Cry1Ab protein from Bacillus thuringiensis and MON810 cry1Ab-transgenic maize exerts no adjuvant effect after airway exposure.

The genetically modified (GM) maize event MON810 has been inserted with a processed version of the transgene, cry1Ab, derived from the soil bacterium Bacillus thuringiensis (Bt) to express proteins with insecticidal properties. Such proteins may introduce new allergens and also act as adjuvants that promote allergic responses. While focus has been on safe consumption and hence the oral exposure to GM food and feed, little is known regarding inhalation of pollen and desiccated airborne plant material from GM crops. The aim of this study was to investigate whether plant material from the Cry1Ab-expressing maize variety MON810, or trypsin-activated Cry1Ab (trypCry1Ab) protein produced in recombinant bacteria, may act as adjuvants against the allergen ovalbumin (OVA) in a mouse model of airway allergy. A clear proallergic adjuvant effect of the mucosal adjuvant cholera toxin (CT) was demonstrated, determined as increased specific IgE, eosinophils and Th2 cytokines in MLN cell supernates, while no elevation in OVA-specific antibodies or cytokine release from MLN cells after stimulation with OVA were observed in mice receiving Cry1Ab-containing plant materials or the trypCry1Ab protein. Our data suggest that Cry1Ab proteins had no detectable systemic adjuvant effect in mice after airway exposure. Further experiments with purified plant proteins, as well as long-term exposures needs be conducted to further evaluate exposures experienced in real-life situations.

Compositional differences in soybeans on the market: glyphosate accumulates in Roundup Ready GM soybeans.

This article describes the nutrient and elemental composition, including residues of herbicides and pesticides, of 31 soybean batches from Iowa, USA. The soy samples were grouped into three different categories: (i) genetically modified, glyphosate-tolerant soy (GM-soy); (ii) unmodified soy cultivated using a conventional "chemical" cultivation regime; and (iii) unmodified soy cultivated using an organic cultivation regime. Organic soybeans showed the healthiest nutritional profile with more sugars, such as glucose, fructose, sucrose and maltose, significantly more total protein, zinc and less fibre than both conventional and GM-soy. Organic soybeans also contained less total saturated fat and total omega-6 fatty acids than both conventional and GM-soy. GM-soy contained high residues of glyphosate and AMPA (mean 3.3 and 5.7 mg/kg, respectively). Conventional and organic soybean batches contained none of these agrochemicals. Using 35 different nutritional and elemental variables to characterise each soy sample, we were able to discriminate GM, conventional and organic soybeans without exception, demonstrating "substantial non-equivalence" in compositional characteristics for 'ready-to-market' soybeans.

Human endothelial cells allow passage of an archetypal BK virus (BKV) strain--a tool for cultivation and functional studies of natural BKV strains.

The ubiquitous human polyomavirus BK (BKV) causes the serious condition BKV-nephropathy in an increasing number of renal-transplant patients. The lack of authentic cell cultures for multiplication of naturally occurring strains has hampered cultivation and functional studies of BKV. Here we demonstrate that the most common urine shed BKV strain, the archetype, multiplies in the human endothelial cell line HUV-EC-C. Additional variants with deletions in the non-coding control region (NCCR) appear upon prolonged propagation. Although the titer produced was low, at the present HUV-EC-C is the only cell line shown to allow propagation of archetypal BKV. HUV-EC-C may therefore be a useful tool for BKV cultivation as well as functional studies.

Analysis and comparison of the 4b core protein gene of avipoxviruses from wild birds: evidence for interspecies spatial phylogenetic variation.

Avipoxviruses have been isolated from a wide variety of avian hosts, and yet little is known regarding the host-virus species variation of the genus Avipoxvirus. We have investigated the variations in the viral 4b core protein gene from six different avipoxviruses based on PCR, Southern blot and nucleotide sequence analysis to evaluate the suitability of this region for differentiation between avipoxvirus isolates. Southern blot and nucleotide sequence analysis revealed considerable interspecies variation between the different virus isolates. In the deduced amino acid sequences (of 142 residues) of the 4b core protein gene, fowlpox virus vaccine strain (FPV-VR250) was found to be similar to the three poxvirus isolates from great tit (GTV-A310, GTV-A311 and GTV-A256), sparrowpox virus (SPV-A468), and pigeonpox virus (PPV-B7) with similarities of 79.6%, 81%, 81%, 64.8% and 84.5%, respectively. Furthermore, comparative phylogenetic analysis of the aligned DNA sequences revealed divergence among the different viruses that can be consistently correlated to the host.

