Hartwig P. Huemer

Epidemiology of Enterovirus Types Causing Neurological Disease in Austria 1999-2007: Detection of Clusters of Echovirus 30 and Enterovirus 71 and Analysis of Prevalent Genotypes

Between 1999 and 2007 1,388 stool specimens from patients with acute flaccid paralysis or aseptic meningitis were submitted to the Austrian reference laboratory for poliomyelitis. Samples (201) yielded non‐poliovirus enterovirus in culture. One hundred eighty‐one viruses were available for typing and 78 isolates which remained serologically untyped were further analyzed by CODEHOP‐PCR and sequencing of the VP1 gene and the 5′‐untranslated region (5′‐UTR). Typing revealed an Echovirus 30 outbreak in northwestern Austria in 2000, which was in accordance with the situation in Europe, and no dramatic seasonal changes of Coxsackie viruses were observed. In 2002/2003 a small outbreak of enterovirus 71 (EV71), affected 12 patients in the province of Styria. This virus was identified as genotype C1 and appeared to be genetically distinct from the isolates observed in 2001/2002 in Vienna. In 2004 two unrelated cases occurred in Lower Austria, which were identified as genotype C4, which has been described associated with high mortality most recently in China. In contrast to the situation in Asia the detected EV71 cases were not associated with hand–foot–mouth disease, but with serous meningitis only. This was surprising as a recent publication suggested a reduced neurovirulence of C1 genotype in children in Norway, presumably due to alterations in 5′‐UTR and polymerase gene. However, comparing the 5′‐UTR of the Austrian isolates and established virulent reference strains to the Norwegian isolate and an attenuated EV71 laboratory strain we did not find an indication that the genotype C1 possesses a RNA structure in its 5′‐UTR leading to reduced neurovirulence. J. Med. Virol. 81:317–324, 2009.

Determination of soluble CD21 as a parameter of B cell activation.

In this study we established a novel solid‐phase immunoassay for CD21 using the time‐resolved fluorescence of lanthanide chelates. The capture assay was able to detect concentrations of as low as 100 pg of CD21 antigen per millilitre of sample and was used for quantitative determination of CD21 in lysates of different cell lines as well as in patient serum specimens. CD21 was measured in lysates of tonsils and cell lines of B. T cell and myelomonocyte lineage, and appeared to consist of monomeric antigen under the detergent conditions used. Elevated levels of soluble CD21 were observed in serum of patients with Epstein Barr virus (EBV) infection, a disease known to be associated with polyclonal B cell activation, and in infection with the lymphotropic rubella virus. Significantly increased levels were also found in malignancies which are associated with EBV. In patients with nasopharyngeal carcinoma (NPC), a correlation with the titre of EBV‐specific IgA was observed, thus supporting a possible role of soluble CD21 as a marker for disease activity in certain malignancies. Our data suggest that measurement of soluble CD21 could serve as a marker for activation of the immune system and diseases involving the B cell lymphoid system. Possible mechanisms and functions of soluble CD21 are discussed.

Prevalence of respiratory viruses, including newly identified viruses, in hospitalised children in Austria

The aim of this epidemiological study was to determine the prevalence of respiratory viruses, including new viruses, in hospitalised children in Austria. Two hundred fourteen nasopharyngeal samples from hospitalised children were tested for the presence of viruses using cell culture and PCR and/or viral antigen assays. The results revealed a parainfluenza virus 1 (PIV1) outbreak that ended right before the onset of the influenza season, with nearly no overlapping, moderate respiratory syncytial virus (RSV) activity, and only a few adenoviruses. Human metapneumovirus (hMPV) was present in 14.5% of the total samples but was detected in combination with other viruses in only five cases: with PIV1 in three cases and with RSV in two cases. There were no cases of dual infection with hMPV and flu or adenovirus. This suggests that hMPV alone is a leading cause of hospitalisation in children under 1xa0year of age. Interestingly, hMPV, in contrast to RSV, coincided with PIV1 but was absent during the community outbreak of the flu. Samples were also tested for Mimiviridae, a group of newly described DNA viruses that are similar to Legionella spp., replicate in water amoebae, and also have been found in alveolar cells. However, mimivirus was detected neither in respiratory samples nor in amoebae-containing water samples, indicating that this particular type of virus is either not abundant or does not contribute to paediatric respiratory illnesses.

Structural and functional relationships among receptors and regulators of the complement system

The classical and alternative pathway of complement activation are regulated by a series of fluid phase and cell-bound factors, some of which at the same time serve as receptors for fragments of C3 and C4. These molecules are factor H, CR1 (C3b/C4b receptor), CR2 (C3d/EBV receptor), C4BP (C4b binding protein), DAF (decay accelerating factor), MCP (membrane cofactor protein; earlier designated p45/70), CR3 (iC3b receptor or Mac-1) and CR4 (protein 150/95). Due to structural, genetic and functional features these factors are members of one or several newly recognized large families of proteins: (1) molecules with 60 amino acids long repeats (H, CR1, CR2, C4BP, DAF); (2) proteins with 1,2-diacylglycerol membrane anchoring (DAF); (3) proteins with a heterodimer structure and preference for ligands containing the tripeptide arginine-glycine-asparagine (CR3, CR4). Recognizing the above mentioned regulators and receptors of the complement system as belonging to these protein families opens new perspectives for further genetic and functional research of mutual interest to complement and noncomplement scientists.

