K. E. Schneweis

Pathogenesis of genital herpes simplex virus infection in mice. III: Comparison of the virulence of wild and mutant strains

This study was undertaken to establish the role of virulence of various herpes simplex virus (HSV) strains in the course of infection when applying the virus to the non-injured mucous membranes of mice.Wild-type HSV-type 1 (HSV-1) strains with marked differences in their neurovirulence following intracerebral inoculation showed minor differences in virulence after vaginal inoculation, but essentially their neurovirulence in cerebral infection corresponded to their virulence on the mucous membranes.In comparison with the wild-types, however, there were pronounced differences among syn- and TK−-mutants of HSV-1 and HSV-2 in the degree of virulence at different sites in the course of virus infection. Whereas syn-mutants proved avirulent on the mucous membranes but not in neural tissues, TK−-mutants were avirulent both on mucous membranes and in neural tissues.Ts-mutants of HSV-2 were not found to establish themselves when administered to the non-injured mucous membranes, nor did they induce neutralizing antibodies, but a later challenge with the wild-type virus at the same site lead only to an attenuated course of infection.

The Prevalence of Antibody to Parvovirus B19 in Hemophiliacs and in the General Population

The prevalence of antibodies to parvovirus B19 (B19) was measured in the sera of 566 hemophiliacs and 524 individuals of the general population by immunofluorescence assays, using antigen expressed by the baculovirus system. In the general population, anti-B19 IgG seroprevalence was found to continuously decline from 64 percent at birth to 0 percent in the age of 9-11 months and thereupon to increase to 61 percent in the age of 12 years. In younger adults and older people, IgG seroprevalence only slowly increased with age, reaching 77 percent in people aged 60 and above. In contrast, in hemophilic children treated exclusively with virally inactivated clotting factor concentrates, neither decrease nor increase of B19 IgG antibody was detectable and the overall seroprevalence was 92 percent. In the group of hemophiliacs older than 12 years and treated before 1984 with non-inactivated clotting factor concentrates, 98 percent showed antibody to B19. Anti-B19 IgM seroprevalence was significantly higher in hemophilic than in non-hemophilic individuals older than 12 years. Since it seems to be unlikely that the high seroprevalence in hemophiliacs is acquired by immunization with inactivated viral antigen, the results suggest that infection with B19 is transmitted by clotting factor concentrates, even if subjected to virucidal methods.

Protective effect of an oral infection with herpes simplex virus type 1 against subsequent genital infection with herpes simplex virus type 2.

The problem of whether oral Herpes simplex virus type 1 (HSV-1) infection provides protection against subsequent genital infection by Herpes simplex virus type 2 (HSV-2) was investigated. Mice were used as models. Following conditions in man, both the oral and genital infections applied were noninjurious. Mice infected orally with HSV-1 were weakly protected against virus ‘take’ following vaginal challenge with HSV-2. Genital ‘takes’ were found in 67% of the immunized mice, as compared with 83% of the controls (protection rate 20%,P=0.002). The course of genital infection in the immunized mice, however, was relatively mild: Lethality decreased from 97% in the controls to 35% in the immunized mice (protection rate 63%,P<0.001). Furthermore, local and neurologic symptoms occurred less frequently. Attempts to isolate the virus from homogenized brain and spinal cord of immunized mice that died after genital challenge with HSV-2 failed in most cases. Also virus could not be recovered from the liver of infected mice, irrespective of the experimental group.

The influence of different modes of immunization on the experimental genital Herpes simplex virus infection of mice

Previous investigations, which simulated the usual sequence of the humanHerpes simplex virus (HSV) infections, had shown that the oral infection of mice with HSV-1 caused only weak protection from genital infection with HSV-2, although the course of infection was attenuated and lethality diminished. This heterologous, heterotopic model was compared with a homologous, heterotopic and a heterologous, homotopic model. The results did not differ very much, although the homologous immunization protected best from lethal outcome, the homotopic immunization best from local infection. Three different preparations of a killed vaccine from purified HSV-1 virion had little effect on the course of the local infection, although protection from lethal outcome was as good as with live virus. In contrast, a crude UV-inactivated vaccine protected nearly completly from local infection.Latent infection in the lumbosacral ganglia was significantly inhibited by immunization with live virus, but only sligthly prevented by killed vaccine. The prevalence of latent infection correlated with the extent of vaginal infection.The results show that neither the viral type nor the inoculation site used for immunization with live virus are very critical. Moreover, they allow the conclusion that generalized type-dependent immune factors seem to be engaged in protection against lethal disease; these may be circulating humoral antibodies. On the other hand, locally induced immune factors (probably cellular) are apparently of prime importance for the protection from acute local and latent ganglionic infection.

Immunological mechanisms giving rise to latency of herpes simplex virus in the spinal ganglia of the mouse

In the model of genital herpes simplex virus (HSV)-infection of mice, early latency could be induced by passive immunization with HSV-specific antibodies and, to a lesser degree, by adoptive transfer of immune lymphocytes prepared from spleen and draining lymph nodes of genitally infected syngeneic mice. Conversely, spontaneously occurring latency was inhibited by treatment of the animals with cyclophosphamide (Cph) and, to a lesser degree, with cyclosporin A (CyA). Whereas the effect of CyA could be compensated by passively administered HSV-specific antibodies, that of Cph could not. Apparently specific antibodies cooperate with a non-specific proliferating cell type, probably macrophages and/or NK-cells, as could be demonstrated by significantly reduced antibody effect in silica-treated mice. Moreover, F(ab)2 fragments, in contrast to complete antibody molecules, were inactive. HSV-specific antibodies and also immune lymphocytes had little effect on virus production in the mucous membranes, immune lymphocytes being at least as active as antibodies. It is therefore not probable that latency is induced by attenuation of the peripheral disease. It can rather be concluded that the neuron itself is the target for the action of specific antibodies, cooperating in turn with macrophages and/or NK cells.