Lactoferrin and cyclic lactoferricin inhibit the entry of human cytomegalovirus into human fibroblasts.

Lactoferrin is mainly produced by polymorphonuclear leukocytes and has been demonstrated in mammalian milk and external secretions. Lactoferrin is an iron-binding, multifunctional protein and may play an important role in immune regulation and in defense mechanisms against bacteria, fungi and viruses. Lactoferricin is a potent antimicrobial peptide generated from the N-terminal part of lactoferrin by pepsin cleavage. We demonstrate that lactoferrins from different species and its N-terminal peptide lactoferricin (particularly the cyclic form) inhibit expression of early and late antigens, as well as production of infectious viral progeny during human cytomegalovirus (HCMV) infection in vitro. Iron-saturated lactoferrin did not affect HCMV antigen expression. Heparin had the same effects as iron-depleted lactoferrin. Yet, mixtures of lactoferrin and heparin did not inhibit HCMV multiplication i.e. lactoferrin and heparin seemed to mutually block each other's antiviral activities. HCMV-infected cells exposed to lactoferrin and cyclic lactoferricin contained less intracellular virus than unexposed cells. The antiviral activity of cyclic lactoferricin was more than seven-fold weaker than that of the maternal molecule. Lactoferrin and cyclic lactoferricin prevented HCMV entrance into the host cell.

Human polyomavirus BK (BKV) and neuroblastoma: mechanisms of oncogenic action and possible strategy for novel treatment.

BACKGROUND: We reported previously that nearly all human neuroblastomas analyzed contain and express genomic DNA sequences deriving from the human polyomavirus BK (BKV) [Flaegstad et al.: Cancer Res 59:1160-1163, 1999]. PROCEDURE: Here we show that the BKV large T antigen is expressed and bound to p53 in neuroblastoma cells and that this interference compromises the tumor suppressor function of p53. RESULTS: Treatment of neuroblastoma cells with large T antigen antisense constructs relocated active p53 to the nucleus. The relocation event was accompanied by enhanced p21(waf1/cip1) expression as well as induced apoptosis. CONCLUSIONS: Continuous antisense oligonucleotide treatment of nude rats with human neuroblastoma xenografts resulted in a significant but incomplete reduction of tumor growth compared to rats treated with saline.

Comparison of thymidine kinase and A-type inclusion protein gene sequences from Norwegian and Swedish cowpox virus isolates.

During the last decades, cowpox virus, a member of the genus Orthopoxvirus within the Poxviridae family, has appeared as a pathogen in domestic cats, zoo animal species, and humans. At the same time, vaccinia virus, another orthopoxvirus, has been used as a recombinant vaccine vector with foreign genes inserted in the thymidine kinase (TK) gene. By PCR and cycle sequencing, we have determined the nucleotide sequences of the TK gene and the A-type inclusion protein (ATIP) gene of virus isolates from two human cowpox cases in Sweden, as well as a human and a feline case from Norway. We also obtained the corresponding sequences from ectromelia virus (strain Moscow), cowpox virus (strain Brighton) and vaccinia virus (strain Western Reserve). The new virus isolates differed from ectromelia virus and vaccinia virus, and were confirmed to be cowpox virus strains. Isolates originating from the same country had nearly identical TK sequences and fully identical ATIP sequences. They probably represent local geographical strains of cowpox virus.

A possible contributory role of BK virus infection in neuroblastoma development.

The tumor suppressor protein p53 is aberrantly localized to the cytoplasm of neuroblastoma cells, compromising the suppressor function of this protein. Such tumors are experimentally induced in transgenic mice expressing the large tumor (T) antigen of polyomaviruses. The oncogenic mechanisms of T antigen include complex formation with, and inactivation of, the tumor suppressor protein p53. Samples from 18 human neuroblastomas and five normal human adrenal glands were examined. BK virus DNA was detected in all neuroblastomas and none of five normal adrenal glands by PCR. Using DNA in situ hybridization, polyomaviral DNA was found in the tumor cells of 17 of 18 neuroblastomas, but in none of five adrenal medullas. Expression of the large T antigen was detected in the tumor cells of 16 of 18 neuroblastomas, but in none of the five adrenal medullas. By double immunostaining BK virus T antigen and p53 was colocalized to the cytoplasm of the tumor cells. Immunoprecipitation revealed binding between the two proteins. The presence and expression of BK virus in neuroblastomas, but not in normal adrenal medulla, and colocalization and binding to p53, suggest that this virus may play a contributory role in the development of this neoplasm.

