planning for steerable needle based LiTT. This study suggests that steerableneedles have the potential to improve LiTT procedure efficacy whilst improving the safety and should thus be investigated further.

Complete genome sequence of Newcastle disease virus mesogenic vaccine strain R2B from India.

Mesogenic vaccine strains of Newcastle disease virus (NDV) are widely used in many countries of Asia and Africa to control the Newcastle disease of poultry. In India, the mesogenic strain R2B was introduced in 1945; it protects adult chickens that have been preimmunized with a lentogenic vaccine virus and provides long-lasting immunity. In this article, we report the complete genome sequence of the hitherto unsequenced Indian vaccine virus strain R2B. The viral genome is 15,186 nucleotides in length and contains the polybasic amino acid motif in the fusion protein cleavage site, indicating that this vaccine strain has evolved from a virulent virus. Phylogenetic analysis of this mesogenic vaccine virus classified it with the viruses belonging to genotype III of the class cluster II of NDV.

Prospective study of outcome of foramen magnum decompressions in patients with syrinx and non-syrinx associated Chiari malformations.

OBJECTIVE: To assess the outcome in patients with syrinx and non-syrinx associated Chiari malformations undergoing Foramen Magnum Decompression (FMD). METHODS: Sixty-one patients undergoing FMD for Chiari malformations were prospectively studied with disease specific, generic (SF 36) and subjective (patient reported) outcomes. Of these, 34 patients had objective outcome data including SF36, visual analogue pain scores, Neck and Myelopathy disability indices and the Hospital anxiety and depression score. SF 36 scores were compared to normative data. Data were collected pre-operatively, at 3 months and during long-term follow up (12-60 months). RESULTS: Patient reported improvements in headache and neck pain post-operatively was reported in both syrinx and non-syrinx associated patients. Visual Analogue scores showed improvements in arm pain, paresthesia and hand tingling at 3 months in the syrinx group only. Non-syrinx patients showed significant improvements post-operatively in the Neck disability index and the SF-36 domains for physical function, role physical and bodily pain. Comparison with SF-36 normative data indicates that patients still have significantly impaired quality of life 12 months post-operatively. CONCLUSION: FMD is able to relieve symptoms and improve quality of life in patients with both syrinx and non-syrinx associated Chiari malformations. In syrinx patients we observed symptomatic improvement in limb symptoms as well as radiological resolution of the syrinx. The use of SF-36 allows the health gain associated with FMD to be quantified. SF 36 is not adequate as a stand alone measure of outcome in this complex disorder and we advocate the concurrent use of disease specific measures and post-operative imaging of the syrinx.

Immune responses elicited in rainbow trout through the administration of infectious pancreatic necrosis virus-like particles.

Virus like particles (VLPs) against viral pathogens not only constitute a novel approach for the development of antiviral vaccines for an specific virus, but also for the creation of multivalent vaccines in which antigens from other pathogens may be expressed on the surface of these VLPs. Despite positive results on protection for many of these VLPs in both fish and mammals, not many studies have focused on the immune response triggered by these particles; studies that may provide hints for the identification of immune mechanisms responsible for antiviral protection, which are mostly unknown in fish. In the current work, we have studied the levels of transcription of several immune genes in the spleen of rainbow trout (Oncorhynchus mykiss) intraperitoneally injected with VLPs from infectious pancreatic necrosis virus (IPNV) focusing on the chemokine response as well as the response of genes related to interferon (IFN) production. Surprisingly, the capacity of VLPs to induce chemokines differed from that of live IPNV, suggesting a direct effect of viral replication on the chemokine response in this organ. While VLPs up-regulated the transcription of CK3, CK10 and CXCd and down-modulated CK5B, CK6 and CK9 transcription, a previous study in which the transcription of γIP, CXCd, CK1, CK3, CK5B, CK6, CK7A, CK9 and CK12 had been studied demonstrated that IPNV only significantly up-regulated CK6 and down-modulated CK3 in the spleen. On the other hand, the administration of VLPs produced a strong mobilization to the peritoneum of CD4(+), IgM(+), IgT(+) and CD83(+) leukocytes similar to that induced by the live viral infection. In both cases, this leukocyte recruitment seemed to be greatly mediated through CK3, CK5B, CK9 and CK10 chemokine production. These results together with the fact that VLPs strongly induced non-specific lymphocyte proliferation and specific anti-IPNV antibody production point to VLPs as excellent candidates for vaccine development.

