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J Clin Microbiol 2003 Mar;41(3):1091-100.
Hepatitis C Virus Subtyping by a Core-Envelope 1-Based Reverse
Transcriptase PCR Assay with Sequencing and Its Use in Determining Subtype
Distribution among Danish Patients.
Corbet S, Bukh J, Heinsen A, Fomsgaard A.
Department of Virology, Statens Serum Institut, Denmark..
A reverse transcriptase PCR (RT-PCR) assay using conserved primers
deduced from the core-envelope 1 (C-E1) region of the hepatitis C virus
(HCV) genome was developed for subtyping purposes. The sensitivity and
specificity of this assay tested against two HCV reference panels
containing genotype 1 through 5 subtypes were similar to those of an
RT-PCR assay from the 5'-untranslated region (5'-UTR). The sensitivity
of the RT-PCR typing assay in the more variable C-E1 region was,
however, lower than that of the RT-PCR in the highly conserved 5'-UTR
when testing multiple clinical samples. Thus, 71 (88%) of 81 consecutive
samples from hospitalized Danish patients positive for HCV antibodies
and RNA (5'-UTR) were positive also in the C-E1 RT-PCR assay.
Phylogenetic analysis of the E1 sequences obtained by direct sequencing
of HCV from two reference panels and 71 Danish patients allowed us to
readily distinguish the subtypes. In contrast, phylogenetic analysis of
their corresponding 5'-UTR sequences was able to predict only major
genotypes. Three different genotypes and four subtypes were identified
among Danish samples: 1a (43%), 1b (11%), 2b (6%), and 3a (39%). An isolate
from a Somalian refugee was identified as a new HCV type related to Somalian
isolates described as subtype 3h. The most common genotype in Denmark is
genotype 1 (53%), which is the most difficult to treat. However, Denmark had
the highest prevalence in Europe of subtype 3a, which responds more
favorably to treatment. The described C-E1 RT-PCR with sequencing is
suggested as an easy routine assay for definitive genotyping and subtyping
of HCV.
Vet Microbiol 2003 Apr 2;92(3):197-212
Experimental infection with the Paderborn isolate of classical swine fever virus in 10-week-old pigs: determination of viral replication kinetics by quantitative RT-PCR, virus isolation and antigen ELISA.
Uttenthal A, Storgaard T, Oleksiewicz MB, de Stricker K.
Danish Veterinary Institute, Lindholm, DK-4771, Kalvehave, Denmark
We performed experimental infection in 10-week-old pigs with the Paderborn isolate of classical swine fever virus (CSFV). Despite being epidemiologically linked to the major CSFV outbreak in The Netherlands in 1997, the in vivo replication kinetics of this isolate have to our knowledge not been described in detail previously. We found that oronasal infection with 10(4.7) TCID(50) produced mortality in three out of five pigs after 29-31 days, and severe clinical symptoms in one out of five pigs, while one out of five pigs exhibited no clinical symptoms. At this infection dose, pigs had viral RNA (monitored by quantitative reverse transcription (RT)-PCR) in serum as soon as 2 days post-infection, and excretion of infectious virus (monitored by sentinel pigs) appeared to be virtually concomitant with viremia onset. While virus RNA was cleared from the serum of most pigs after 1-2 weeks, some pigs had viral RNA in serum for more than 30 days, and exhibited only mild clinical symptoms. We observed an excellent correlation between clinical symptoms and viral RNA loads in serum, while serum antibody levels were low. Clinically affected pigs had up to 1000-fold higher serum viral RNA loads than did pigs without clinical symptoms. At this level of infection, and this age group, the Paderborn isolate exhibited a strikingly wide range of replication patterns, which might be relevant to the spread of the virus through susceptible pig populations, and the severity of the 1997-1998
outbreak.
Tjornehoj K, Uttenthal A, Viuff B, Larsen LE, Rontved C, Ronsholt L.
Danish Veterinary Institute, Lindholm, DK-4771, Kalvehave, Denmark
Res Vet Sci 2003 Feb;74(1):55-65
An experimental infection model for reproduction of calf pneumonia with bovine respiratory syncytial virus
(BRSV) based on one combined exposure of calves.