Clinical cowpox cases in Norway.

In 1994, a human and a feline case of cowpox virus infection appeared in the western part of Norway. Cowpox has not been diagnosed with certainty in Norway since the beginning of this century, when it was associated with the use of cowpox virus as a vaccine against smallpox. The human infection manifested as a spontaneously emerged, severe ulceration at the medial angle of the right eye in a 37-y-old woman, and developed into a relatively severe dermatitis. The ulcer healed slowly, leaving a scar. The feline infection was represented by a febrile, dehydrated and anorectic 6-months-old non-pedigree short-hair, with crater-like ulcers all over the body. After antibiotic and fluid therapy, revision of the skin lesions and amputation of a gangrenous toe, the cat recovered. Electron microscopy of the isolates and cultivation of virus on chorioallantoic membrane of chicken embryos confirmed the suspicion of cowpox virus infection.

Antibodies to orthopoxvirus in domestic cats in Norway.

The prevalence of antibodies to orthopoxvirus in 217 sera collected from domestic cats in the western part of Norway was 10.1 per cent as measured by a competitive ELISA. In one of the seropositive cats antibodies were also detected by an immunofluorescence assay. The average age of the cats sampled was 4.9 years, but the average age of the seropositive individuals was 7.3 years, higher than the average age of clinical cowpox virus cases in Britain (4.2 years), and in Germany (3.9 years). Antibodies against feline immunodeficiency virus (FIV) were detected in nine of 30 (30 per cent) of the seropositive cats, and in five of 30 (17 per cent) of the seronegative cats, which suggests that FIV infection may influence the susceptibility of domestic cats to orthopoxvirus, or vice versa. Orthopoxvirus infections, have recently been detected in rodent populations in several areas of Norway, and the infection may therefore be present in cats all over the country; cat owners and animal handlers should be aware of this (re)emerging zoonosis.

Characteristics of four cowpox virus isolates from Norway and Sweden.

We report the first isolation of cowpox virus from a domestic cat in Norway, and the first confirmed isolation of cowpox virus from a human case in Norway. These two Norwegian cowpox virus isolates, as well as two Swedish human isolates, were partially characterized and compared with each other and with cowpox virus Brighton and vaccinia virus strain Western Reserve. Restriction enzyme analysis of the genomes revealed differences between all six viruses examined, but suggested that the two Norwegian isolates are closely related, as are the two Swedish isolates. Restriction endonuclease digestion of genomic DNA demonstrated that one of the Swedish isolates and the two Norwegian isolates have larger genomes than vaccinia virus strain Western Reserve, but smaller than cowpox Brighton. All four Scandinavian isolates lacked a 72 base-pair region within the A-type inclusion body protein gene which is present in the prototype cowpox virus Brighton.

Naturally occurring orthopoxviruses: potential for recombination with vaccine vectors.

Orthopoxviruses are being increasingly used as live recombinant vectors for vaccination against numerous infectious diseases in humans, domestic animals, and wildlife. For risk assessments and surveillance, information about the occurrence, distribution and ecology of orthopoxviruses in western Europe is important but has mainly been based on serological investigations. We have examined kidneys, lungs, spleens, and livers of Norwegian small rodents and common shrews (Sorex araneus) for the presence of orthopoxvirus DNA sequences by PCR with primers complementary to the viral thymidine kinase (TK) gene. PCR amplicons were verified as orthopoxvirus specific by hybridization with a vaccinia virus TK-specific probe. A total of 347 animals (1,388 organs) from eight locations in different parts of Norway, collected at different times of the year during 1993 to 1995, were examined. Fifty-two animals (15%) from five locations, up to 1,600 km apart, carried orthopoxvirus DNA in one or more of their organs, most frequently in the lungs. These included 9 of 68 (13%) bank voles (Clethrionomys glareolus), 4 of 13 (31%) gray-sided voles (Clethrionomys rufocanus), 3 of 11 (27%) northern red-backed voles (Clethrionomys rutilus), 16 of 76 (21%) wood mice (Apodemus sylvaticus), and 20 of 157 (13%) common shrews. The previous isolation of cowpox virus from two clinical cases of infection (human and feline) at two of the locations investigated suggests that the viruses detected are cowpox and that some of the virus-carrying small mammalian species should be included among the cowpox virus natural reservoir hosts in Scandinavia and western Europe.

