Temporal relationship between environmental influenza A and Epstein-Barr viral infections and high multiple sclerosis relapse occurrence.

BACKGROUND: Multiple sclerosis (MS) relapses have been associated with viral and bacterial infection epidemics in MS patients who have not used interferon. OBJECTIVES: We studied whether environmental viral infections in the general population can be associated with increased MS relapse occurrence using retrospective data from 1986 to 1995 when interferons were not yet available. METHODS: Logistic regression modelling was used to compare retrospectively the monthly relapse occurrence from 407 MS patients in Turku University hospital archives and data on ten different specifically diagnosed viral infection epidemics in the general population of Southwestern Finland from 1986 to 1995. The outcome was the odds ratio (OR) of very high relapse occurrence versus low relapse occurrence, or moderate versus low relapse occurrence. RESULTS: After a peak in diagnosed influenza A cases in the general population, the MS relapse occurrence was 6.5 times more likely to be very high (95% CI 1.8-24.0) and 7.1 times more likely to be moderately high (95% CI 1.5-33.2). An increase in MS relapse counts also followed Epstein-Barr virus (EBV) infections (OR 4.4, 95% CI 1.3-15.1), but we found no significant association with adenovirus infections and MS relapses. The MS relapse occurrence was lowest in the summer months July-August (Chi-square test, p<0.01). CONCLUSIONS: Our findings suggest that influenza A and EBV viral infections in the general population are associated with a higher occurrence of exacerbations in MS patients, and thus environmental infection data should be included in epidemiological models on MS relapses.

Microbiology of acute otitis media in children with tympanostomy tubes: prevalences of bacteria and viruses.

BACKGROUND: Bacteria are found in 50%-90% of cases of acute otitis media (AOM) with or without otorrhea, and viruses are found in 20%-49% of cases. However, for at least 15% of patients with AOM, the microbiological etiology is never determined. Our aim was to specify the full etiology of acute middle ear infection by using modern microbiological methods concomitantly for bacterial and viral detection. METHODS: The subjects were 79 young children having AOM with new onset (<48 h) of otorrhea through a tympanostomy tube. Middle ear fluid samples were suctioned from the middle ear through the tympanostomy tube. Bacteria were sought by culture and polymerase chain reaction; viruses were analyzed by culture, antigen detection, and polymerase chain reaction. RESULTS: At least 1 respiratory tract pathogen was noted in 76 children (96%). Bacteria were found in 73 cases (92%), and viruses were found in 55 (70%). In 52 patients (66%), both bacteria and viruses were found. Bacteria typical of AOM were detected in 86% of patients. Picornaviruses accounted for 60% of all viral findings. CONCLUSIONS: In the great majority of children, AOM is a coinfection with bacteria and viruses. The patent tympanostomy tube does not change the spectrum of causative agents in AOM. A microbiological etiology can be established in practically all cases.

A randomized, double-blind study of the safety, transmissibility and phenotypic and genotypic stability of cold-adapted influenza virus vaccine.

BACKGROUND: Live attenuated influenza vaccine (LAIV; FluMist) is a trivalent vaccine containing cold-adapted influenza vaccine viruses that infect and replicate in cells lining the nasopharynx to induce immunity. Recovery of viruses (shedding) is measured by culture of nasal specimens. Shedding of vaccine viruses is not equated with transmission because transmission requires more virus than is detected in many nasal swabs. Previous studies with LAIV did not detect transmission to close contacts. The primary objective of this study was to estimate the probability of transmission to placebo contacts in a day care setting. METHODS: One hundred ninety-seven healthy children aged 9 to 36 months attending day care were randomized to receive vaccine or placebo. Postvaccination viral shedding, safety, genotype and phenotype of shed viruses and probability of transmission were assessed. RESULTS: Eighty percent of 98 vaccine recipients shed at least one vaccine strain. No clinically significant differences in solicited adverse events attributable to vaccine occurred; safety profiles were similar in both groups. Vaccine virus isolates retained their phenotypic characteristics (cold adaptation and temperature sensitivity) and did not revert at nucleotides known to confer an attenuating phenotype. There was one confirmed transmission of a vaccine strain to a single placebo recipient. According to the Reed-Frost model, the calculated probability of transmission to a child after contact with a single vaccinated child was 0.58% (95% confidence interval, 0-1.7%). There was no increased reactogenicity or other safety concerns in the recipient child. CONCLUSIONS: Young children in a day care setting had a high rate of shedding and a low rate of transmission. No clinically significant illness occurred among children who received vaccine or placebo or in the child to whom the vaccine virus was transmitted.