gp13 (EHV-gC): a complement receptor induced by equine herpesviruses☆

Equine herpesviruses type 1 (EHV-1) and type 4 (EHV-4) induce a complement receptor protein on the surface of infected cells capable of binding to the third component of complement (C3). The protein mediating the binding to the C3 component of complement was identified as glycoprotein 13 (gp13, EHV-gC), as expression of the cloned viral gene under the control of a CMV promoter induced C3 binding activity at the transfected cell surface. Comparable to glycoprotein C (gC) from herpes simplex virus type 1 (HSV-1-gC), glycoprotein III from pseudorabiesvirus (gIII, PRV-gC) and bovine herpesvirus-1 (gIII, BHV-1-gC), gp13 derived from EHV-infected cell lysates bound to C3 fixed to solid phase, showing preferential binding to the appropriate host complement component. Similar to wild-type isolates, a highly attenuated vaccine EHV-1 strain also displayed complement receptor activity despite apparent differences of the gp13 gene in restriction enzyme digest pattern and reactivity with monoclonal antibodies. In addition, other structural proteins were altered in the vaccine strain as compared to wild-type strains, which might contribute to its attenuated phenotype. In contrast to the situation observed with HSV-1-gC, the interaction of gp13 (EHV-gC) with horse complement was not inhibited by polyanionic substances like heparin or dextran sulfate. These results suggest structural differences in the particular binding mechanism of the respective viral envelope proteins.

Chronic active Epstein-Barr virus disease in a case of persistent polyclonal B-cell lymphocytosis

Summary. Persistent polyclonal B‐cell lymphocytosis (PPBL) is a rare haematological disorder. It is characterized by activated and morphologically atypical B lymphocytes and polyclonal IgM production and has been associated with female sex, cigarette smoking, and HLA‐DR7 expression.

Fatal Infection of a Pet Monkey with Human herpesvirus 1

Concerns have been raised about pet monkeys as a potential threat to humans. We report the opposite situation, a danger to pets that arises from humans. Similar to herpesvirus B (Cercopithecine herpesvirus 1), which endangers humans but not its host species, Human herpesvirus 1 can act as a “killer virus” when crossing the species barrier to New World monkeys.

Role of Epstein-Barr virus and soluble CD21 in persistent polyclonal B-cell lymphocytosis.

Summary. The expression of EBV proteins and immunological properties were studied in the first stable cell line (SM) established from a patient presenting with persistent polyclonal B‐cell lymphocytosis (PPBL).

Pseudorabies virus glycoprotein III derived from virions and infected cells binds to the third component of complement

Glycoprotein III (gIII) of pseudorabies virus (PRV) was shown to bind to the third component of complement (C3). This was observed only with porcine C3 whereas human C3 showed negligible binding under the conditions tested. PRV virion proteins could be precipitated from supernatants and cell lysates of PRV-infected cells by means of swine-C3 coupled to sepharose. According to their molecular size and their reactivity with anti-gIII monoclonal antibodies, the precipitated PRV proteins represented the fully glycosylated and smaller forms of the gIII protein. Precipitation from PRV virions yielded predominantly the fully glycosylated form of gIII whereas infected cell lysates also contained lower molecular weight gIII proteins. The observed specificity of the virus protein for porcine C3 correlates well with the known host tropism of PRV. Our findings suggest that PRV gIII may exhibit more functions than solely providing attachment to heparin-like moieties on target cell surfaces. As the complement cascade is an important defense mechanism against a variety of pathogens, the interaction with the host C3, the pivotal component of the complement activation, might be a virulence factor of PRV.

Correlation of hepatitis C virus antibodies with HIV-1 seropositivity in intravenous drug addicts

Since Choo, Kuo, and co-workers [1, 2] used a yeast expression system to synthesize a recombinant polypeptide of the hepatitis C virus (HCV) (a major etiologic virus of blood-borne non-A, non-B hepatitis (NANBH) many investigators have studied the prevalence of antibodies to HCV. High percentages of antibodies were detected in patients with post-transfusion NANBH [2, 3], in patients with hepatocellular carcinoma, hepatic cirrhosis [4, 5] and in haemophiliacs [3, 6]. In healthy blood donors the percentage of antibodies detected against HCV varied from 0.42% [7], 0.5% [2], 0.68% [8], to 7.3% [4]. We studied HCV-antibody prevalence in anti-H/V-1 positive (referred to as HIV +) and negative (HIV-) intravenous drug addicts (IVDA) in Western Austria. Antibodies to HCV were determined by ELISA purchased from Ortho Diagnostic Systems Inc., Raritan, N.Y., USA; code 933440, lot# hcvl03. All tests for detection of Hepatitis B markers were obtained from ABBOTT LAB., North Chicago, I1, USA: HBs-antigen Auszyme monoclehal, # 32281 M201), anti-HBc (~orzyme # 32565 M301), and anti-HBs (Ausab-eia, #32591 M202). In the sere of 30 HIV-positive and 36 H/V-negative IVDA we found 20 (66.6%) and 13 (36.1%), respectively, positive for anti-HCV in an ELISA system obtained from ORTHO whereas only three (4.1%) out of 73 healthy, young volunteers reacted to this test (Table 1 ). In addition, the prevalence of hepatitis B in these persons Was tested by the determination of hepatitis virus B surface antigen, of antiHBc, and anti-HBs antibodies. HBs-antigen and/or anti-HBs could be detected in 17 (56.6%) of HIV +, in 19 (52.7%) of H/VIVDA and in two (2.7%) of the healthy volunteers. Anti-HBc could be detected in 22 (73.3%) of HIV + and in 14 (38.8%) of the HIVIVDA, but in only one (1.37%) of the healthy volunteers. Our findings indicate that: a high prevalence of anti-HCV exists in IVDA, which is in agreement with data previously reported [3]; a higher prevalence of anti-HCV in H/V + IVDA is detectable when compared to HIVIVDA. A similar observation was made by Mortimer et al. [9] studying HCV seropositivity in H/V + and HIVhomosexual men;