Antibodies against orthopoxviruses in wild carnivores from Fennoscandia.

Two hundred and three sera obtained in 1993-96 from red foxes (Vulpes vulpes), lynx (Lynx lynx), brown bears (Ursus arctos) and wolverines (Gulo gulo) in Fennoscandia (Norway, Sweden, and Finland) were examined for the presence of anti-orthopoxvirus antibodies by a competition enzyme linked immunosorbent assay (ELISA). High prevalences were found for the red foxes in Norway (7/62, 11%) and Finland (7/14, 50%). While only one of 73 (1%) lynx from Finland had anti-orthopoxvirus antibodies, a high prevalence was found in sera from the Sarek National Park in Sweden (5/17, 29%). In addition, anti-orthopoxvirus antibodies were found in one brown bear from the same area (1/45, 2%), whereas none of the 14 wolverines were seropositive. This is the first report of anti-orthopoxvirus antibodies in the brown bear and the lynx, and the first screening for such antibodies in Sweden and Finland. These results indicate that orthopoxviruses are distributed in Sweden and Finland as well as in Norway, and that the red fox and the European lynx may serve as indicator species for the presence of orthopoxviruses in the local populations of small mammals.

The agnogene of the human polyomavirus BK is expressed.

Primate polyomavirus genomes all contain an open reading frame at the 5' end of the late coding region called the agnogene. A simian virus 40 agnoprotein with unknown functions has previously been demonstrated. We now show that a BK virus agnoprotein appears in the perinuclear area and cytoplasm late in the infectious cycle. It is phosphorylated in vivo and coimmunoprecipitates with a subset of host cell proteins.

Serosurvey for orthopoxviruses in rodents and shrews from Norway.

Two hundred and twenty one blood samples representing eight different rodent species and the common shrew (Sorex araneus), collected in Norway between 1993 and 1995, were examined for anti-orthopoxvirus antibodies by a competition enzyme linked imunnosorbent assay (ELISA) and, when possible, an indirect immunofluorescence assay. The serological results indicated that the bank vole (Clethrionomys glareolus), woodmouse (Apodemus sylvaticus) and Norway lemming (Lemmus lemmus) may be reservoir species for orthopoxviruses in Norway, with antibody prevalences of 17 (12/69), 30 (24/81) and 56% (19/34), respectively. Orthopoxvirus infection in lemmings has not been reported previously. On some other small rodent species such as field voles (Microtus agrestis), common rats (Rattus norvegicus), and common shrews, seropositive individuals were detected. However, the total number of tested animals was low, and the role of these species in the epidemiology of orthopoxvirus infections remains unclear. Attempts to isolate orthopoxviruses from these small mammals failed, although orthopoxvirus specific DNA sequences were detected previously in the same animals by the polymerase chain reaction (PCR). The serological results were compared with and discussed in the context of the occurrence of orthopoxvirus-specific DNA sequences, and it is concluded that orthopoxviruses are widely distributed among wildlife in Norway.

Experimental expression in mice and spontaneous expression in human SLE of polyomavirus T-antigen. A molecular basis for induction of antibodies to DNA and eukaryotic transcription factors.

