Stephan W. Aberle

Single versus dual respiratory virus infections in hospitalized infants : Impact on clinical course of disease and interferon-γ response

Background: Dual respiratory viral infections are frequently associated with lower respiratory tract illness in infants. This study aimed to determine the impact of a dual respiratory viral infection on specific aspects of the infants immune response and the clinical course of illness. Methods: A prospective study was performed with 772 infants hospitalized from October 2000 through July 2004. Sensitive polymerase chain reaction methodology revealed the presence of a single respiratory virus in 443 (57%) of 772 cases, whereas dual infections were identified in 153 (20%) of cases. From 250 infants with confirmed respiratory viral infection, fresh heparinized blood was analyzed for interferon-γ (IFN-γ) responses by flow cytometry. Of these, 191 patients had a single infection with respiratory syncytial virus (RSV), rhinoviruses, adenoviruses or influenza viruses; and 59 patients had a dual infection with RSV and rhinoviruses, RSV and adenoviruses, influenza viruses and rhinoviruses or adenoviruses and rhinoviruses. The clinical features and peripheral lymphocyte IFN-γ responses were compared among infants with single or dual infections. Results: It was found that dual infections with non-RSV respiratory viruses induced peripheral blood mononuclear cell IFN-γ responses that mimic those of single infections, whereas coinfection with RSV was associated with reduced IFN-γ responses and a more severe clinical course of lower respiratory tract disease. Conclusions: The results indicate that the clinical characteristics and the IFN-γ response differ significantly in single and dual respiratory viral infection, depending on the nature of the simultaneously detected viruses. In dual infections, RSV involvement was associated with a decreased IFN-γ response in peripheral blood mononuclear cell and an increase in severity of illness.

Effectiveness of Reverse Transcription-PCR, Virus Isolation, and Enzyme-Linked Immunosorbent Assay for Diagnosis of Influenza A Virus Infection in Different Age Groups

ABSTRACT The degrees of effectiveness of reverse transcription (RT)-PCR, virus isolation, and antigen enzyme-linked immunosorbent assay (ELISA) for the detection of influenza A virus were evaluated with nasopharyngeal swabs from 150 patients (1 week to 86 years old) with influenza A virus infection. RT-PCR had a sensitivity for influenza A virus in stock virus preparations 103 times higher than virus isolation and 106 to 107 times higher than ELISA. The detection rate achieved by RT-PCR in clinical samples was clearly higher (93%) than that by virus isolation (80%) and ELISA (62%). Despite low overall detection rates achieved by antigen ELISA, samples from patients younger than 5 years old yielded higher-than-average rates in this rapid assay (88%). The likelihood of negative results in the ELISA increased significantly with increasing age of the patient (P < 0.01). The degrees of effectiveness of RT-PCR and virus isolation were not influenced by the age of the patient. Neither influenza immunizations nor the interval between onset of symptoms and laboratory investigation (mean, 4.7 days; standard deviation, 3.3 days) affected results obtained by the three test systems. Our results demonstrate that the ELISA is reliable for rapid laboratory diagnosis of influenza in infants and young children, but for older patients application of RT-PCR or virus isolation is necessary to avoid false negative results.

Tick-borne Encephalitis from Eating Goat Cheese in a Mountain Region of Austria

We report transmission of tick-borne encephalitis virus (TBEV) in July 2008 through nonpasteurized goat milk to 6 humans and 4 domestic pigs in an alpine pasture 1,500 m above sea level. This outbreak indicates the emergence of ticks and TBEV at increasing altitudes in central Europe and the efficiency of oral transmission of TBEV.

In vitro-synthesized infectious RNA as an attenuated live vaccine in a flavivirus model.

Live virus vaccines have in many cases proven to be an extremely effective tool for the prevention of viral diseases. However, the production of conventional live vaccines in eukaryotic cell cultures has many disadvantages, including the potential for contamination with adventitious agents and genetic alterations during propagation, making it necessary to do extensive testing before distribution. Based on results obtained with a flavivirus (tick-borne encephalitis virus) in an experimental animal system, we propose a novel live attenuated virus vaccination strategy consisting of the application of in vitro-synthesized infectious RNA instead of the live virus itself. When administered using the GeneGun, less than 1 ng of RNA was required to initiate replication of virus that was attenuated by a specifically engineered deletion and this induced a protective immunity in laboratory mice. Because this approach uses RNA, it does not have the potential drawbacks of DNA vaccines and thus combines the advantages of conventional live virus vaccines (for example, mimicking natural infection and inducing long-lasting immunity) with those of nucleic acid-based vaccines (for example, ease of production without a requirement for eukaryotic cell culture, stability and purity).

