A. D. M. E. Osterhaus

Diagnosing Herpesvirus Infections by Real-Time Amplification and Rapid Culture

ABSTRACT Procedures using real-time technique were developed to demonstrate the presence of herpes simplex virus type 1 (HSV-1) and HSV-2, varicella zoster virus (VZV), and cytomegalovirus (CMV) in miscellaneous clinical specimens. The assays were compared to rapid culture using centrifugation followed by detection with monoclonal antibodies. A total of 711 consecutive samples were collected from different patient groups. Throat swabs were obtained from transplant patients; dermal or oral specimens were collected from patients suspected for VZV or HSV infection. Genital specimens were taken from patients who attended the Clinic for Sexually Transmitted Diseases at the Dijkzigt Hospital Rotterdam presenting with symptoms of a primary genital ulcer. Nucleic acid extraction was carried out using a MagnaPure LC instrument. The amplification steps were performed on the ABI Prism 7700 sequence detection system. To monitor the process of extraction and amplification, a universal control consisting of seal herpesvirus type 1 (PhHV-1) was added to the clinical specimens. By culture 127 of 668 (19%) samples were positive for HSV-1, 72 of 668 (10.8%) specimens were positive for HSV-2, and 17 of 366 (4.6%) were positive for VZV. Using real-time amplification the numbers of positive specimens were 143 of 668 (21.4%), 97 of 668 (14.5%), and 27 of 366 (7.4%), respectively. Eighty-six specimens were tested for CMV, 12 (14.0%) were positive by culture, and 17 (19.8%) were positive by real-time PCR. The clinical data of the patients with discrepant results were reviewed thoroughly. In all cases the patients with only real-time PCR-positive results could be considered as truly infected. We concluded that the real-time amplification technique is suitable for the detection of human herpesvirus infection. It offers a semiquantitative and reliable assay with a quick result that is more sensitive than rapid culture, especially for the diagnosis of HSV-2 and VZV infections.

Clinical impact and diagnosis of human metapneumovirus infection.

Background. Acute respiratory infections are the most common illnesses experienced by people of all ages worldwide. A portion of hitherto unexplained viral respiratory tract illnesses (RTIs) can now be attributed to the human metapneumovirus (hMPV), which was discovered in 2001. Several surveys on the burden of disease of hMPV infection have been conducted in various study groups and with different diagnostic assays. To estimate the impact of hMPV infection in a hospital setting and in the community at large, we reviewed these surveys to establish the burden of disease of hMPV infection. Methods. Published data and our own additional unpublished data on the clinical impact of hMPV infection were reviewed. Results and conclusion. Worldwide, hMPV infections account for at least 5 to 7% of the RTI in hospitalized children, but immunocompromised and elderly individuals are also at risk. In the general community hMPV infections account for at least 3% of patients who visit a general practitioner for RTI. The seasonality of hMPV infections resembles that for respiratory syncytial virus and influenza virus infections, with recurrent epidemics during the winter months. Clinical symptoms and laboratory findings associated with hMPV infection exhibit a spectrum virtually indistinguishable from those associated with respiratory syncytial virus disease. The development of diagnostic assays must take into account the existence of two hMPV serotypes.

Avian flu: multiple introductions of H5N1 in Nigeria.

As the avian influenza virus H5N1 swept from Asia across Russia to Europe, Nigeria was the first country in Africa to report the emergence of this highly pathogenic virus. Here we analyse H5N1 sequences in poultry from two different farms in Lagos state and find that three H5N1 lineages were independently introduced through routes that coincide with the flight paths of migratory birds, although independent trade imports cannot be excluded.