Herpes simplex virus type 1 infection induces upregulation of interleukin-23 (p19) mRNA expression in trigeminal ganglia of BALB/c mice.

We investigated the expression kinetics of several cytokines in trigeminal ganglia (TG) and in brains of BALB/c mice during the course of ocular herpes simplex virus type 1 (HSV-1) infection. All mice recovered from the infection within 2 weeks. The quantitative rapid real-time RT-PCR method was used to analyze interleukin-4 (IL-4), interferon-gamma (IFN-gamma), IL-12p35, IL-12p40, and the recently described IL-23 (p19) mRNA in TG, brain, and splenocyte samples. In TG, we found elevated expression of mRNA for IL-23 (p19) from early acute infection (day 3) to the beginning of the latent phase (day 14). The increase was not detected in brain or in the spleen. IL-4 expression occurred in both TG and brain from the beginning of the experiment to the latent phase. During the latent phase (days 14 and 31), IL-4 expression was significantly elevated in the brain when compared with the uninfected controls (p < 0.05). Considerable expression of IFN-gamma mRNA was detected in TG of mice during acute HSV-1 infection. The expression of IL-23 was detected also in the brains of the mice, even though no significant changes were found during the acute HSV-1 infection. This is, to our knowledge, the first report to show elevated expression of IL-23 (p19) mRNA (p < 0.05) during viral infection in TG of mice.

Cytokines in experimental herpes simplex virus infection.

Herpes simplex virus (HSV) causes productive and latent forms of infection in humans and experimental animals. The primary infection and reactivation of the latent infection evoke an immune response in the host organism, involving activities of macrophages, CD4+ and CD8+ lymphocytes, and B lymphocytes. Strong cytokine responses are associated with the acute and recurrent phases of HSV infection. Also, during the latent phase of HSV infection in the sensory ganglia, expression of certain cytokines can be detected. The cytokine response to HSV infection is dominated by proinflammatory and Th1 type cytokines; however, Th2 type cytokines such as interleukin-4 also are expressed in the infected tissue. The use of novel HSV-derived, cytokine-expressing gene therapy vectors necessitates studies on the possible modulation of the host responses by the virus-encoded cytokine transgenes. This review focuses on the roles of certain Th1 and Th2 type cytokines in different phases of the experimental HSV infections.

Low copy number detection of herpes simplex virus type 1 mRNA and mouse Th1 type cytokine mRNAs by Light Cycler quantitative real-time PCR.

The real-time principle of reverse transcriptase polymerase chain reaction (RT-PCR) provides a quantitative and reproducible method to detect low copy number transcripts. The quantitative detection of cytokines from tissue samples is complicated by the low expression rates and the short half-lives of the cytokine proteins. The methods have been insensitive and labor-intensive. The LightCycler technique provides a 30-min PCR system with continuous fluorescent detection, analysis of the melting points of the products and user-friendly software for the analysis of the unknown samples. External copy number standards enable the measurement of amounts of the desired targets. We demonstrate the dynamic range of the RT-PCR system from a 100 to 10(7) mRNA copies of the mouse Th1 cytokines interleukin- (IL-) 12p35, 12p40 and IL-23p19 as well as gamma interferon (IFN-gamma) and the housekeeping gene beta-actin, with the usage of fluorescent hybridization probes. The cytokine quantitation was exemplified in murine nervous system samples. A viral transcript, mRNA of alpha trans-inducing factor (alphaTIF), or VP16 gene, of herpes simplex virus (HSV) type 1 was used to quantitate the viral replication in infected cells and in murine nervous system samples. For this viral transcript the linear dynamic range spanned from ten copies to one million copies (highest tested). For all tested cytokine transcripts, the detection level with the dsDNA binding dye SYBR Green I was one log lower than with the hybridizing fluorescent probes. The viral transcript was detected even with the SYBR Green I system at the level of ten copies. The specificity of the PCR was reached with the use of TaqStart antibody, by careful design of primers and probes, by melting temperature analysis and comparison with the gel electrophoresis and Southern blot analysis.