Genetic analysis of aquabirnaviruses isolated from wild fish reveals occurrence of natural reassortment of infectious pancreatic necrosis virus.

In this study, we report the sequencing of the whole genome [including the 5' and 3' non-coding regions (NCR) of both segments A and B] of seven birnavirus strains isolated from wild fish from the Flemish Cap (FC) fishery at Newfoundland, Canada. From analysis and comparison of the sequences, most of the FC isolates clustered with the North American reference strains West Buxton (WB), Dry Mill and Jasper. One strain was included in the same genotype as the European strain Ab. In addition, at least in one case cohabitation of both type strains in an individual fish was demonstrated. These results clearly suggest the acquisition of the viruses from two different sources. The prevalence of the American type is easily explained by the close proximity of this fishing bank to the American coast whereas, although surprising, the presence of the European type strain could be because of migration of fish from European waters. In one strain, segment A and B sequences were typed differently (WB and Ab, respectively). These findings indicate natural reassortment between two strains of aquabirnaviruses in a host.

Development of a subunit vaccine for infectious pancreatic necrosis virus using a baculovirus insect/larvae system.

Various attempts to develop a vaccine against infectious pancreatic necrosis virus (IPNV) have not yielded consistent results. Thus, at present, no commercial vaccine is available that can be used with confidence to immunize fry of salmon and trout. We generated a cDNA clone of the large genome segment A of an IPNV Sp strain and expressed all structural protein genes in insect cells and larvae using a baculovirus expression system. Green fluorescent protein was also coexpressed as a reporter molecule. High yields of IPNV proteins were obtained and the structural proteins self assembled to form virus-like particles (VLPs). We tested the immunogenicity of the putative VLP antigen in immersion vaccine experiments (two concentrations) in rainbow trout (Oncorhynchus mykiss) fry, and by intraperitoneal immunisation of Atlantic salmon (Salmo salar) pre-smolts using an oil adjuvant formulation. Rainbow trout were challenged by immersion using either the Sp or the VR-299 strain of IPNV two or three weeks post-vaccination, while Atlantic salmon were bath challenged with Sp strain after two months, after parr-smolt transformation. In the rainbow trout fry challenged two weeks post-immunization, cumulative mortality rates three weeks post challenge were 14 % in the fry that had received the highest dose versus 8 % in the control groups. No indication of protection was seen in repeated trials using a lower dose of antigen and challenge three weeks post-immunisation. The cumulative mortality rate of intraperitoneally immunised Atlantic salmon post-smolts four weeks post challenge was lower (56%) than in the control fish (77%), showing a dose-response pattern.

Molecular characterization of Sp serotype strains of infectious pancreatic necrosis virus exhibiting differences in virulence.