Effects of genetic resistance against Herpes simplex virus in vaginally infected mice.

In order to take the conditions of naturalHerpes simplex virus (HSV) infection into consideration, the genetic resistance of C57-B1 mice, which was established by intraperitoneal HSV-1 infection [Lopez, 1975], was investigated in vaginally infected mice. The course of infection in the mucous membranes did not differ in sensitive (NMRI) and resistant (C57-B1) mice: both number of takes and virus elimination from the vagina were equal, and no difference in viral titer produced in the vagina was detected. Viral titer in the productively infected lumbosacral ganglia, however, was less in the resistant mice. An experiment with foot-pad-infected mice confirmed that the number of productively infected ganglia was reduced in resistant mice, and contralateral ganglia were infected only in the sensitive mouse strain. In spite of this, the number of latently infected animals did not vary significantly in the mouse strains. Higher activity of defense mechanisms in resistant mice, apparently localized in the ganglia, resulted in reduced lethality.As to the mechanisms of the resistance, neither antibody nor interferon response were enhanced in C57-B1 mice, but resistance was abolished by depletion of several cellular functions, i.e., lymphocytes by cyclophophamide or X-rays, macrophages by silica or macrophage-antiserum, and M-cells by89Sr.

Typing of Herpes simplex virus strains by analysis of the early proteins

In Herpes simplex virus (HSV) infected cells treated with the arginine analogue canavanine, early (α−) viral proteins accumulate and show a typical pattern in the acrylamide gel. This paper investigates the possibility of using this pattern for typing of HSV strains.Twenty strains were analyzed by this method and results were compared with typing by neutralization test. For 18 strains both methods agreed. Two strains regarded as intermediary strains by neutralization test could be clearly typed by the analysis of the early proteins.

Advances in HIV-PCR in respect to the different fields of diagnosis

infection was proved by detecting B19 DNA in the sera using polymerase chain reaction (PCR) and DNA obtained by proteinase K digestion and phenol-chloroform extraction. However, B19 DNA was also found in 3/69 anti-B19 IgM negative, HIV-infected hemophiliacs (all three patients in CDC [CDC: centers for disease control] stage IV). The observations suggest that B19 is still transmitted by clotting factors treated for virus inactivation and that reinfection can occur. As far as viremic immunocompromised patients are concerned, persistent infection must be considered. Recently, we introduced a new method for detection of B19 by PCR. Magnetic beads coupled with protein G purified IgG from sera with high levels of anti-B 19 antibodies were incubated with the specimen. After magnetic separation the sample was heat denaturated in PCR buffer and the supernatant was used as the substrate in the PCR reaction. This technique proved to be useful because it is time saving, avoids handling of toxic agents and allows the investigation of larger volumes of the specimens.

Detection of parvovirus B19 specific antibodies and DNa in sera of hemophiliacs

important. PCR complements traditional serological and other detection methods by allowing discrimination between and detection of specific nucleic acid sequences. In viral hepatitis for example, PCR is used to distinguish between viremic and non-viremic infection, and to monitor viral replication in patients undergoing interferon therapy. However, PCR analysis of biological samples such as blood and other body fluids, is often unreliable due to the presence of amplification inhibitors. These inhibitors are often difficult to separate from the nucleic acids, requiring long and inconvenient purification procedures for their removal. Even then, amplification may be compromised. The authors have developed a fast, safe and easy protocol to efficiently purify viral nucleic acids from serum for reliable PCR. It is shown, that the sensitivity of the PCR strongly depends on template quality and that our new extraction method provides better quality nucleic acids than standard acid-phenol extractions. Data are presented demonstrating that RNA from as little as 0.01 gl HCV (Hepatitis C virus) positive serum can efficiently be amplified after purification by our procedure. The new procedure combines the handling advantages of spin-column technology with the ability of silica to specifically bind nucleic acids. After sample lysis in a special lysis buffer containing a chaotropic salt, samples are loaded onto a spin-column by microcentrifugation. Nucleic acids are selectively bound to a silica membrane, and contaminants are washed away by two brief washes. Purified nucleic acid is then eluted in water or buffer, ready for direct addition to the PCR reaction. Moreover, this new method requires less hands-on time, uses no organic solvents or alcohol precipitations and minimizes the danger of contamination by infectious agents. It is ideal for simultaneous handling of multiple samples, enabling the preparation of 24 samples within 30 min. So far the method has successfully been tested for samples as different as fresh, frozen or dried whole blood in the presence of all common anti-coagulants, plasma, serum, buffy coat, bone marrow, mucus, cell suspensions, urine and tissue.

Kombinierter Bacitracin- und Hämolyse-Hemmungstest zur praktischen Grruppendifferenzierung hämolysierender Streptokokken

ZusammenfassungUm Streptokokken der Gruppe A vonβ-hämolysierenden Streptokokken anderer Gruppen abzugrenzen, wird der Bacitracin-Test mit dem Hämolyse-Versuch auf einer Dextrose-Wasserblau-Blutplatte kombiniert. Dadurch werden die Fehler, die jeder der beiden Methoden anhaften, weitgehend ausgeglichen.