We have previously demonstrated that experimental expression of the polyomavirus transcription factor T-antigen has the potential to induce anti-DNA antibodies in mice. Two sets of independent evidences are presented here that demonstrate a biological relevance for this model. First, we describe results demonstrating that mice inoculated with T-antigen-expressing plasmids produced antibodies, not only to T-antigen and DNA, but also to the DNA-binding eukaryotic transcription factors TATA-binding protein (TBP), and to the cAMP-response-element-binding protein (CREB). Secondly, we investigated whether polyomavirus reactivation occurs in SLE patients, and whether antibodies to T-antigen, DNA, and to TBP and CREB are linked to such events. Both within and among these SLE patients, frequent polyomavirus reactivations were observed that could not be explained by certain rearrangements of the noncoding control regions, nor by corticosteroid treatment. Linked to these events, antibodies to T-antigen, DNA, TBP, and CREB were detected, identical to what we observed in mice. Antibodies recognizing double-stranded DNA were confined to patients with frequent polyomavirus reactivations. The results described here indicate that cognate interaction of B cells recognizing DNA or DNA-associated proteins and T cells recognizing T antigen had taken place as a consequence of complex formation between T ag and DNA in vivo in the context of polyomavirus reactivations.

Human polyomavirus BK and immunogenicity of mammalian DNA: a conceptual framework.

Although the origin of autoimmune antibodies to double-stranded DNA (dsDNA) is not known, the variable-region structures of such antibodies indicate that they are produced in response to antigen-selective stimulation. In accordance with this, results from experiments using artificial complexes of DNA and DNA-binding polypeptides for immunizations have indicated that DNA may induce these antibodies. The immunogenicity of DNA in vivo may therefore depend upon structures, or processes, that render DNA immunogenic. It is therefore crucial to determine the nature of the antigen(s) recognized by anti-DNA antibody-inducing Th cells. We describe here the results of a series of experiments using polyomavirus BK (BKV) inoculation as a model system for initiation of antibodies to DNA, including dsDNA, in animals. From the early observation that BKV had the potential to induce the linked production of antibodies to DNA and histones, we have investigated and described the molecular bases for how this virus may do so. The minimum requirement for DNA to act as an immunogen is the in vivo expression of the BKV early gene encoding the DNA-binding large T-antigen. In the context of in vivo expression of this gene, IgG antibodies to dsDNA, histones, and T-antigen were produced. Monoclonal anti-dsDNA antibodies derived from BKV immunized mice demonstrated variable-region structures highly similar to those of spontaneous anti-DNA antibodies in murine lupus. These results represent a conceptual advance in understanding a potential molecular basis for initiation of autoimmunity in systemic lupus erythematosus and establish a precedent for further studies on polyomavirus expression as one biological origin for anti-dsDNA antibodies in human lupus.

The human polyomavirus BK T antigen induces gene expression in human cytomegalovirus.

Co-infections or co-habitations of cells by two or more viruses may occur in the human organism. Human cytomegalovirus (HCMV) and the human polyomavirus BK (BKV) have common host cells and may both establish lifelong latency/persistence following primary infection. Both viruses may become reactivated by immunosuppression or other conditions which upset host-virus balance, and they encode gene products with the inherent potential of acting as heterologous transacting factors for expression of cellular or viral genes. It has been shown that HCMV induces gene expression and replication of primate polyomaviruses. We now demonstrate that BKV is able to enhance the expression of HCMV immediate early (IE1 and 2) as well as the early (E) protein pp65 during double infections in semi-permissive cells. By transfection experiments it was established that the phenomenon is due to heterologous transcriptional transactivation of the HCMV major IE promoter (MIEP) by the BKV large T antigen, without contribution from the small t antigen.

In vivo expression of a single viral DNA-binding protein generates systemic lupus erythematosus-related autoimmunity to double-stranded DNA and histones.

Although the origin of autoimmune antibodies to double-stranded DNA is not known, the variable-region structures of such antibodies indicate that they are produced in response to antigen-selective stimulation. In accordance with this, results from experiments using artificial complexes of DNA and DNA-binding polypeptides for immunizations have indicated that DNA may induce these antibodies. Hence, the immunogenicity of DNA in vivo may depend upon other structures or processes that may render DNA immunogenic. We report that in vivo expression of a single DNA-binding protein, the polyoma virus T antigen, is sufficient to initiate production of anti-double-stranded DNA and anti-histone antibodies but not a panel of other autoantigens. Expression of a mutant, non-DNA-binding T antigen did result in strong production of antibodies to the T antigen, but only borderline levels of antibodies to DNA and no detectable antibodies to histones. Nonexpressing plasmid DNA containing the complete cDNA sequence for T antigen did not evoke such immune responses, indicating that DNA by itself is not immunogenic in vivo. The results represent a conceptual advance in understanding a potential molecular basis for initiation of autoimmunity in systemic lupus erythematosus.