Epstein-Barr virus and disease activity in multiple sclerosis

Objectives: To study in relapsing–remitting (RR) multiple sclerosis (MS) whether exacerbations and brain activity as measured by magnetic resonance imaging (MRI) are associated with plasma levels of anti-Epstein Barr (EBV) antibodies and EBV DNA. Methods: This was a prospective study with 73 RR MS patients followed for an average of 1.7 years with frequent neurological examination and blood sampling. Antibodies to various EBV proteins were measured by ELISA and plasma EBV DNA was measured by PCR. Results: All MS patients had IgG antibodies to EBV (viral capsid antigen (VCA) and/or EBV nuclear antigen (EBNA)), irrespective whether samples were taken at stable disease or exacerbation. A significantly elevated percentage of the patients (48%) had antibodies against EBV antigens (early antigen, EA) that indicate active viral replication, compared with the age matched healthy controls (25%). Antibodies against a control herpesvirus, cytomegalovirus, were similar between the two groups. The percentage of EA positive individuals and EA titres did not differ between stable disease or exacerbation. Anti-VCA IgM was positive in three cases, unrelated to disease activity. Using a highly sensitive PCR on 51 samples taken at exacerbation visits, only three patients were found to have one timepoint with viraemia, and this viraemia was unrelated to disease activity. Of special note was the fact that anti-EA seropositive patients remained seropositive during follow up, with stable titres over time. We hypothesised that these patients may constitute a subgroup with higher disease activity, due to the triggering effect of a chronic attempt of the virus to reactivate. The EA positive group did not differ from the EA negative with respect to clinical disease activity or other characteristics. However, in the EA positive group, analysis with gadolinium enhanced MRI indicated more MRI disease activity. Conclusions: There was no evidence for increased clinical disease activity in the subgroup of MS patients with serological signs of EBV reactivation. However, the observation that chronic EBV reactivation may be associated with increased inflammatory activity as assessed by gadolinium enhanced MRI lesions should be reproduced in a larger and independent dataset.

Epizootic of morbilliviral disease in common dolphins (Delphinus delphis ponticus) from the Black Sea

Forty-seven common dolphins (Delphinus delphis ponticus) were stranded on the northem shores of the Black Sea between mid-July and early September 1994, more than in previous or subsequent years. Two of the 47 dolphins were examined in detail to try to determine the cause of the increased stranding rate. Their lesions included broncho-interstitial pneumonia with type 11 epithelial cell hyperplasia and multinucleate syncytial cells, neuronal necrosis, gliosis, and non-suppurative meningitis of the brain, necrotic stomatitis, gastroenteritis and cholangitis, and lymphoid depletion of the spleen and lymph nodes. The diseased tissues stained positive in an immunoperoxidase test, using a polyclonal antiserum to measles virus as the primary antibody, and electron microscopy showed that they contained regularly-shaped intranuclear particles about 22 nm in diameter. They were positive by the polymerase chain reaction (PcR) for the nucleoprotein gene of morbillivirus. However, there was no evidence of morbillivirus in frozen tissues either by virus isolation or by antigen capture EUSA. The concentration of ΣDDTS in the blubber of both dolphins was about 50 to 100 times higher than the levels in toothed cetaceans from the North Sea, North Atlantic Ocean, and Baltic Sea. The lesions were consistent with those found in other species with morbilliviral disease, and the positive immunoperoxidase test, PCR and electron microscopical examination confirmed a morbillivirus as the primary cause of these lesions.

Norovirus in a Dutch tertiary care hospital (2002–2007): frequent nosocomial transmission and dominance of GIIb strains in young children

We report a retrospective analysis of norovirus (NoV) infections occurring in patients of a tertiary care hospital during five winter seasons (2002/03 to 2006/07). Data were compared with national surveillance data and with corresponding data for rotavirus. Between July 2002 and June 2007, faecal specimens from 221 (9.0%) of 2458 hospital patients with diarrhoea tested positive for NoV. The incidence in children varied from 2.52 per 1000 admissions in 2004/05 (when testing began to be performed routinely) to 11.9 per 1000 admissions in 2006/07, while the incidence in adults remained stable (mean: 1.49 per 1000 admissions). Two genotypes predominated during the study period: GIIb strains occurred mainly in children below the age of two-and-a-half years [odds ratio (OR): 14.7; P<0.0001] whereas GII.4 strains affected all age groups. Compared with rotavirus infections, NoV infections in children were more often hospital-acquired (59% vs 39%, OR: 2.29; P<0.01). Among these cases we identified 22 clusters of NoV infection among inpatients. Twelve of 53 patients from whom follow-up samples were available demonstrated long-term virus shedding. We report a dynamic pattern of sporadic NoV infections in large hospitals, with frequent nosocomial transmission and with the predominance of GIIb-related strains in children. Effective prevention strategies are required to reduce the impact of sporadic NoV infection in vulnerable patients.