Infectious pancreatic necrosis virus (IPNV), a prototype virus of the family Birnaviridae, exhibits a high degree of antigenic variability, pathogenicity and virulence in salmonid species. The Genomic Segment A encodes all the structural (VP2 and VP3) and nonstructural (NS) proteins, whereas Segment B encodes the viral RNA-dependent RNA polymerase (VP1). We tested 3 different IPNV isolates (Sp103, Sp116 and Sp122) isolated during field outbreaks in Norway for their ability to cause mortality in fry and post-smolt of Atlantic salmon Salmo salar L. The cumulative mortality following experimental challenge in fry was 29% for Sp122 followed by 19% for Sp116 and 15% for Sp103. In post-smolt, the corresponding mortality rates were 79, 46 and 16%, respectively. Comparisons of the deduced amino acid sequences of Segments A and B of all 3 Sp strains revealed substitutions of residues in 13 positions, of which 6 are in VP2, 2 in VP3, and 5 in VP1. Our results suggest that these residues, especially those in the outer capsid VP2, may be involved in the virulence of IPNV. Genome Segment A of the Sp serotype is 3097 nucleotides long and contains a major open reading frame (ORF) encoding a polyprotein of 972 amino acids, which initiates at the second in-frame start codon at Position 119. This was ascertained by making mutants of Segment A clone using site-directed mutagenesis, followed by in vitro transcription-coupled translation reaction and immunoprecipitation analyses. In addition, Segment A also encodes a 15 kDa arginine-rich non-structural protein from a small ORF, preceding and partially overlapping the polyprotein ORF, which is truncated to 12 kDa in the virulent Sp122 strain. Moreover, Segment A could encode a novel, putative 25 kDa protein from another ORF between VP2 and VP4 coding regions, which is only detected in the Sp serotype. Segment B is 2777 nucleotides long and encodes in a single large ORF (a polypeptide of 844 amino acid residues), VP1.

A novel transcutaneous plasmid-dimethylsulfoxide delivery technique for avian nucleic acid immunization.

In this report, we show that dimethylsulfoxide (DMSO) enhances liposome-mediated transfection of nucleic acid in chicken macrophage cells and that this could be exploited for the transcutaneous delivery of naked DNA through the intact skin of chickens. We found that DMSO enhanced transfection efficiencies of lipofectamine and polyethyleneimine in HD-11 chicken macrophage cells. Based on this principle, we showed that transcutaneous delivery of a DNA plasmid-dimethylsulfoxide mixture (1:1) to untreated skin of chickens results in a wide distribution of the plasmid in the body. Distribution studies were done using plasmids encoding enhanced green fluorescent protein (EGFP) reporter gene and a bivalent DNA vaccine coding for infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV) immunogenic protein genes. This bivalent vaccine induced mucosal and systemic immune responses, as evidenced by IgA and IgM production in the tears and serum of vaccinated chickens. Mucosal immune responses in the tears after topical vaccination were significantly higher (P < 0.05) than after i.m. delivery of the same DNA vaccine and were characterized by the absence of an IgG response. The biodistribution of plasmid indicated that topical delivery with DMSO resulted in a wide distribution and persistence of the plasmid until 15 weeks post-primary vaccination. Both delivery methods resulted in insert-specific message being made in several body tissues, but after topical delivery the virus-specific mRNA could be detected in the bone marrow of one out of three chickens until 15 weeks post-primary vaccination. Furthermore, transcutaneous delivery of this DNA vaccine using DMSO conferred protection from challenge with virulent IBDV (86% survival) and NDV (86% survival). This novel transcutaneous method of delivery of a DNA vaccine shows promise as being an easy and effective way to deliver nucleic acids through intact skin for vaccination or therapeutic purposes.

Molecular determinants of virulence, cell tropism, and pathogenic phenotype of infectious bursal disease virus.

