Roland Kehm

Is the major capsid protein of iridoviruses a suitable target for the study of viral evolution

Iridoviruses are large cytoplasmic DNA viruses that are specific for different insect or vertebrate hosts. The major structural component of the non-enveloped icosahedral virus particles is the major capsid protein (MCP) which appears to be highly conserved among members of the family Iridoviridae, Phycodnaviridae, and African swine fever virus. The amino acid sequences of the known MCPs were used in comparative analyses to elucidate the phylogenic relationships between different cytoplasmic DNA viruses including three insect iridoviruses (Tipula iridescent virus, Simulium iridescent virus, Chilo iridescent virus), seven vertebrate iridoviruses isolated either from fish (lymphocystis disease virus, rainbow trout virus, European catfish virus, doctor fish virus), amphibians (frog virus 3), or reptiles (turtle virus 3, turtle virus 5), one member of the family Phycodnaviridae (Paramecium bursaria Chlorella virus type 1), and African swine fever virus. These analyses revealed that the amino acid sequence of the MCP is a suitable target for the study of viral evolution since it contains highly conserved domains, but is sufficiently diverse to distinguish closely related iridovirus isolates. Furthermore the results suggest that a substantial revision of the taxonomy of iridoviruses based on molecular phylogeny is required.

New ecological aspects of hantavirus infection : a change of a paradigm and a challenge of prevention-a review

In the last decades a significant number of so far unknown or underestimated pathogens have emerged as fundamental health hazards of the human population despite intensive research and exceptional efforts of modern medicine to embank and eradicate infectious diseases. Almost all incidents caused by such emerging pathogens could be ascribed to agents that are zoonotic or expanded their host range and crossed species barriers. Many different factors influence the status of a pathogen to remain unnoticed or evolves into a worldwide threat. The ability of an infectious agent to adapt to changing environmental conditions and variations in human behavior, population development, nutrition, education, social, and health status are relevant factors affecting the correlation between pathogen and host. Hantaviruses belong to the emerging pathogens having gained more and more attention in the last decades. These viruses are members of the family Bunyaviridae and are grouped into a separate genus known as Hantavirus. The serotypes Hantaan (HTN), Seoul (SEO), Puumala (PUU), and Dobrava (DOB) virus predominantly cause hemorrhagic fever with renal syndrome (HFRS), a disease characterized by renal failure, hemorrhages, and shock. In the recent past, many hantavirus isolates have been identified and classified in hitherto unaffected geographic regions in the New World (North, Middle, and South America) with characteristic features affecting the lungs of infected individuals and causing an acute pulmonary syndrome. Hantavirus outbreaks in the United States of America at the beginning of the 10th decade of the last century fundamentally changed our knowledge about the appearance of the hantavirus specific clinical picture, mortality, origin, and transmission route in human beings. The hantavirus pulmonary syndrome (HPS) was first recognized in 1993 in the Four Corners Region of the United States and had a lethality of more than 50%. Although the causative virus was first termed in connection with the geographic name of its outbreak region the analysis of the individual viruses indicate that the causing virus of HPS was a genetically distinct hantavirus and consequently termed as Sin Nombre virus. Hantaviruses are distributed worldwide and are assumed to share a long time period of co-evolution with specific rodent species as their natural reservoir. The degree of relatedness between virus serotypes normally coincides with the relatedness between their respective hosts. There are no known diseases that are associated with hantavirus infections in rodents underlining the amicable relationship between virus and host developed by mutual interaction in hundreds of thousands of years. Although rodents are the major reservoir, antibodies against hantaviruses are also present in domestic and wild animals like cats, dogs, pigs, cattle, and deer. Domestic animals and rodents live jointly in a similar habitat. Therefore the transmission of hantaviruses from rodents to domestic animals seems to be possible, if the target organs, tissues, and cell parenchyma of the co-habitat domestic animals possess adequate virus receptors and are suitable for hantavirus entry and replication. The most likely incidental infection of species other than rodents as for example humans turns hantaviruses from harmless to life-threatening pathogenic agents focusing the attention on this virus group, their ecology and evolution in order to prevent the human population from a serious health risk. Much more studies on the influence of non-natural hosts on the ecology of hantaviruses are needed to understand the directions that the hantavirus evolution could pursue. At least, domestic animals that share their environmental habitat with rodents and humans particularly in areas known as high endemic hantavirus regions have to be copiously screened. Each transfer of hantaviruses from their original natural hosts to other often incidental hosts is accompanied by a change of ecology, a change of environment, a modulation of numerous factors probably influencing the pathogenicity and virulence of the virus. The new environment exerts a modified evolutionary pressure on the virus forcing it to adapt and probably to adopt a form that is much more dangerous for other host species compared to the original one.

