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Biomedical subjects

L Haas

Publications and source records attributed to L Haas.

At least 55 records · Page 3Linked to original sources
PubMed1993

RNA insertions and gene duplications in the nonstructural protein p125 region of pestivirus strains and isolates in vitro and in vivo.

Sixteen cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strains/isolates were screened for the existence of RNA insertions in the p125 gene region and/or for p80 gene duplications using the polymerase chain reaction after reverse transcription. Three strains/isolates were shown to contain insertions, and in three others gene duplications were demonstrated. One strain was shown to contain a gene duplication in addition to an insertion. Insertions or duplications were absent in the genomes of 10 non-cp BVD virus strains/isolates, in 4 hog cholera (HC) virus strains, and in 9 of the 16 cp BVD virus strains/isolates analyzed. These results indicate that RNA insertions and gene duplications, although significant, are not the only mechanism contributing to cytopathogenicity in vitro. The RT-PCR was also applied to RNA isolated from tissues of a calf suffering from mucosal disease. Insertion-containing amplification products could be demonstrated in an organ-specific pattern.

Animals↗
PubMed1993

Distribution of cytopathogenic and noncytopathogenic bovine virus diarrhea virus in tissues from a calf with experimentally induced mucosal disease using antigenic and genetic markers.

A comparative analysis of the distribution of cytopathogenic (cp) and noncytopathogenic (ncp) bovine virus diarrhea disease (BVD) virus in tissues from a calf with experimentally induced mucosal disease was performed using immunohistology and polymerase chain reaction after reverse transcription (RT-PCR) of viral RNA. For immunohistology, an antigenic marker on the superinfecting cp BVD virus defined by a monoclonal antibody (mab) was used, and overall presence of antigen was assessed with a pestivirus specific mab. The primers selected for RT-PCR detected the genomic insertion in the p125 region of the superinfecting cp BVD virus. Both methods gave consistent results.

Animals↗
PubMed1993

Detection of bovine viral diarrhea virus RNA in formalin-fixed, paraffin-embedded brain tissue by nested polymerase chain reaction.

Isolation and amplification of RNA from formalin-fixed, paraffin-embedded tissues is delicate due to its fragility and ubiquitous ribonucleases. For retrospective studies, however, a convenient procedure for the detection of RNA in archived material is of great value. Bovine viral diarrhea (BVD) virus is a member of the pestivirus genus in the family Flaviviridae. Different protocols for the isolation of BVD virus RNA from fresh and autolytic as well as from routinely formalin-fixed and paraffin-embedded brain tissue of BVDV-infected calves were compared. The polymerase chain reaction (PCR) after reverse transcription (RT-PCR) was carried out subsequently for the detection of viral RNA. Using proteinase K digestion of deparaffinized tissue sections without additional ribonuclease inhibitors and subsequent nested PCR, a 803 bp fragment of the gene coding for the nonstructural protein p125 of BVD virus could be consistently detected. In addition, BVD virus RNA was detected by RT-PCR from non-fixed brain tissue after 10 days of autolysis.

Animals↗
PubMed1993

Prolonged persistence of cytopathogenic bovine viral diarrhea virus (BVDV) in a persistently viremic cattle.

A bull persistently viremic with noncytopathogenic (ncp) BVDV was inoculated with the cytopathic (cp) BVDV strain TGAC, which had been found to be antigenically different from the endogenous ncpBVDV (ncpW8). Neutralizing antibodies against strains NADL and TGAC were detectable 12 days and four weeks post infection, respectively. The animal developed fever and diarrhea 15 weeks post infection. On days 3 and 8 after onset of diarrhea a cpBVDV (cpX) was isolated from feces. Antigenic analysis using monoclonal antibodies (MoAbs) showed that cpX and the endogenous ncpBVDV (ncpW8) had identical reactivity patterns except for one epitope that was neither expressed on TGAC nor on ncpW8. Using polymerase chain reaction analysis it was shown that both TGAC and cpX contained a p8 phi gene duplication combined with genomic insertions of identical size. Restriction enzyme analysis of the TGAC and cpX amplicons using four enzymes showed an identical cleavage pattern, except for HaeIII digestion where an additional fragment was observed with cpX. These results suggest that cpBVDV strain TGAC persisted in the viremic animal and apparently caused disease after 15 weeks.