Infectious bursal disease viruses (IBDVs), belonging to the family Birnaviridae, exhibit a wide range of immunosuppressive potential, pathogenicity, and virulence for chickens. The genomic segment A encodes all the structural (VP2, VP4, and VP3) and nonstructural proteins, whereas segment B encodes the viral RNA-dependent RNA polymerase (VP1). To identify the molecular determinants for the virulence, pathogenic phenotype, and cell tropism of IBDV, we prepared full-length cDNA clones of a virulent strain, Irwin Moulthrop (IM), and constructed several chimeric cDNA clones of segments A and B between the attenuated vaccine strain (D78) and the virulent IM or GLS variant strain. Using the cRNA-based reverse-genetics system developed for IBDV, we generated five chimeric viruses after transfection by electroporation procedures in Vero or chicken embryo fibroblast (CEF) cells, one of which was recovered after propagation in embryonated eggs. To evaluate the characteristics of the recovered viruses in vivo, we inoculated 3-week-old chickens with D78, IM, GLS, or chimeric viruses and analyzed their bursae for pathological lesions 3 days postinfection. Viruses in which VP4, VP4-VP3, and VP1 coding sequences of the virulent strain IM were substituted for the corresponding region in the vaccine strain failed to induce hemorrhagic lesions in the bursa. In contrast, viruses in which the VP2 coding region of the vaccine strain was replaced with the variant GLS or virulent IM strain caused rapid bursal atrophy or hemorrhagic lesions in the bursa, as seen with the variant or classical virulent strain, respectively. These results show that the virulence and pathogenic-phenotype markers of IBDV reside in VP2. Moreover, one of the chimeric viruses containing VP2 sequences of the virulent strain could not be recovered in Vero or CEF cells but was recovered in embryonated eggs, suggesting that VP2 contains the determinants for cell tropism. Similarly, one of the chimeric viruses containing the VP1 segment of the virulent strain could not be recovered in Vero cells but was recovered in CEF cells, suggesting that VP1 contains the determinants for cell-specific replication in Vero cells. By comparing the deduced amino acid sequences of the D78 and IM strains and their reactivities with monoclonal antibody 21, which binds specifically to virulent IBDV, the putative amino acids involved in virulence and cell tropism were identified. Our results indicate that residues Gln at position 253 (Gln253), Asp279, and Ala284 of VP2 are involved in the virulence, cell tropism, and pathogenic phenotype of virulent IBDV.

Induction of apoptosis in vitro by the 17-kDa nonstructural protein of infectious bursal disease virus: possible role in viral pathogenesis.

Infectious bursal disease virus (IBDV) causes severe immunodeficiency in young chickens by destroying the precursors of antibody-producing B cells in the bursa of Fabricius. It has been shown that IBDV infection induces apoptosis in chicken embryo and tissue culture cells. We previously reported that an IBDV mutant lacking the expression of 17-kDa nonstructural (NS) protein exhibited decreased apoptotic effects in cell culture as compared to the parental IBDV, suggesting that the NS protein may be involved in induction of apoptosis. Here, we report that the NS protein of IBDV alone is capable of inducing apoptosis in cell culture. Transfection of chicken B-lymphocyte cell line (RP9) and chicken embryo fibroblast cells with a plasmid DNA, containing the NS protein gene under the control of the immediate-early promoter-enhancer region of human cytomegalovirus, induced programmed cell death in both cell lines. Apoptosis changes, such as chromatin condensation, DNA fragmentation, and the appearance of apoptotic nuclear bodies, were observed in cell cultures 48-h posttransfection. As reported earlier, the mutant IBDV grew to lower titers with slower replication kinetics and lower cytopathogenicity when compared to that of the parental virus. Here, we demonstrate that the mutant virus is closely associated with cells and its yield from the supernatant was approximately 30-fold lower than the wild-type due to increased cell association, indicating a deficiency in lysis of virus-infected cells. Taken together, our results indicate that the NS protein of IBDV is highly cytotoxic, which brings about the release of the viral progeny from cells, and thus play an important role in viral pathogenesis.

A recombinant Eimeria protein inducing interferon-gamma production: comparison of different gene expression systems and immunization strategies for vaccination against coccidiosis.

A rabbit antiserum against an 18- to 27-kD native protein fraction (F3) from Eimeria acervulina merozoites identified a cDNA (3-1E) containing a 1086-base pair insertion with an open reading frame of 170 amino acids (predicted molecular weight, 18,523). The recombinant 3-1E cDNA expressed in Escherichia coli produced a 60-kD fusion protein and a 23-kD protein after factor Xa treatment of the fusion protein. Both proteins were reactive with the F3 antiserum by western blot analysis. A rabbit antiserum against a synthetic peptide deduced from the amino acid sequence of the 3-1E cDNA reacted with a 27-kD recombinant 3-1E protein expressed in Sf9 insect cells and a 20-kD native protein expressed by E. acervulina sporozoites and Eimeria tenella sporozoites and merozoites. By immunofluorescence staining, a monoclonal antibody produced against the recombinant 3-1E protein reacted with sporozoites and merozoites of E. acervulina, E. tenella, and Eimeria maxima. Spleen lymphocytes from E. acervulina-immune chickens showed antigen-specific proliferation and interferon (IFN)-gamma production upon stimulation with the recombinant 3-1E protein, indicating that the protein activates cell-mediated immunity during coccidiosis. Immunization of chickens with either the E. coli- or Sf9-expressed recombinant 3-1E protein with adjuvant, or direct injection of the 3-1E cDNA, induced protective immunity against live E. acervulina. Simultaneous injection of the recombinant 3-1E protein, or the 3-1E cDNA, with cDNAs encoding chicken IFN-gamma or interleukin (IL)-2/15 further enhanced protective immunity. These results indicate that the recombinant E. acervulina 3-1E cDNA or its polypeptide product may prove useful as vaccines against avian coccidiosis.