Acute and long-term alteration of chemokine mRNA expression after anti-viral and anti-inflammatory treatment in herpes simplex virus encephalitis

Mortality and morbidity rates remain high among patients with herpes simplex virus encephalitis (HSVE). Chemokine-mediated recruitment and activation of leukocytes to focal areas of viral CNS infection are crucial steps in antiviral response and clearance. However, the inflammatory reaction and cellular antiviral response may enhance collateral damage to neurons and account for chronic progressive brain damage. We identified a specific mRNA expression of the interferon-gamma-inducible chemokines (CXCL9, CXCL10 and CXCL11), and RANTES (CCL5) in the acute course and long-term of experimental HSVE. This pattern was substantially altered by anti-viral and anti-inflammatory treatment. Our findings indicate a pivotal role of these chemokines in the immunopathogenesis of HSVE.

Herpes simplex virus encephalitis: long-term comparative study of viral load and the expression of immunologic nitric oxide synthase in mouse brain tissue

In the brain tissue of 21 mice infected with herpes simplex virus type 1 (HSV-1) strain F we determined the expression of immunologic nitric oxide synthase (iNOS) as a potential mediator of neuronal injury with a semiquantitative reverse transcription polymerase chain reaction. Viral burden in brain tissue was quantitated with a dilutional polymerase chain reaction assay. Viral burden and iNOS-expression peaked at day 7 following infection. Thereafter viral burden declined to a low baseline value at 6 months following infection, whereas iNOS-expression was still 4-fold increased compared to baseline levels. In experimental herpes simplex virus encephalitis iNOS, as one potent mediator of neuronal injury, is upregulated in the acute and chronic disease. In future, in addition to antiviral treatment, inhibitors of iNOS might offer new therapeutic strategies in herpes simplex virus encephalitis.

Detection of antibodies against viral capsid proteins of human herpesvirus 8 in AIDS-associated Kaposi's sarcoma

Abstract Sequences of a new herpesvirus with homology to gammaherpesvirinae were recently identified in AIDS-associated Kaposi’s sarcoma (KS). Subsequently this novel virus, called KS-associated virus (KSHV) or human herpesvirus (HHV) 8 was detected in classical KS and AIDS-associated body cavity based lymphomas by polymerase chain reaction. In this report major and minor capsid proteins of HHV-8 were molecularly cloned and produced as recombinant proteins in Escherichia coli. Sera from 69 HIV-1 infected patients with KS, 30 HIV-1 infected patients without KS and 106 control individuals were tested by enzyme-linked immunosorbent assay for anti-HHV-8 capsid IgM and IgG antibodies. Sera from four patients were tested over periods ranging from 18 months to 6 years. IgG antibodies directed against HHV-8 capsid antigens were detected in patients with AIDS-associated KS and in some AIDS patients without KS. Seroconversion with IgM and IgG antibodies directed against HHV-8 capsid proteins occurred more than 1 year prior to diagnosis of KS. In a considerable portion of KS patients no IgM or IgG antibodies against HHV-8 capsid proteins were detected. In these patients there was an inverse relationship between antibodies against HHV-8orf26 and the CD4/CD8 ratio, suggesting that the inconsistency of anti-HHV-8orf26 antibodies is due at least partly to an impaired immune response. No reactivity against HHV-8 capsid antigens was detected in the vast majority of sera from HIV-negative control individuals. Our findings indicate that a specific humoral immune response against capsid proteins is raised in HHV-8 infected individuals, and that anti-capsid antibodies can be used to diagnose HHV-8 infection. The correlation between occurrence of anti-HHV-8 antibodies and KS supports the hypothesis of a causative role of HHV-8.

