First detection of tick-borne "Candidatus Neoehrlichia mikurensis" in Denmark 2011.

This is the first reporting of the tick-borne zoonotic bacterium "Candidatus Neoehrlichia mikurensis" in Denmark. A total of 2,625 Ixodes ricinus ticks from 58 locations in Denmark were collected and analysed for "Ca. Neoehrlichia mikurensis". A nested PCR revealed the presence of the bacterium at three geographically separate locations, which indicates that it is widely established in ticks.

Cases of travel-acquired dengue fever in Denmark 2001-2009.

Dengue fever (DF) remains one of the most important emerging infectious diseases. Whereas DF is well recognized in endemic countries, there are indications that the disease is underdiagnosed among travellers to endemic regions. Here, we present the first descriptive survey on cases of travel-acquired DF imported to Denmark diagnosed at the national reference laboratory for dengue virus diagnostics during a 9-year period. In our study, 16 - 46 travel-acquired dengue virus infections were diagnosed per year. DF is mainly imported by adults, mostly men, returning from Southeast Asian countries. The minimum incidence of dengue virus infection among Danish travellers is estimated to be 4.9 per 100,000 travellers. Our results confirm and expand studies from other European countries, and underline the importance of surveillance based on relevant diagnostic analyses.

First identification of tick-borne encephalitis in Denmark outside of Bornholm, August 2009.

The incidence of tick-borne encephalitis (TBE) in Scandinavia is increasing and spreading geographically. Following two clinical cases of TBE hospitalised after tick bites in northern Zealand, Denmark, specific IgM and IgG antibodies against tick-borne encephalitis virus (TBEV) were demonstrated in acute serum samples of these patients. TBEV was identified by RT-PCR in ticks collected from the same location. This is the first report of TBEV in Ixodes ricinus leading to clinical cases in Denmark outside of Bornholm island.

Genome characterisation of the newly discovered avian influenza A H5N7 virus subtype combination.

In Denmark, in 2003, a previously unknown subtype combination of avian influenza A virus, H5N7 (A/Mallard/Denmark/64650/03), was isolated from a flock of 12,000 mallards. The H5N7 subtype combination might be a reassortant between recent European avian influenza A H5, H7, and a third subtype, possibly an H6. The haemagglutinin and the acidic polymerase genes of the virus were closely related to a low-pathogenic Danish H5N2 virus A/Duck/Denmark/65041/04 (H5N2). The neuraminidase gene and the non-structural gene were most similar to the highly pathogenic A/Chicken/Netherlands/1/03 (H7N7) and the human-fatal A/Netherlands/219/03 (H7N7), respectively. The basic polymerase 1 and 2 genes were phylogenetically equidistant to both A/Duck/Denmark/65047/04 (H5N2) and A/Chicken/Netherlands/1/03 (H7N7). The nucleoprotein and matrix gene had highest nucleotide sequence similarity to the H6 subtypes A/Duck/Hong Kong/3096/99 (H6N2) and A/WDk/ST/1737/2000 (H6N8), respectively. All genes of the H5N7 strain were of avian origin, and no further evidence of pathogenicity to humans has been found.

An emerging avian influenza A virus H5N7 is a genetic reassortant of highly pathogenic genes.

We full genome characterised the newly discovered avian influenza virus H5N7 subtype combination isolated from a stock of Danish game ducks to investigate the composition of the genome and possible features of high pathogenicity. It was found that the haemagglutinin and the acidic polymerase genes were closely related to a low pathogenic H5 strain (A/Duck/Denmark/65047/04 H5N2). The neuraminidase and the non-structural genes were closely related to the highly pathogenic H7N7 strains from The Netherlands 2003. The basic polymerase genes 1 and 2 were shared between the Danish H5N7 and H5N2 and the H7N7 from The Netherlands. The nucleoprotein and the matrix genes were closely related to H6 strains. Thus, the new H5N7 subtype share genes with H5, H7 and H6 subtypes and possesses internal genes originating from highly pathogenic strains. The findings emphasize the need for surveillance presumed low pathogenic avian influenza A viruses.

New avian influenza A virus subtype combination H5N7 identified in Danish mallard ducks.