Possible involvement of the double-stranded RNA-binding core protein sigmaA in the resistance of avian reovirus to interferon.

Treatment of primary cultures of chicken embryo fibroblasts with a recombinant chicken alpha/beta interferon (rcIFN) induces an antiviral state that causes a strong inhibition of vaccinia virus and vesicular stomatitis virus replication but has no effect on avian reovirus S1133 replication. The fact that avian reovirus polypeptides are synthesized normally in rcIFN-treated cells prompted us to investigate whether this virus expresses factors that interfere with the activation and/or the activity of the IFN-induced, double-stranded RNA (dsRNA)-dependent enzymes. Our results demonstrate that extracts of avian-reovirus-infected cells, but not those of uninfected cells, are able to relieve the translation-inhibitory activity of dsRNA in reticulocyte lysates, by blocking the activation of the dsRNA-dependent enzymes. In addition, our results show that protein sigmaA, an S1133 core polypeptide, binds to dsRNA in an irreversible manner and that clearing this protein from extracts of infected cells abolishes their protranslational capacity. Taken together, our results raise the interesting possibility that protein sigmaA antagonizes the IFN-induced cellular response against avian reovirus by blocking the intracellular activation of enzyme pathways dependent on dsRNA, as has been suggested for several other viral dsRNA-binding proteins.

Insect larval expression process is optimized by generating fusions with green fluorescent protein.

The insect larvae/baculovirus protein production process was dramatically simplified by expressing fusion proteins containing green fluorescent protein (GFP) and the product-of-interest. In this case, human interleukin-2 (hIL-2) and chloramphenicol acetyl-transferase (CAT) were model products. Specifically, our fusion construct was comprised of a histidine affinity ligand for simplified purification using immobilized metal affinity chromatography (IMAC), the UV-optimized GFP (GFPuv) as a marker, an enterokinase cleavage site for recovery of the product from the fusion, and the product, hIL-2 or CAT. Both the approximately 52 kDa GFPuv/hIL-2 and approximately 63 kDa GFPuv/CAT fusions were expressed in Trichoplusia ni larvae at 9.0 microg-hIL-2 and 24.1 microg-CAT per larva, respectively. The GFP enabled clear identification of the infection process, harvest time, and more importantly, the quantity of product protein. Because the GFP served as a marker, this technique obviates the need for in-process Western analyses (during expression, separation, and purification stages). As a purification marker, GFP facilitated rapid identification of product-containing elution fractions (Cha et al., 1999b), as well as product-containing waste fractions (e.g., cell pellet). Also, because the fluorescence intensity was linear with hIL-2 and CAT, we were able to select the highest-producing larvae. That is, three fold more product was found in the brightest larva compared to the average. Finally, because the GFP is attached to the product protein and the producing larvae can be selected, the infection and production processes can be made semi-continuous or continuous, replacing the current batch process. These advantages should help to enable commercialization of larvae as expression hosts.

Chimeric infectious bursal disease virus-like particles expressed in insect cells and purified by immobilized metal affinity chromatography.