Stable and Long-Lasting Immune Response in Horses after DNA Vaccination against Equine Arteritis Virus

Equine arteritis virus (EAV) is the causative agent of the equine viral arteritis. It is a small RNA virus with a linear, non-segmented plus RNA genome. EAV is a member of the Arteriviridae family that includes porcine reproductive and respiratory syndrome virus (PRSSV), simian haemorrhagic fever virus (SHFV) and lactate dehydrogenase virus (LDV). The viral transmission is via respiratory and reproductive routes. Clinical signs in horses vary, and severe infection can lead to abortions in pregnant mares or neonatal foal death. The aim of this study was to investigate the development of the immune response in horses after immunization with a DNA vaccine harbouring and expressing EAV Open Reading Frames (ORF) 2, 5, and 7, in combination with equine interleukin 2 (eqIL2). Three boosters followed the basic immunization in two-week intervals. Each immunization was a combination of gene gun and intramuscular injection. All horses developed a high titer of neutralizing antibodies after basic immunization within 2 weeks. Remarkably, this immune response was found to be independent of the age of animals. The youngest horse was six-years old, and the oldest twenty-two years old. A remarkable difference in the immune response between the young and old were not observed. The duration of immunity was investigated during a period of one year. After 12 months, neutralizing antibodies were still detectable in all the vaccinated horses.

Efficacy of anise oil, dwarf-pine oil and chamomile oil against thymidine-kinase-positive and thymidine-kinase-negative herpesviruses.

The effect of anise oil, dwarf‐pine oil and chamomile oil against different thymidine‐kinase‐positive (aciclovir‐sensitive) and thymidine‐kinase‐negative (aciclovir‐resistant) herpes simplex virus type 1 (HSV‐1) strains was examined. Clinical HSV‐1 isolates containing frameshift mutations in the thymidine kinase (TK) gene, an insertion or a deletion, yield a non‐functional thymidine kinase enzyme resulting in phenotypical resistance against aciclovir. The inhibitory activity of three different essential oils against herpes simplex virus isolates was tested in‐vitro using a plaque reduction assay. All essential oils exhibited high levels of antiviral activity against aciclovir‐sensitive HSV strain KOS and aciclovir‐resistant clinical HSV isolates as well as aciclovir‐resistant strain Angelotti. At maximum noncytotoxic concentrations of the plant oils, plaque formation was significantly reduced by 96.6–99.9%, when herpesviruses were preincubated with drugs before attachment to host cells. No significant effect on viral infectivity could be achieved by adding these compounds during the replication phase. These results indicate that anise oil, dwarf‐pine oil and chamomile oil affected the virus by interrupting adsorption of herpesviruses and in a different manner than aciclovir, which is effective after attachment inside the infected cells. Thus the investigated essential oils are capable of exerting a direct effect on HSV and might be useful in the treatment of drug‐resistant viruses. Chamomile oil did not reveal any irritating potential on hens egg chorioallantoic membrane, demonstrated the highest selectivity index among the oils tested and was highly active against clinically relevant aciclovir‐resistant HSV‐1 strains.