During the past years increasing incidences of influenza A zoonosis have made it of uppermost importance to possess methods for rapid and precise identification and characterisation of influenza A viruses. We present here a convenient one-step RT-PCR method that will amplify full-length haemagglutinin (HA) and neuraminidase (NA) directly from clinical samples and from all known subtypes of influenza A. We applied the method on samples collected in September 2003 from a Danish flock of mallards with general health problems and by this a previously undescribed influenza A subtype combination, H5N7, was identified. The HA gene showed great sequence similarity to the highly pathogenic avian influenza A virus (HPAIV) A/Chicken/Italy/312/97 (H5N2); however, the cleavage site sequence between HA1 and HA2 had a motif typical for low pathogenic avian influenza viruses (LPAIV). The full-length NA sequence was most closely related to the HPAIV A/Chicken/Netherlands/01/03 (H7N7) that infected chickens and humans in the Netherlands in 2003. Ten persons with direct or indirect contact with the Danish mallard ducks showed signs of influenza-like illness 2-3 days following the killing of the ducks, but no evidence of influence infections was detected. To our knowledge this is the first report of an H5N7 influenza A virus.

Sensitive quantitative predictions of peptide-MHC binding by a 'Query by Committee' artificial neural network approach.

We have generated Artificial Neural Networks (ANN) capable of performing sensitive, quantitative predictions of peptide binding to the MHC class I molecule, HLA-A*0204. We have shown that such quantitative ANN are superior to conventional classification ANN, that have been trained to predict binding vs non-binding peptides. Furthermore, quantitative ANN allowed a straightforward application of a 'Query by Committee' (QBC) principle whereby particularly information-rich peptides could be identified and subsequently tested experimentally. Iterative training based on QBC-selected peptides considerably increased the sensitivity without compromising the efficiency of the prediction. This suggests a general, rational and unbiased approach to the development of high quality predictions of epitopes restricted to this and other HLA molecules. Due to their quantitative nature, such predictions will cover a wide range of MHC-binding affinities of immunological interest, and they can be readily integrated with predictions of other events involved in generating immunogenic epitopes. These predictions have the capacity to perform rapid proteome-wide searches for epitopes. Finally, it is an example of an iterative feedback loop whereby advanced, computational bioinformatics optimize experimental strategy, and vice versa.

No association of HIV-1 envelope (C2-V3-C3) sequence pattern with long-term nonprogression.

We previously identified a group of 10 long-term nonprogressors (LTNP) with HIV-1 infection. In this study, we have sequenced the envelope gene (C2-V3-C3) from the 10 LTNPs and from a control group of 9 people with rapidly progressing infection (RPI). The 19 individuals' CCR5 genotype and virus phenotype (i.e., syncytium-inducing/non-syncytium-inducing [SI/NSI]) were obtained from a previous study. A phylogenetic tree was constructed containing the 19 envelope sequences together with 42 local control env sequences obtained from other studies. Analysis of the phylogenetic tree did not reveal any relation between the envelope gene (C2-V3-C3) from LTNPs versus RPIs. When data from the CCR5 genotype and the virus phenotype were assembled in the phylogenetic tree, no significant clustering was observed. From alignment of the protein sequences, we found a possible N-glycan in position aa294 in env that was conserved in only 1 of 10 LTNPs; however, it was conserved in 6 of 9 RPIs. Our study could not demonstrate any association between LTNPs and the sequenced envelope gene segment (C2-V3-C3). This lack of association could be due to the relatively small sample size of the data set. Nor did we find any relation between the CCR5 genotype or the SI/NSI phenotype with the sequenced envelope genes from the 19 participants. The possible N-glycan position we have described is an interesting observation that may require further investigation.

HSV-1--induced acute retinal necrosis syndrome presenting with severe inflammatory orbitopathy, proptosis, and optic nerve involvement.

OBJECTIVE: To present a unique case in which orbital inflammation, proptosis, and optic neuritis were the initial symptoms of acute retinal necrosis (ARN). The clinical presentation of ARN, as well as the currently recommended diagnostic procedures and guidelines for medical treatment of ARN, are summarized. DESIGN: Interventional case report. TESTING: Polymerase chain reaction (PCR) techniques were made on the vitreous for cytomegalovirus, Epstein-Barr virus, herpes simplex virus (HSV), varicella zoster virus, and toxoplasmosis. A full laboratory evaluation was made together with HLA-typing and serologic tests measuring convalescent titers for HSV and other micro-organisms. Magnetic resonance imaging scan, computed tomography (CT) scan, and fluorescein angiographic examination were performed. The patient was treated with acyclovir and oral prednisone. MAIN OUTCOME MEASURES: The patient was evaluated for initial and final visual acuity and for degree of proptosis, periocular edema, and vitreitis. RESULTS: The first symptoms and signs of ARN were eye pain, headache, proptosis, and a swollen optic nerve on CT scan. Other than increased C-reactive protein, all blood samples were normal. PCR was positive for HSV-type I in two separate vitreous biopsies. The patient had the strongly ARN-related specificity HLA-DQ7. CONCLUSIONS: This is the first report of HSV-induced ARN presenting with inflammatory orbitopathy and optic neuritis. Polymerase chain reaction for HSV-1 was positive more than 4 weeks after debut of symptoms, which is a new finding. The combination of severe vitreitis and retinal whitening, with or without proptosis, should alert the clinician to the possibility of herpes infection and treatment with intravenous acyclovir started promptly.