Chimeric virus-like particles (VLPs) of infectious bursal disease virus (IBDV) were produced by coinfecting Spodoptera frugiperda (Sf-9) insect cells with two recombinant baculoviruses, vIBD-7 and vEDLH-22. vIBD-7 encodes VP2, VP3, and VP4 of the IBDV structural proteins. vEDLH-22 encodes VP2 with five histidine residues at the carboxy-terminus (VP2H). Coinfection produced hybrid VLPs composed of VP2, VP2H, and VP3. The additional histidine residues on VP2H enabled the efficient purification of VLPs based on immobilized metal affinity chromatography (IMAC). These results demonstrated that the VLPs formed are comprised of chimeric subunits with attached affinity ligands, and further, that sufficient His5 ligand was available for binding to the IMAC metal-chelating resin. Additionally, these novel particles were fully characterized for antigenicity by a series of monoclonal antibodies, and appeared identical to the two wild-type IBDV strains contributing subunits to the chimeric VLP. IMAC purification provides a promising low-cost and simple scheme to purify VLPs as vaccines.

Expression and purification of human interleukin-2 simplified as a fusion with green fluorescent protein in suspended Sf-9 insect cells.

A fusion protein of human interleukin-2 (hIL-2) and green fluorescent protein (GFP) was expressed in insect Sf-9 cells infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was comprised of a histidine affinity ligand for simplified purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv) as a marker, an enterokinase cleavage site for recovery of hIL-2 from the fusion, and the model product hIL-2. Successful production of hIL-2 as a fusion protein (approximately 52,000 Da) with GFPuv was obtained. GFPuv enabled rapid monitoring and quantification of the hIL-2 by simply checking the fluorescence, obviating the need for Western blot and/or ELISA assays during infection and production stages. There was no increased 'metabolic burden' due to the presence of GFPuv in the fusion product. The additional histidine residues at the N-terminus enabled efficient one-step purification of the fusion protein using IMAC. Additional advantages of GFP as a fusion marker were seen, particularly during separation and purification in that hIL-2 containing fractions were identified simply by illumination with UV light. Our results demonstrated that GFP was an effective non-invasive on-line marker for the expression and purification of heterologous protein in the suspended insect cell/baculovirus expression system.

Green fluorescent protein as a noninvasive stress probe in resting Escherichia coli cells.

We constructed and characterized three stress probe plasmids which utilize a green fluorescent protein as a noninvasive reporter in order to elucidate Escherichia coli cellular stress responses in quiescent or resting cells. Cellular stress levels were easily detected by fusing three heat shock stress protein promoter elements, those of the heat shock transcription factor sigma32, the protease subunit ClpB, and the chaperone DnaK, to the reporter gene gfpuv. When perturbed by a chemical or physical stress (such as a heat shock, nutrient [amino acid] limitation, or addition of IPTG [isopropyl-beta-D-thiogalactopyranoside], acetic acid, ethanol, phenol, antifoam, or salt [osmotic shock]), the E. coli cells produced GFPuv, which was easily detected within the cells as emitted green fluorescence. Temporal and amplitudinal mapping of the responses was performed, and the results revealed regions where quantitative delineation of cell stress was afforded.

Induction of protective immunity in chickens immunised with plasmid DNA encoding infectious bursal disease virus antigens.

Direct DNA inoculations were used to determine the efficacy of gene immunisation of chickens to elicit protective immune responses against infectious bursal disease virus (IBDV). The vp2 gene of IBDV strains GP40 and D78, and the vp2-vp4-vp3 encoding segment of strain D78 were cloned in an expression vector which consisted of human cytomegalovirus (HCMV) immediate early enhancer and promoter, adenovirus tripartite leader sequences and SV40 polyadenylation signal. For purification of vaccine-quality plasmid DNA from E. coli, an effective method was developed. Chickens were vaccinated by inoculation of DNA by two routes (intramuscular and intraperitoneal). Two weeks later, chickens were boosted with DNA, and at 2 weeks post-boost, they were challenged with virulent IBDV strain. Low to undetectable levels of IBDV-specific antibodies and no protection were observed with DNA encoding VP2. However, plasmids encoding VP2-VP4-VP3 induced IBDV-specific antibodies and protection in the chickens. DNA immunisation opens a new approach to the development of gene vaccines for chickens against infectious diseases.