Herpes simplex virus encephalitis: cranial magnetic resonance imaging and neuropathology in a mouse model

We performed a long-term magnetic resonance imaging (MRI) study in a mouse model of herpes simplex virus encephalitis. Mice were infected with herpes simplex virus type 1 (HSV-1) strain F. A 1.5-T cranial MRI scanner with standard spin-echo sequences was used. Neuropathological studies included immunohistochemistry. The presence of HSV DNA in brain tissue was determined with a polymerase chain reaction assay. Clinical assessment was performed daily: within the first 2 weeks the animals were severely affected and recovered thereafter. MRI and histopathological abnormalities corresponded well. HSV DNA was detectable initially and at 6 months. Extent and severity of structural abnormalities increased at 6 months. MRI offers a new in vivo approach for the detection of structural changes in the disease course of experimental herpes simplex virus encephalitis.

Restitution of the UL56 gene expression of HSV-1 HFEM led to restoration of virulent phenotype; deletion of the amino acids 217 to 234 of the UL56 protein abrogates the virulent phenotype

Recently it was shown that the avirulent phenotype of HSV-1 strain HFEM is correlated to the lack of DNA sequences of the promoter region of the UL56 gene. In order to investigate the role of the UL56 gene of HSV-1 in the process of viral pathogenicity in more detail, a complete copy of the UL56 gene of the virulent HSV-1 strain 17 was inserted within the DNA sequences of the incomplete UL56 gene of the genome of HSV-1 strain HFEM. The UL56 gene of HSV-1 strain 17 comprises 1428 bp corresponding to the nucleotide positions (NP) 11,5967-117,395 of the genome of HSV-1 strain 17 (SacII-DNA fragment) containing the promoter region and the entire UL56 gene with identical transcription termination signals. This particular DNA fragment was inserted into the corresponding region of the genome of HSV-1 strain HFEM by co-transfection experiments in which the beta-galactosidase gene served as reporter gene. Those recombinant viruses with the ability to express the UL56 gene were tested for their pathogenicity in vivo. The results of these experiments indicate that the restoration of the viral UL56 gene expression led to the restitution of the virulent phenotype of HSV-1 strain HFEM. The UL56 protein which has been shown to be a component of the virion possesses several characteristic signatures e.g. a hydrophobic domain at the carboxy-terminus between amino acid residues 217 and 234 (VFGVVAIVVVIILVFLWR). In order to investigate the role of this particular signature of the UL56 protein in the process of viral pathogenicity, site-specific mutagenesis was performed for removing the carboxy-terminus of the UL56 protein. The deleted region of the DNA sequences of the UL56 gene between NP 1122-1175 corresponds to NP 116 220-116 373 of the viral genome. The DNA sequences of the UL56 gene of virulent HSV-1 strain 17 and F were replaced by DNA sequences of the truncated UL56 gene by co-transfection experiments in which the beta-galactosidase gene served as a reporter gene. Those recombinant viruses with the ability to express the truncated UL56 gene were examined for their pathogenicity in vivo. The analysis revealed that the expression of the truncated UL56 protein (without hydrophobic domain 217-234 aa) was not sufficient for the maintenance of the virulent phenotype of HSV-1 strains.

Experimental herpes simplex virus encephalitis: inhibition of the expression of inducible nitric oxide synthase in mouse brain tissue

In the brain tissue of 36 mice infected with herpes simplex virus type 1, strain F, we determined the expression of inducible nitric oxide synthase (iNOS) with semiquantitative reverse transcription polymerase chain reaction. The viral burden was quantitated by polymerase chain reaction. Nitric oxide, induced by iNOS, may contribute to neuronal cell damage following virus infection. As the experimental therapeutic strategy in herpes simplex virus encephalitis (HSVE), we used: N-nitro-L-arginin (L-NA), a selective inhibitor of iNOS; and combination therapies of either methylprednisolone/acyclovir or L-NA/acyclovir. The viral burden peaked in acute disease, and then returned to a low baseline value, except in untreated controls. The expression of iNOS mRNA was suppressed by L-NA and by acyclovir/corticosteroids. INOS inhibition may provide an additional therapeutic strategy targeted specifically to suppress iNOS expression as a potential secondary mechanism of tissue damage in acute and chronic HSVE.