Construction, biological activity, and immunogenicity of synthetic envelope DNA vaccines based on a primary, CCR5-tropic, early HIV type 1 isolate (BX08) with human codons.

So far codon-optimized HIV-1 envelope genes have been investigated for the T cell line-adapted strain MN, which differs in several aspects from primary isolates. Envelopes of primary isolates may be more relevant for vaccine purposes. This article describes for the first time the engineering and characterization of four "humanized" genes encoding the secreted gp120/gp140, or the membrane-bound gp150/gp160, of a primary CCR5 tropic, clade B, clinical isolate HIV-1(BX08). The genes were built in fragments for easy cassette exchange of regions important for immunogenicity, function, and expression. The transcription and expression of the synthetic genes in mammalian cell lines were Rev independent and highly increased. Increased expression of membrane-bound gp160 induced a high cytopathic effect in U87.CD4.CCR5 cells. Gene gun and intramuscular DNA vaccination in mice induced a strong specific cytotoxic T lymphocyte response independent of the gene construct, expression level, or DNA immunization route. In contrast, the highest anti-gp120 antibody levels were induced by synthetic genes encoding the secreted glycoproteins followed by gp160/gp150. Unlike HIV-1(MN), HIV-1(BX08) V3 was not immune dominant. Despite the high antibody response only low and inconsistent neutralizing titers to the homologous HIV-1 isolate were measured. However, neutralization of SHIV89.6P could be obtained. Thus, the neutralizing epitopes on the cell line-adapted SHIV89.6P and HIV-1(MN) may be more antigenically available for the cross-neutralizing antibodies induced. In conclusion, complete "humanization" of the DNA vaccine genes failed to induce a consistent neutralizing antibody response, albeit expression and immunogenicity of the primary HIV-1 glycoproteins were greatly improved. Optimization in terms of improving neutralization may require further modifications of the DNA vaccine gene. The synthetic cassette construct described is a convenient tool developed to investigate this further.

A phylogenetic analysis elucidating a case of patient-to-patient transmission of hepatitis C virus during surgery.

A phylogenetic hepatitis C virus (HCV) assay based on the core-Envelope 1 (C-E1) region was developed and used to elucidate a case of a patient-to-patient transmission. The index patient showed clinical symptoms of hepatitis seven weeks after surgery for hallux valgus under general anaesthesia. She progressed to a chronic persistent infection as indicated by positive HCV PCR results two years after surgery. Before her operation, a patient with HCV antibodies and positive HCV PCR had undergone surgery in the same room. There were two possibilities whereby the index patient could have been infected with hepatitis C, either through her work as a nurse or by transmission during surgery. By sequencing the 5' non-coding region PCR product, we found that both patients were infected with genotype 1a. Phylogenetic analysis with the variable C-E1 region suggested that the two patients clustered together with a bootstrap 100% in a tree with 75 sequence references. We further performed a phylogenetic analysis in this region with the genotype 1a reference sequences and an additional 25 genotype 1a sequences consecutively collected from Danish patients with HCV. The two patients still clustered together, supported by a high bootstrap 1000 value of 999. Homology analyses combined with the epidemiological findings indicate that the patient operated on in the same room before the index case was the most likely source of transmission. The mode of transmission could not be conclusively established, but a reusable part of the anaesthetic respiratory circuit is a possibility and a well known risk.

Identifying cytotoxic T cell epitopes from genomic and proteomic information: "The human MHC project.".

Complete genomes of many species including pathogenic microorganisms are rapidly becoming available and with them the encoded proteins, or proteomes. Proteomes are extremely diverse and constitute unique imprints of the originating organisms allowing positive identification and accurate discrimination, even at the peptide level. It is not surprising that peptides are key targets of the immune system. It follows that proteomes can be translated into immunogens once it is known how the immune system generates and handles peptides. Recent advances have identified many of the basic principles involved. The single most selective event is that of peptide binding to MHC, making it particularly important to establish accurate descriptions and predictions of peptide binding for the most common MHC variants. These predictions should be integrated with those of other steps involved in antigen processing, as these become available. The ability to translate the accumulating primary sequence databases in terms of immune recognition should enable scientists and clinicians to analyze any protein of interest for the presence of potentially immunogenic epitopes. The computational tools to scan entire proteomes should also be developed, as this would enable a rational approach to vaccine development and immunotherapy. Thus, candidate vaccine epitopes might be predicted from the various microbial genome projects, tumor vaccine candidates from mRNA expression profiling of tumors ("transcriptomes") and auto-antigens from the human genome.