Animals↗
PubMed1992

Molecular and serological studies on the recent seal virus epizootics in Europe and Siberia.

The virus epizootics which occurred in seals in both Europe and Siberia during 1987/1988 were caused by two different morbilliviruses, referred to as phocid distemper virus (PDV) 1 and 2, respectively. Molecular and serological studies have shown that the European virus is quite distinct from canine distemper virus (CDV), its closest relative in the morbillivirus group. Analysis of tissues obtained from infected seals from a wide geographical distribution over Northern Europe showed that the infectious agent (PDV 1) was identical in all cases. Nucleotide sequence analysis of one of the virus genes suggested that this virus has evolved away from CDV over a long time period and is most probably an enzootic virus of marine mammals. In contrast, the virus (PDV 2) which caused the deaths of many Siberian seals was indistinguishable, both serologically and at the molecular level, from CDV and must have originated from a land source.

Animals↗
PubMed1992

Bovine leukemia virus gene expression in vivo.

The in vivo transcriptional status of bovine leukemia virus was assessed at three stages of infection during the progression of the disease: aleukemic stage, persistent lymphocytosis, and leukemia/lymphosarcoma. Bovine leukemia virus transcripts could be amplified from total or cytoplasmic enriched lymphocyte RNA by reverse transcription polymerase chain reaction in cells from all but a few aleukemic animals. With primer pairs diagnostic for differentially spliced transcripts (full length-genomic, envelope, tax/rex, and alternatively spliced), a trend toward exclusion of both full-length and envelope RNAs, with retention of the tax/rex message, appears as leukemia/lymphosarcoma develops.

Base Sequence↗
PubMed1991

Detection of phocid distemper virus RNA in seal tissues using slot hybridization and the polymerase chain reaction amplification assay: genetic evidence that the virus is distinct from canine distemper virus.

Slot hybridization and the polymerase chain reaction (PCR) after reverse transcription (RT) were used to detect RNA extracted from tissues of seals after naturally occurring disease and experimental infection with phocid distemper virus (PDV). A phosphoprotein (P) gene-specific cDNA served as a probe for both slot hybridization and the identification of PCR-generated fragments by Southern blotting. As primers for the PCR assay PDV P gene-derived oligonucleotides were used. Hybridization, PCR and partial nucleic acid sequence analysis clearly demonstrated that PDV is a distinct virus (most closely related to canine distemper virus) within the morbillivirus group. PCR, when combined with Southern blot hybridization, was clearly superior to slot hybridization and more sensitive than cell culture isolation and immunofluorescence assays for the detection of virus in tissues. Considerable amounts of viral RNA could be demonstrated in the lungs and spleens. In experimentally infected animals a large quantity of virus-specific RNA was additionally found in colon samples. Using RT-PCR in combination with Southern blotting. PDV could be demonstrated in buffy coat cells using a simple and fast cell lysis procedure.

Animals↗
PubMed1990

The monomer covalently closed linear replicative form DNA is an intermediate of Aleutian disease parvovirus DNA replication.

In this report we present data indicating that the recently detected monomer covalently closed linear replicative form DNA (Mccl RF DNA) is an intermediate of Aleutian disease virus (ADV) DNA replication. This DNA molecule is characterized by covalently closed terminal hairpins on either end of the linear ds genomic molecule. Its first detection early after infection in vitro, the association with ADV-specific replication complexes and the de novo synthesis of Mccl RF DNA by isolated replication complexes point to an important role in parvoviral DNA replication. The presence of the Mccl RF DNA in extracts of virus-producing bone marrow cells of naturally infected mink rules out the possibility that this DNA form represents a mere cell culture artefact. The detection of the ADV non-structural protein 1 (NS1) in replication complexes supports the view that this protein is involved in ADV DNA replication.

Aleutian Mink Disease Virus↗
PubMed1990

Violet mink develop an acute disease after experimental infection with Aleutian disease virus (ADV) isolate ADV SL3.