IL-4 is the key regulator in herpes simplex virus-based gene therapy of BALB/c experimental autoimmune encephalomyelitis.

Local delivery of cytokines or other immunomodulatory components has been applied as a potential therapy for experimental autoimmune encephalomyelitis (EAE), which is used as a model of human multiple sclerosis. We have used herpes simplex virus-based vectors expressing Th2 cytokines IL-4 and IL-10 and have previously shown a significant abolishment of disease symptoms by the virus expressing IL-4 (R8306), but not by the one expressing IL-10 (R8308). In the present study, the aim was to investigate the local and systemic cytokine response after HSV-based gene therapy. We show that the local expression of IL-4 from an HSV vector delivered to the brain converts the cytokine environment from the disease-promoting Th1-prominent to the disease-limiting IL-4 expressing type. We measured the expression of cytokines IL-4, IL-10, IFN-gamma, IL-12p35, IL-12p40 and the novel IL-23p19 from the brain by quantitative LightCycler RT-PCR. We also investigated the systemic cytokine response from the mouse sera. The results indicate that an increase in the Th2 cytokine IL-4 is observed if the diseased mice are treated with IL-4-expressing virus R8306. Surprisingly, the IL-23 expression of R8306 treated mice was at the same level as in the untreated EAE mice. On the contrary, in the R8308 (IL-10 expression) treated mice, the expression of IL-23 was decreased (P < 0.05). We conclude that the favorable effect of IL-4 on the disease development is more important than the downregulation of the Th1 type cytokines (like IL-23), and that IL-4 would be the key mediator of disease abolishment during gene therapy using these vectors.

Nasal swab versus nasopharyngeal aspirate for isolation of respiratory viruses.

To determine the usefulness of nasal swabs as a simple method for detection of respiratory viruses, we compared nasal swabs and nasopharyngeal aspirates obtained at the same time from the opposite nostrils of 230 children with upper respiratory infection. The sensitivity of nasal swabs was comparable to that of nasopharyngeal aspirates for the detection of all major respiratory viruses except respiratory syncytial virus.

Prefusion rearrangements resulting in fusion Peptide exposure in Semliki forest virus.

Semliki Forest virus (SFV), like many enveloped viruses, takes advantage of the low pH in the endosome to convert into a fusion-competent configuration and complete infection by fusion with the endosomal membrane. Unlike influenza virus, carrying an N-terminal fusion peptide, SFV represents a less-well understood fusion principle involving an endosequence fusion peptide. To explore the series of events leading to a fusogenic configuration of the SFV, we exposed the virus to successive acidification, mimicking endosomal conditions, and followed structural rearrangements at probed sensor surfaces. Thus revealed, the initial phase involves a transient appearance of a non-linear neutralizing antibody epitope in the fusion protein, E1. Concurrent with the disappearance of this epitope, a set of masked sequences in proteins E1 and E2 became exposed. When pH reached 6.0-5.9 the virion transformed into a configuration of enlarged diameter with the fusion peptide optimally exposed. Simultaneously, a partly hidden sequence close to the receptor binding site in E2 became fully uncovered. At this presumably fusogenic stage, maximally 80 fusion peptide-identifying antibody Fab fragments could be bound per virion, i.e. one ligand per three copies of the fusion protein. The phenomena observed are discussed in terms of alphavirus structure and reported functional domains.