Induction of cytotoxic T-cell responses by gene gun DNA vaccination with minigenes encoding influenza A virus HA and NP CTL-epitopes.

Cytotoxic T-lymphocyte (CTL) response is an important component of anti-viral immunity. CTLs are specific to short peptides presented by MHC-I molecules and immunisation with the exact peptide sequence introduced in the cytosol is therefore a minimal approach, which potentially affords a high degree of controllability. We have examined the induction of murine CTL's by this approach using DNA plasmid minigene vaccines encoding known mouse K(k) minimal CTL epitopes (8 amino acids) from the influenza A virus hemagglutinin and nucleoprotein. We here report that such an approach is feasible and that wild type influenza virus flanking amino acid sequences can influence the CTL response but are not essential for optimal CTL induction. We also examined the effect of different new amino acid sequences flanking the CTL epitopes. In one version, two CTL epitopes were linked together as 'string of beads'. This did not improve CTL induction. In another version, one CTL epitope was inserted into a known T-helper protein (HBsAg). This did significantly augment the response probably due to immunological help from HBsAg Th epitopes. Finally, the CTL inducing minigene DNA vaccines were compared with Flu-induced CTL responses and tested for their protective effect against a lethal influenza A virus infection in mice and no effect was found. We conclude that a specific and highly directed CTL induction is possible by unlinked minigene DNA immunisation, but that CTL induction solely is not always sufficient to provide protection.

Complete protection against lethal Toxoplasma gondii infection in mice immunized with a plasmid encoding the SAG1 gene.

Infection with the protozoan parasite Toxoplasma gondii is transmitted to humans from infected animals by tissue cysts and oocysts excreted by cats. Immunization with inactivated parasites or recombinant proteins has at best shown partial protection. We constructed a plasmid expressing the SAG1 surface antigen of T. gondii, p1tPASAG1, and showed that animals immunized with the plasmid produce anti-SAG1 antibodies which recognize the native SAG1. Mice immunized with p1tPASAG1 showed 80 to 100% protection against challenge with the non-cyst-producing, virulent RH isolate, compared to an 80% mortality in mice immunized with empty plasmid, which is the greatest efficacy of any vaccine against T. gondii produced so far. The SAG1 molecule was analyzed for potential cytotoxic T-lymphocyte (CTL) epitopes, and four peptides with the best fit were synthesized. The ability of the peptides to stimulate gamma interferon production by CD8(+) T cells from p1tPASAG1-immunized mice was tested in an ELISPOT assay, and one new CTL epitope was identified. Adoptive transfer of CD8(+) T cells from p1tPASAG1-immunized to naïve mice showed partial protection. In conclusion, DNA vaccination with p1tPASAG1 gave effective protection in mice against T. gondii infection and the protection could be adoptively transferred by purified CD8(+) T cells.

Transmission of a minor variant of transcriptionally active HIV-1.

We investigated the genotypic variation of pro-viral human immunodeficiency virus type 1 (HIV-1) DNA in a virus donor-recipient pair by comparing sequences from the HIV-1 V3 region of the gp120 gene and the p17gag gene. We found that the transmitted virus was a minor variant in the donor's virus population in blood. Possible selection mechanisms within the host (neutralization by antibodies) were studied in order to investigate whether antibodies could explain the conserved HIV genotype found in the recipient. In conclusion, our data indicate that a minor variant of pro-viral transcriptionally active HIV-1 found in PBMC was transmitted from donor to recipient. Development of a homogeneous genotype regarding the V3 region (V3d-B1(1)) of pro-viral DNA in the recipient's PBMC and a partially homogeneous genotype regarding the HIV-RNA was possibly caused by an envelope associated selection that is not dependent upon neutralizing antibodies.

[Hepatitis G virus or hepatitis GB virus C]. / Hepatitis G-virus eller GB virus-C.

In 1995 two novel human virae were identified independently, GB virus C and hepatitis G virus, as possible etiological agents for non-A-E viral hepatitis. GBV-C and HGV are two isolates of the same virus. GBV-C/HGV is an RNA virus of app. 9 kb belonging to the Flaviviridae family. GBV-C/HGV is transmitted intravenously, sexually and vertically from mother to child. GBV-C/HGV can be detected in blood by RT-PCR and serological tests based on the envelope 2 protein. Infection by GBV-C/HGV is frequent and 1-10% of blood donors in Western countries have been found positive by PCR. Acute GBV-C/HGV infections are in some cases associated with mild hepatitis. About 10-20% of the infections become chronically persistent but are not associated with chronic hepatitis or other known disease. GBV-C/HGV is not found in hepatocytes and is not infectious in the chimpanzee model, therefore GBV-C/HGV is not a classical hepatitis virus. Detection of GBV-C/HGV by PCR may be indicated in the diagnosis of acute hepatitis.