Six-Aleutian (aa)-genotype violet mink were infected intraperitoneally with the Aleutian Disease Virus (ADV) bone marrow derived isolate ADV SL3. All animals developed virus-specific antibodies and hypergammaglobulinaemia. Mortality during the fourteen week duration of the infection was 50%. The virus induced (histo)pathological lesions typical for Aleutian Disease. By immunohistochemical examination using a virus capsid-specific monoclonal antibody viral antigen was detected in lymph nodes, spleen, kidneys and once in hepatic Kupffer cells. By Southern blot and in situ hybridization studies with strand-specific RNA probes able to distinguish viral replicative forms from merely sequestered genomic DNA, ADV replication was detected in mesenteric lymph nodes and spleen. In one mink DNA replicative forms were also found in bone marrow cells or mononuclear cells of the peripheral blood, respectively. Only single-stranded viral DNA was detected in liver, kidney, gut and lung of infected animals. From Southern blot hybridization results a different, possibly organ-specific permissiveness of ADV in vivo is suggested.

Acute Disease↗
PubMed1990

Antigen distribution in organs of mink with Aleutian disease parvovirus infection.

On tissues from naturally infected non-Aleutian mink an immunohistological study was performed using monoclonal antibodies and the immunoperoxidase method. Structural proteins of ADV were demonstrated in cryosections and in ethanol-fixed and paraffin-embedded material which provide antigen detection in a similar amount together with good histological structure. In lymphoid organs viral antigen was restricted to B-cell areas, particularly lymphoid follicles. The pattern of antigen distribution was typical for follicular dendritic cells which are capable to retain immune complexes. Beside macrophages in the interior of lymphoid follicles most likely proliferating B-lymphoblasts reveal nuclear and cytoplasmatic presence of structural proteins indicating viral replication. Cells of the mononuclear phagocyte system such as cells of lymphatic sinuses and hepatic Kupffer cells harbor viral protein in the cytoplasm, probably resulting from phagocytosis of immune complexes. Renal glomeruli were consistently negative for virus antigen whereas in interstitial infiltrates cells resembling macrophages stained positive for ADV structural proteins.

Aleutian Mink Disease↗
PubMed1990

Use of biotechnical methods in veterinary medicine.

Biotechnological methods offer promising approaches for improved diagnostic and prophylactic purposes. The following biotechnological techniques are used in the Institute of Virology at the Hanover Veterinary School:--Production of monoclonal antibodies directed against viral and bacteria-specific antigens such as bovine virus diarrhoea virus, classical swine fever (hog cholera) virus, feline leukaemia virus, animal parvoviruses, Alphavirus, Brucella and Francisella--Establishment of improved and sensitive diagnostic enzyme immunoassays (ELISA) using monoclonal antibodies--Molecular cloning and sequencing of classical swine fever virus RNA and parvovirus DNA--Development of diagnostic hybridisation techniques (dot, slot, Southern and Northern blot, in situ, oligonucleotides)--Detection of viral genomes in tissues of infected animals--Development of synthetic oligopeptides as diagnostic antigens and as potential immunogens for vaccines. Currently available techniques used in basic research (e.g. pathogenesis studies) will be tested for their application in routine diagnosis of viral diseases, e.g. by molecular hybridisation. Some techniques need to be simplified (e.g. RNA extraction procedures) and, particularly, alternative labelling schedules must be developed (e.g. biotin or sulfone labelling instead of radionuclides).

Aleutian Mink Disease Virus↗
PubMed1990

Mechanisms contributing to the virus persistence in Aleutian disease.

In this review published results and further studies concerning the persistence of Aleutian disease virus (ADV) isolate SL3 are presented. By Southern blot and in situ hybridization with strand-specific RNA probes focal replication of ADV-DNA was demonstrated in spleen, mesenteric lymph nodes, sporadically in mononuclear cells of the peripheral blood and bone marrow cells. These findings further support the concept of the lymphotropism of ADV. All cell culture-adapted ADV strains appear to have a ts-defect. Our in vitro studies indicate that the ADV isolate G(orham) induced the synthesis of comparable amounts of viral replicative DNA and viral proteins VP1 and VP2 at the non-permissive temperature of 37 degrees C. However, the viral progeny DNA synthesis was about threefold less at 37 degrees C compared to the permissive temperature of 32 degrees C. These findings suggest that the reduced level of viral progeny DNA at 37 degrees C accounts for the reduced production of infectious ADV. Finally, we provided experimental evidence that the apparent lack of neutralizing antibodies in AD is due to the masking of critical viral epitopes by cellular phospholipids.

