Lothar Zöller

A Major Antigenic Domain of Hantaviruses is Located on the Aminoproximal Site of the Viral Nucleocapsid Protein

Hantavirus nucleocapsid protein has recently been shown to be an immunodominant antigen in hemorrhagic with renal syndrome (HFRS) inducing an early and long-lasting immune response. Recombinant proteins representing various regions of the nucleocapsid proteins as well as segments of the G1 and the G2 glycoproteins of hantavirus strains CG18-20 (Puumala serotype) and Hantaan 76-118 have been expressed in E. coli. The antigenicity of these proteins was tested in enzyme immunoassays and immunoblots. These studies revealed that human IgG immune response is primarily directed against epitopes located within the amino acid residues 1 to 119 of the amino terminus of viral nucleocapsid proteins. This fragment was recognized by all HFRS patient sera tested (n=128). The corresponding enzyme immunoassays proved to be more sensitive than the indirect immunofluorescence assays. Furthermore, the majority of bank vole monoclonal antibodies raised against Puumala virus reacted specifically with this site. A recombinant G1 protein (aa 59 to 401) derived from the CG 18-20 strain was recognized by 19 out of 20 sera from HFRS patients.

Antigenicity of hantavirus nucleocapsid proteins expressed in E. coli

DNA clones representing the small genomic segment of Nephropathia epidemica virus strain Hällnäs B1 (NEV) and Hantaan virus strain 76-118 (HTV) encoding their nucleocapsid proteins were inserted into the E. coli vector pIN-III-ompA for secretion of proteins into the periplasmic space. The complete HTV and NEV nucleocapsid proteins and two truncated versions of the NEV nucleocapsid proteins were expressed as fusion proteins. Unexpectedly, all products accumulated as insoluble aggregates. Most of the ompA signal peptide remained uncleaved. However, nucleocapsid fusion proteins could be purified from the insoluble fraction by extraction with 8 M urea followed by separation on SDS-PAGE and electroelution. Rabbits were immunized with the eluted proteins and the resulting antibodies reacted specifically with authentic viral nucleocapsid proteins of HTV and NEV. The recombinant nucleocapsid proteins were found to react specifically with various hantavirus-immune sera, but not with human control sera, indicating their suitability as potential diagnostic antigens. This is the first report on the expression of a protein of a NEV serotype strain of hantaviruses by use of recombinant DNA techniques.

Rapid detection of genomic variations in different strains of hantaviruses by polymerase chain reaction techniques and nucleotide sequence analysis

The polymerase chain reaction (PCR) with subsequent nucleotide sequence analysis was employed to rapidly detect genomic variations among different Hantavirus strains. Using synthetic oligonucleotide primers derived from the M and S segment RNAs of nephropathia epidemica virus strain Hällnäs B1 (NEV) we succeeded in amplifying the corresponding sequences of Hantaan and Puumala viruses. The nucleotide sequences of the cDNAs derived from the Puumala M and S RNA segments were analyzed. It was found that the particular nucleotide sequences of Puumala M and S segments were 81% and 82% homologous to the corresponding genomic segments of NEV, respectively. The amino acid homology was 94% for both segments. In contrast, the degree of homology to the corresponding Hantaan M and S genomic RNA segments was 63% at the nucleotide level for both segments and 53 and 55% at the deduced amino acid level, respectively. This demonstrates that Puumala virus is very similar to NEV and significantly different from Hantaan virus at both the nucleotide and protein level.

Expression of human immunodeficiency virus type 1gag gene using genetically engineered herpes simplex virus type 1 recombinants

Infectious herpes simplex virus type 1 (HSV-1) recombinants were constructed by inserting the cDNA sequence of the human immunodeficiency virus type 1 (HIV-1)gag gene (from nucleotide position 675 [SacI] to 3859 [Asp 718] of the cDNA sequences of HIV-1 strain BH-10) within the DNA sequences of theBamHI DNA fragment B of the genome of an apathogenic HSV-1 strain HFEM. This HSV-1 strain possesses a 4.1-kbp deletion within theBamHI DNA fragment B between 0.762 and 0.789 map units of the viral genome, which allows the insertion of at least 4 kbp of foreign genetic material into this particular region. The DNA sequences of the immediate early promoter (IE4) of HSV-1 that were inserted upstream from thegag gene were used as a promoter. The screening of 205 virus stocks derived from individual plaques revealed that 46 recombinant viruses harbor HIV-1gag-specific DNA sequences. However, it was found that only six of the recombinant viruses are able to express thegag gene product of HIV-1. This indicates that the ratio of the positive recombination events is about 2.9%.