Mimicking live flavivirus immunization with a noninfectious RNA vaccine

Flaviviruses are human pathogens of world-wide medical importance. They have recently received much additional attention because of their spread to new regions (such as West Nile virus to North America), highlighting their potential as newly emerging disease agents. Using tick-borne encephalitis virus, we have developed and evaluated in mice a new genetic vaccine based on self-replicating but noninfectious RNA. This RNA contains all of the necessary genetic information for establishing its replication machinery in the host cell, thus mimicking a natural infection. However, genetic modifications in the region encoding the capsid protein simultaneously prevent the assembly of infectious virus particles and promote the secretion of noninfectious subviral particles that elicit neutralizing antibodies. These characteristics demonstrate that a new generation of flavivirus vaccines can be designed that stimulate the same spectrum of innate and specific immune responses as a live vaccine but have the safety features of an inactivated vaccine.

Incorporation of Tick-Borne Encephalitis Virus Replicons into Virus-Like Particles by a Packaging Cell Line

ABSTRACT RNA replicons derived from flavivirus genomes show considerable potential as gene transfer and immunization vectors. A convenient and efficient encapsidation system is an important prerequisite for the practical application of such vectors. In this work, tick-borne encephalitis (TBE) virus replicons and an appropriate packaging cell line were constructed and characterized. A stable CHO cell line constitutively expressing the two surface proteins prM/M and E (named CHO-ME cells) was generated and shown to efficiently export mature recombinant subviral particles (RSPs). When replicon NdΔME lacking the prM/M and E genes was introduced into CHO-ME cells, virus-like particles (VLPs) capable of initiating a single round of infection were released, yielding titers of up to 5 × 107/ml in the supernatant of these cells. Another replicon (NdΔCME) lacking the region encoding most of the capsid protein C in addition to proteins prM/M and E was not packaged by CHO-ME cells. As observed with other flavivirus replicons, both TBE virus replicons appeared to exert no cytopathic effect on their host cells. Sedimentation analysis revealed that the NdΔME-containing VLPs were physically distinct from RSPs and similar to infectious virions. VLPs could be repeatedly passaged in CHO-ME cells but maintained the property of being able to initiate only a single round of infection in other cells during these passages. CHO-ME cells can thus be used both as a source for mature TBE virus RSPs and as a safe and convenient replicon packaging cell line, providing the TBE virus surface proteins prM/M and E in trans.

Diagnosis of herpesvirus infections of the central nervous system

BACKGROUND Human herpesviruses may cause infections of the central nervous system (CNS). The early diagnosis of herpesvirus-associated neurological diseases is of high importance. OBJECTIVES The objective of this paper is to summarize the experience gained with the diagnosis of herpesvirus infections of the CNS at our institute by polymerase chain reaction (PCR)-based assays within the past few years. STUDY DESIGN A retrospective analysis of herpesvirus desoxy ribonucleic acid (DNA)-positive cerebrospinal fluid (CSF) samples was performed, with particular emphasis on data obtained by quantification of virus DNA in CSF by newly established real-time quantitative PCR assays. RESULTS Herpesviruses were found in 26.6% of all virus-positive CSF samples detected at our institute between 1995 and 2001. The overall broad testing for different herpesviruses from CSF has led to an increase in the detection rate, especially in relation to varicella zoster virus (VZV)-associated CNS disease. The herpesvirus DNA load in CSF was investigated by TaqMan real-time PCR assays that were established for the individual herpesviruses. The amount of virus varied among the individual diseases, associated with herpes simplex virus type 1, herpes simplex virus type 2, VZV and cytomegalovirus, while for Epstein-Barr virus and human herpesvirus type 6 only low levels of virus were detectable in CSF. CONCLUSIONS A generally broad testing for different herpesviruses in CSF samples is highly recommended. In addition, determination of the virus DNA level in CSF by quantitative assays seems to be of high importance for elucidating aspects concerning the prognosis of disease, the prediction of distinct CNS manifestations, and possibly the differentiation between specific virus-associated disease and unspecific presence of virus in CSF, especially in immunocompromised patients.