Aleutian Mink Disease↗
PubMed1990

[Equine viral arteritis].

Equine viral arteritis (EVA) caused by the equine arteritis virus, member of the genus arterivirus within the family of Togaviridae was recently isolated from the seminal plasma of two stallions indicating that the virus infection is also prevalent in the Federal Republic of Germany despite the apparent lack of acute clinical symptoms in the horse population. These findings are further supported by data from serological screenings. Out of 739 horse sera, 28 (3.8%) were found to have EVA virus-specific antibodies with titers greater than or equal to 4. It is important to note that the percentage of sero-positive horses was found to be increased from 1.8% in 1987-1988 to 6.8% in 1989. A voluntary prophylactic programme based on virological and serological tests is proposed to prevent further spread of the EVA virus infection. The pathogenesis, clinical symptoms and diagnosis of EVA are also reviewed.

Animals↗
PubMed1990

Detection of phocine distemper virus using the polymerase chain reaction.

During the fatal seal epizootics in the North and Baltic Seas in summer 1988 a virus was isolated which was shown to be the causal agent. It was subsequently classified as morbillivirus by neutralization assays, reaction with monoclonal antibodies and nucleic acid hybridization studies. The virus (tentatively called Phocine Distemper Virus, PDV) is difficult to grow in culture making rapid diagnosis difficult. We have used the Polymerase Chain Reaction (PCR) as an alternative and fast method to detect the presence of virus-specific nucleic acid and we describe here the amplification of cell culture derived PDV RNA in a "one-tube" reaction using heterologous (Rinderpest Virus cDNA derived) F gene primers. The resulting 370 bp DNA fragment was shown to be morbillivirus derived by Southern blot hybridization using cloned RPV F gene as probe.

Animals↗
PubMed1989

Herpesvirus in harbour seals (Phoca vitulina): isolation, partial characterization and distribution.

From post-mortem material (liver and lung) and leucocytes of four (3.6%) out of 112 examined harbour seals during the seal epizootic in 1988 six cytopathogenic viral isolates were obtained which were provisionally classified as herpes-like viruses. Results of physico-chemical and electron microscopic investigations suggested their relationship to the herpesvirus family. Serological examinations were carried out with sera from wildlife as well as captive animals using herpesvirus isolates from four different seals. The neutralization tests revealed as only moderate distribution of seropositive reagents up to 53% of the wildlife seal population. Amongst the seals in the orphanage of Norddeich a very small number of seropositive animals was found. The results obtained indicated a minor role of herpesviruses as primary cause of seal mortality in the North Sea during the 1988 season.

Animals↗
PubMed1989

[Probe seeks gene: nucleic acid hybridization as a current contribution to virologic diagnosis].

Molecular nucleic acid hybridization is based on the ability of single-stranded DNA/RNA to form hybrids with complementary labeled nucleic acids. This review shortly describes the components of this technique and presents the most important hybridization methods (spot-, Southern blot-, in situ-hybridization). The (potential) applications of nucleic acid hybridization as a diagnostic tool are discussed (e.g., for viruses which grow insufficiently or not at all in cell culture; virus latency; viruses with labile or antigenically variable envelope proteins, resp.; for virus classification) and yet existing limitations are indicated. An impetus for this technique in means of diagnostic application is expected to result from the Polymerase Chain Reaction (PCR) in the next future.

Animals↗
PubMed1989

[Review article: actual danger to the domestic animal populations of exotic animal epidemics].

Most of the formerly important virus diseases like foot-and-mouth disease and enzootic bovine leukosis were eradicated in the Federal Republic of Germany during the recent decades. However, there is a continuous menace of our domestic animal population by exotic virus epidemics related to the concentration of animals in large farms, the intensified international trade of animals and their meat or milk products, and the introduction of a common European market starting in 1992. This view is emphasized by the recent outbreaks of African horse sickness in Spain in 1987/1988. In this article, foot-and-mouth disease and African horse sickness will be described as potentially dangerous virus epidemics. Furthermore, the occurrence of formerly unknown diseases has to be considered. Haemorrhagic disease of rabbits which was recently introduced in Germany is an example of new developments in virus epidemics. These three diseases, their epidemiology and the biology of the corresponding viruses will be discussed in detail.

African Horse Sickness↗