Generation of infectious pancreatic necrosis virus from cloned cDNA.

We developed a reverse genetics system for infectious pancreatic necrosis virus (IPNV), a prototype virus of the Birnaviridae family, with the use of plus-stranded RNA transcripts derived from cloned cDNA. Full-length cDNA clones of the IPNV genome that contained the entire coding and noncoding regions of RNA segments A and B were constructed. Segment A encodes a 106-kDa precursor protein which is cleaved to yield mature VP2, nonstructural protease, and VP3 proteins, whereas segment B encodes the RNA-dependent RNA polymerase VP1. Plus-sense RNA transcripts of both segments were prepared by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of chinook salmon embryo (CHSE) cells with combined transcripts of segments A and B generated infectious IPNV particles 10 days posttransfection. Furthermore, a transfectant virus containing a genetically tagged sequence was generated to confirm the feasibility of this system. The presence and specificity of the recovered virus were ascertained by immunofluorescence staining of infected CHSE cells with rabbit anti-IPNV serum and by nucleotide sequence analysis. In addition, 3'-terminal sequence analysis of RNA from the recovered virus showed that extraneous nucleotides synthesized at the 3' end during in vitro transcription were precisely trimmed or excluded during replication, and hence these were not incorporated into the genome. An attempt was made to determine if RNA-dependent RNA polymerase of IPNV and infectious bursal disease virus (IBDV), another birnavirus, can support virus rescue in heterologous combinations. Thus, CHSE cells were transfected with transcripts derived from IPNV segment A and IBDV segment B and Vero cells were transfected with transcripts derived from IBDV segment A and IPNV segment B. In either case, no infectious IPNV or IBDV particles were generated even after a third passage in cell culture, suggesting that viral RNA-dependent RNA polymerase is species specific. However, the reverse genetics system for IPNV that we developed will greatly facilitate studies of viral replication and pathogenesis and the design of a new generation of live attenuated vaccines.

Generation of a mutant infectious bursal disease virus that does not cause bursal lesions.

A reverse genetics system for birnavirus, based on synthetic transcripts of the infectious bursal disease virus (IBDV) genome, was recently developed (E. Mundt and V. N. Vakharia, Proc. Natl. Acad. Sci. USA 93:11131-11136, 1996). To study the function of the 17-kDa nonstructural (NS) protein in viral growth and pathogenesis, we constructed a cDNA clone of IBDV segment A in which the first and only initiation codon (ATG) of NS protein was mutated to a stop codon (TAG). Transfection of Vero cells with combined transcripts of either modified or unmodified segment A, and with segment B, generated viable IBDV progeny. When chicken embryo fibroblast cells infected with transfectant viruses were analyzed by immunofluorescence assays using NS-specific antiserum, the mutant virus did not yield a fluorescence signal, indicating a lack of NS protein expression. Furthermore, replication kinetics and cytotoxic effects of the mutant virus were compared with those of the parental attenuated vaccine strain of IBDV (D78) in vitro. The mutant virus grew to slightly lower titers than D78 virus and exhibited decreased cytotoxic and apoptotic effects in cell culture. To evaluate the characteristics of the recovered viruses in vivo, we inoculated 3-week-old chickens with D78 or mutant virus and analyzed their bursa for histopathological lesions. The recovered D78 virus caused microscopic lesions and atrophy of the bursa, while the mutant virus failed to induce any pathological lesions or clinical signs of disease. In both instances, the virus was recovered from the bursa, and the presence or absence of mutation in these viruses was confirmed by nucleotide sequence analysis of NS gene. Although the mutant virus exhibited a delay in replication in vivo, it induced levels of IBDV neutralizing antibodies that were similar to those of D78 virus. In addition, no reversion of mutation was detected in the mutant virus recovered from inoculated chickens. These results demonstrate that NS protein is dispensable for viral replication in vitro and in vivo and that it plays an important role in viral pathogenesis. Thus, generation of such NS protein-deficient virus will facilitate the study of immunosuppression and aid in the development of live-attenuated vaccines for IBDV.