Early detection of acute rhinovirus infections by a rapid reverse transcription-PCR assay.

ABSTRACT The development of a rhinovirus (RV)-RNA-specific reverse transcription (RT)-PCR assay is complicated by the close homology between the RV and enterovirus (EV) genomes in the highly conserved 5′-noncoding region, which is chosen for primer design in most RT-PCR assays. We have developed a sensitive, rapid, and RV-specific nested RT-PCR assay and have used it to test nasopharyngeal aspirates from 556 patients presenting with acute respiratory tract infections. RV RNA was detected by nested RT-PCR not only in all of 52 samples that were RV positive by virus isolation methods but also in 124 of 367 samples that were negative by virus isolation methods and enzyme-linked immunosorbent assay (ELISA). In addition, in 23 of 137 samples that were positive for a different respiratory virus by virus isolation and/or ELISA, RV RNA was detected by RT-PCR. EVs, adenoviruses, respiratory syncytial viruses, coronaviruses, and influenza and parainfluenza viruses, including clinical isolates as well as stock viruses, were not amplified in our RV-specific RT-PCR assay, indicating that this assay was highly specific. The processing time was less than 2 days for the RT-PCR, as opposed to up to 2 weeks for virus isolation. These results indicate that nested RT-PCR is more sensitive than conventional methods for the detection of RV in patients experiencing acute respiratory tract infections and represents the only reliable tool for the early laboratory diagnosis of RV infections. This is especially important in light of new opportunities for therapy currently being developed.

Improved detection of respiratory syncytial virus in nasal aspirates by seminested RT-PCR

A seminested RT‐PCR for amplification of Respiratory syncytial virus (RSV)‐RNA in nasal aspirates has been developed and used to test nasopharyngeal aspirates (NPAs) from 132 infants hospitalized with acute respiratory tract infections during winter epidemics. The results were compared with those obtained by virus isolation in tissue culture and antigen detection with an enzyme‐linked immunosorbent assay (Ag‐ELISA). RSV‐RNA was detected by seminested RT‐PCR in 57 of the 59 samples that were positive by virus isolation and/or ELISA, as well as in 25 of 73 samples negative by virus isolation and ELISA. Eighteen of these 25 samples were obtained from children older than one year of age, 17 of whom were experiencing reinfection, as indicated by the presence of preexisting serum RSV‐IgG antibodies. These results indicate that seminested RT‐PCR is more sensitive than conventional methods for the detection of RSV in patients experiencing reinfections and suggest that this assay might also be useful for rapid diagnosis of RSV infections in older people. J. Med. Virol. 53:366–371, 1997.

Relationship between Cytomegalovirus DNA Load in Epithelial Lining Fluid and Plasma of Lung Transplant Recipients and Analysis of Coinfection with Epstein-Barr Virus and Human Herpesvirus 6 in the Lung Compartment

ABSTRACT Cytomegalovirus (CMV) is a significant cause of morbidity and mortality in lung transplant recipients (LTRs). The aim of the present study was to elucidate the relationship between the CMV DNA load in the lung compartment and that in plasma. For CMV load determination, the level of CMV DNA in plasma and bronchoalveolar lavage (BAL) samples was measured in a total of 97 paired BAL and plasma samples obtained from 25 LTRs. The original virus concentration in the epithelial lining fluid (ELF) was calculated from the BAL samples by correcting for dilution using the urea dilution method. In addition, the load of Epstein-Barr virus (EBV) and that of human herpesvirus 6 (HHV-6) DNA also were determined in BAL samples, recalculated for their concentrations in the ELF, and compared with the CMV DNA load. CMV DNA was found more frequently and at significantly higher levels in the lung compartment than in plasma (P < 0.001, Wilcoxon test), and the CMV load in the ELF was associated with symptomatic CMV disease. EBV and HHV-6 were detected in 43.6% and 21.7% of the ELF samples, respectively. A statistically significant association was found between the CMV and EBV DNA loads in the ELF (P < 0.001; Spearmans rho = 0.651). Thus, in LTRs, determination of the CMV DNA load in the lung compartment may be advantageous compared to monitoring only viremia. The significant relationship between EBV and CMV DNA loads in the ELF of LTRs and its clinical impact require further investigation.