Mutations in CCR5-coding sequences are not associated with SIV carrier status in African nonhuman primates.

African monkeys can be naturally infected with SIV but do not progress to AIDS. Since mutations in the human CCR5 gene have been shown to influence susceptibility to HIV infection and disease progression, we have now investigated whether mutations in CCR5-coding sequences in African nonhuman primates can explain species-specific differences in susceptibility to lentiviral infection. The animals studied comprise chronically infected monkeys corresponding to four natural hosts of SIV (Cercopithecus aethiops, Cercopithecus pygerythrus, Cercopithecus sabaeus, and Cercopithecus tantalus), noninfected animals from three species that are known to be susceptible to SIV infection (Cercopithecus patas, Cercopithecus Ihoesti, and Pan troglodytes), and monkeys of six species that do not carry SIV in the wild (Cercocebus galeritus, Cercocebus aterrimus, Cercopithecus ascanius, Cercopithecus nictitans, Cercopithecus neglectus, and Cercopithecus cephus). We observed a high degree of genetic divergence among the species. The rate of accumulation of amino acid mutations was, however, not higher in SIV carriers than in other nonhuman primates. No homozygous premature stop codons, deletions, or frameshift mutations were detected. In at least two animals, one infected AGM (Cercopithecus tantalus) and one noninfected monkey (Cercocebus aterrimus), the CCR5 alleles identified encode functional proteins, as they were identical in terms of amino acid sequence to that of functional CCR5 reported in the literature. We found no other consistent differences in the genetic variability of CCR5-coding sequences between the nonhuman primates that are carriers of SIV and those that are not.

Gene gun DNA vaccination with Rev-independent synthetic HIV-1 gp160 envelope gene using mammalian codons.

DNA immunization with HIV envelope plasmids induce only moderate levels of specific antibodies which may in part be due to limitations in expression influenced by a species-specific and biased HIV codon usage. We compared antibody levels, Th1/Th2 type and CTL responses induced by synthetic genes encoding membrane bound gp160 versus secreted gp120 using optimized codons and the efficient gene gun immunization method. The in vitro expression of syn.gp160 as gp120 + gp41 was Rev independent and much higher than a classical wt.gp160 plasmid. Mice immunized with syn.gp160 and wt.gp160 generated low and inconsistent ELISA antibody titres whereas the secreted gp120 consistently induced faster seroconversion and higher antibody titres. Due to a higher C + G content the numbers of putative CpG immune (Th1) stimulatory motifs were highest in the synthetic gp160 gene. However, both synthetic genes induced an equally strong and more pronounced Th2 response with higher IgG1/IgG2a and IFNgamma/IL-4 ratios than the wt.gp160 gene. As for induction of CTL, synthetic genes induced a somewhat earlier response but did not offer any advantage over wild type genes at a later time point. Thus, optimizing codon usage has the advantage of rendering the structural HIV genes Rev independent. For induction of antibodies the level of expression, while important, seems less critical than optimal contact with antigen presenting cells at locations reached by the secreted gp120 protein. A proposed Th1 adjuvant effect of the higher numbers of CpG motifs in the synthetic genes was not seen using gene gun immunization which may be due to the low amount of DNA used.

Multicenter quality assessment of PCR methods for detection of enteroviruses.

We conducted a multicenter evaluation of commercial and in-house PCR methods for the detection of enteroviruses. Three coded panels of test and control RNA samples, artificial clinical specimens, and representative enterovirus serotypes were used to assess amplification methods, RNA extraction methods, and reactivities with different enterovirus serotypes. Despite several differences between PCR methods, there was good agreement, although some variation in sensitivity was observed. Most PCR methods were able to detect enterovirus RNA derived from 0.01 50% tissue culture infective dose (TCID50) and were able to detect at least 1 TCID50 of enterovirus in cerebrospinal fluid, stool, or throat swab specimens. Most were also able to detect a wide range of enterovirus serotypes, although serotypic identification was not possible. Some laboratories experienced false-positive results due to PCR contamination, which appeared to result mainly from cross-contamination of specimens during RNA extraction. Provided that this problem is overcome, these PCR methods will prove to be a sensitive and rapid alternative to cell culture for the diagnosis of enterovirus infection.