Rocky Mountain Spotted Fever (RMSF)

Rocky Mountain spotted fever (RMSF) is a disease caused by the bacteria (germ) Rickettsia rickettsii.

Identification and characterization of a Hantavirus strain of unknown origin by nucleotide sequence analysis of the cDNA derived from the viral S RNA segment

The genetic characterization of a serologically Hantaan-like virus but of unknown origin (termedDX) was carried out by molecular cloning and nucleotide sequencing of the corresponding cDNA of the viral S RNA segment. The S RNA was found to be 1765 nucleotides long with 3′ and 5′ termini being complementary for 24 bases. The virus messenger-sense RNA contains one major open reading frame (ORF) encoding 428 amino acids or a 50 kD polypeptide. A comparison of the DX S RNA segment to those of Sapporo rat, Hantaan, Puumala/Hällnäs B1, and Prospect Hill viruses reveals 95.4, 71.3, 55.3, and 60.9% homology at the nucleotide sequence level, and 94.7, 80.1, 58.4, and 59.8% at the deduced amino acid sequence level. Thus Hantavirus strain DX is very closely related to Sapporo rat virus. We also analyzed the S RNA segments of these Hantaviruses for the presence of a second ORF encoding a potential nonstructural NSs protein. All potential second ORFs detected in the different S RNA segments differ substantially in length and position among the viruses, despite the high conservation of the nucleotide sequences and the overall structure of the nucleocapsid proteins. This suggests that the nucleocapsid protein is the only polypeptide encoded by Hanta-virus S RNA segments, setting them apart from the other members of the Bunyaviridae family.

Baculovirus expression of the nucleocapsid protein of a Puumala serotype Hantavirus

Recombinant baculoviruses were generated harboring the entire coding region of the S segment cDNA of Hantavirus strain CG 18–20 that belongs to the Puumala serotype. The recombinant nucleocapsid protein was expressed in Sf9 cells and shown to be antigenically identical with the authentic viral nucleocapsid protein by means of immunoblot analysis. Acute-phase and convalescent sera from European HFRS patients recognized the recombinant nucleocapsid protein in Western blots and the recombinant Baculovirus in indirect immunofluorescence assays. Insect cells infected with the recombinant Baculoviruses proved to be a suitable noninfectious substitute for Hantavirus-infected Vero E6 cells as an antigen source for immunodiagnostic assays allowing the detection of antibodies in HFRS patients.

Kyasanur Forest Disease (KFD)

National Center for Emerging and Zoonotic Infectious Diseases Division of High-Consequence Pathogens and Pathology (DHCPP) Kyasanur Forest disease (KFD) is caused by Kyasanur Forest disease virus (KFDV), a member of the virus family Flaviviridae. KFDV was identified in 1957 when it was isolated from a sick monkey from the Kyasanur Forest in Karnataka (formerly Mysore) State, India. Since then, between 400-500 humans cases per year have been reported.

Neu-Delhi Metallo-Beta-Laktamase 1 (NDM-1) und andere Carbapenemasen: Resistenzmechanismen multiresistenter „Superbakterien“

Jedes Jahr infizieren sich weltweit mehrere Millionen Menschen mit Keimen, die gegen die bisher bekannten Antibiotika teilweise oder sogar vollstandig resistent sind. Fur die Bundesrepublik Deutschland wird eine Zahl von 500.000 bis 1 Mio. Personen pro Jahr genannt [5]. Es wird geschatzt, dass ca. 50.000 davon an diesen bakteriellen Infektionen versterben. In den Vereinigten Staaten von Amerika sind etwa 70% der Erreger nosokomialer Infektionen resistent gegen mindestens ein Antibiotikum. Haufig sind Patienten mit multiresistenten Bakterienstammen infiziert, die gegen mehrere Antibiotika resistent sind [1, 7, 8, 9].

El-Moro-Canyon-Virus

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