Bovine respiratory syncytial virus (BRSV) has been recognised as an important pathogen in calf pneumonia for 30 years, but surprisingly few effective infection models for studies of the immune response and the pathogenesis in the natural host have been established. We present a reproducible experimental infection model for BRSV in 2-5-month-old, conventionally reared Jersey calves. Thirty-four colostrum-fed calves were inoculated once by aerosol and intratracheal injection with BRSV. Respiratory disease was recorded in 91% of the BRSV-inoculated calves, 72% had an accompanying rise in rectal temperature and 83% exhibited >5% consolidation of the lung tissue. The disease closely resembled natural outbreaks of BRSV-related pneumonia, and detection of BRSV in nasal secretions and lung tissues confirmed the primary role of BRSV. Nine mock-inoculated control calves failed to develop respiratory disease. This model is a valuable tool for the study of the pathogenesis of BRSV and for vaccine efficacy studies.
Vinner L, Wee EG, Patel S, Corbet S, Gao GP, Nielsen C, Wilson JM, Ertl
HC, Hanke T, Fomsgaard A.
Department of Virology, Statens Serum Institut, 5 Artillerivej, 2300 Copenhagen S,
Denmark.
J Gen Virol 2003 Jan;84(Pt 1):203-13.
Immunogenicity in Mamu-A*01 rhesus macaques of a CCR5-tropic human immunodeficiency virus type 1 envelope from the primary isolate (Bx08) after synthetic DNA prime and recombinant adenovirus 5 boost.
Envelopes of primary R5-tropic human immunodeficiency virus type 1 (HIV-1) isolates may be particularly relevant for vaccine purposes and should be evaluated for immunogenicity in animals including macaques before carrying out human vaccine trials. In the present study, the immunogenicities of synthetic HIV-1 env DNA vaccines, which had been derived from the early primary isolate Bx08 and contain humanized codons, were evaluated in mice, guinea pigs and rhesus macaques. Neutralization sensitivity of the HIV-1(Bx08) isolate was found to resemble that of other primary isolate prototypes. Immunogenicity of gp120 delivered as codon-optimized DNA vaccine was comparable to that of recombinant gp120 protein plus adjuvant in mice. Similarly, DNA vaccination of guinea pigs with synthetic gp140(Bx08) and gp150(Bx08) DNA induced a strong antibody response independent of the gene construct and DNA immunization route. Mamu-A*01 rhesus macaques were DNA vaccinated with synthetic gp150(Bx08) or gp140(Bx08) DNA and boosted with a replication-deficient recombinant human adenovirus type 5 expressing a synthetic gp120(Bx08) gene. DNA-vaccinated rhesus macaques developed specific CD8+ T lymphocyte responses and anti-rgp120(IIIb) antibody responses. Both the humoral and cellular responses were significantly improved following intramuscular boosting with the recombinant adenovirus. The demonstrated humoral and cellular immunogenicities of these HIV Bx08 Env vaccines in non-human primates encourages their further development as one component in candidate HIV vaccines for
humans.
Thomsen AR, Nansen A, Madsen AN, Bartholdy C, Christensen JP.
Institute of Medical Microbiology and Immunology, University of Copenhagen,
Copenhagen, Denmark
Immunol Lett 2003 Jan 22;85(2):119-27
Regulation of T cell migration during viral infection: role of adhesion molecules and
chemokines.
T cell mediated immunity and in particular CD8+ T cells are pivotal for the control of most viral infections. T cells exclusively exert their antiviral effect through close cellular interaction with relevant virus-infected target cells in vivo. It is therefore imperative that efficient mechanisms exist, which will rapidly direct newly generated effector T cells to sites of viral replication. In the present report we have reviewed our present knowledge concerning the molecular interactions, which are important in targeting of effector CD8+ T cells to sites of viral infection.
Tove Christensen & Anné Møller-Larsen
Institut for Medicinsk Mikrobiologi og Immunologi, Aarhus Universitet, DK-8000 Århus C.
Endogenous retroviruses in multiple sclerosis. [Humane endogene retrovirus og sygdom?]
Ugeskr Læger 2003;165:556-61.
Endogene retrovirus er efterkommere af »gamle« retrovirusinfektioner, hvor retrovirus har integreret sig i værtens arvemateriale. Disse DNA-sekvenser medvirker i en række processer, såsom artsdannelse, ontogenese, regulering af vævsspecificitet og af genudtryk. Humane endogene retrovirus er i de senere år kommet i fokus i forbindelse med visse typer af cancer samt autoimmune sygdomme. Et ændret udtryk af humane endogene retrovirus er påvist ved systemisk lupus erythematosus, Sjögrens sygdom, reumatoid artrit og muligvis ved insulinkrævende diabetes. Ved multipel sklerose findes produktion af endogene retrovirus, som ellers ikke er replikativt aktive. Det vides endnu ikke, om relationerne mellem humane endogene retrovirus og sygdom er kausale.
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