Transmission studies resume for avian flu

In January 2012, influenza virus researchers from around the world announced a voluntary pause of 60 days on any research involving highly pathogenic avian influenza H5N1 viruses leading to the generation of viruses that are more transmissible in mammals (1). We declared a pause to this important research to provide time to explain the public health benefits of this work, to describe the measures in place to minimize possible risks, and to enable organizations and governments around the world to review their policies (for example, on biosafety, biosecurity, oversight, and communication) regarding these experiments. n nDuring the past year, the benefits of this important research have been explained clearly in publications (2-7) and meetings (8-10). Measures to mitigate possible risks of the work have been detailed (11-13). The World Health Organization has released recommendations on laboratory biosafety for those conducting this research (14), and relevant authorities in several countries have reviewed the biosafety, biosecurity, and funding conditions under which further research would be conducted on the laboratory-modified H5N1 viruses (10, 15-17). Thus, acknowledging that the aims of the voluntary moratorium have been met in some countries and are close to being met in others, we declare an end to the voluntary moratorium on avian flu transmission studies. n n n n n nThe controversy surrounding H5N1 virus transmission research has highlighted the need for a global approach to dealing with dualuse research of concern. Developing comprehensive solutions to resolve all the issues will take time. Meanwhile, H5N1 viruses continue to evolve in nature. Because H5N1 virus transmission studies are essential for pandemic preparedness and understanding the adaptation of influenza viruses to mammals, researchers who have approval from their governments and institutions to conduct this research safely, under appropriate biosafety and biosecurity conditions, have a public health responsibility to resume this important work. Scientists should not restart their work in countries where, as yet, no decision has been reached on the conditions for H5N1 virus transmission research. At this time, this includes the United States and U.S.-funded research conducted in other countries. Scientists should never conduct this type of research without the appropriate facilities, oversight, and all the necessary approvals. We consider biosafety level 3 conditions with the considerable enhancements (BSL-3+) outlined in the referenced publications (11-13) as appropriate for this type of work, but recognize that some countries may require BSL-4 conditions in accordance with applicable standards (such as Canada). We fully acknowledge that this research—as with any work on infectious agents—is not without risks. However, because the risk exists in nature that an H5N1 virus capable of transmission in mammals may emerge, the benefits of this work outweigh the risks.

Serologic survey for selected viral pathogens in free-ranging endangered European mink (Mustela lutreola) and other mustelids from South-Western France

To investigate the possible role of selected pathogens in the decline of endangered European mink (Mustela lutreola) populations and the potential for these pathogens to affect mink survival, a serologic survey was conducted using serum samples collected from March 1996 to March 2003 in eight departments of south-western France. In total, 481 free-ranging individuals of five mustelid species (including the European mink) were tested. Sympatric mustelids can serve as sentinels to determine the presence of antibodies to viruses in the study area that could potentially infect mink. Antibodies to Canine distemper virus (CDV) were detected in all species; 9% of 127 European mink, 20% of 210 polecats (Mustela putorius), 5% of 112 American mink (Mustela vison), 33% of 21 stone marten (Martes foina) and 5% of 20 pine marten (Martes martes). Antibody prevalence was significantly higher in stone marten and polecats, possibly because their ranges overlap more closely with that of domestic species than that of the other species tested. Antibodies to Canine adenovirus were detected in all species but the pine marten; antibody prevalence estimates ranging from 2% to 10%. Antibodies to canine parainfluenza virus were detected in 1% of European mink, 1% of American mink and 5% of tested polecats but were not detected in Martes species. Antibodies to Rabies virus (RV) were detected in three animals, possibly because of interspecies transmission of bat lyssaviruses as the sampling area is considered to be free of RV, or to a lack of test specificity, as antibody titers were low. The high antibody prevalence to potentially lethal CDV suggests that this pathogen could have significant effects on the free-ranging populations and has implications for the conservation efforts for the endangered European mink.

Evidence for influenza virus CNS invasion along the olfactory route in an immunocompromised infant

Central nervous system (CNS) disease is the most common extrarespiratory complication of influenza in humans. However, the pathogenesis, including the route of virus entry, is largely unknown. Here we present, for the first time, evidence of influenza virus entry into the CNS via the olfactory route in an immune-compromised infant. Since the nasal cavity is a primary site of influenza virus replication and is directly connected to the CNS via the olfactory nerve, these results imply that influenza virus invasion of the CNS may occur more often than previously believed.

Altered Protein Networks and Cellular Pathways in Severe West Nile Disease in Mice

Background The recent West Nile virus (WNV) outbreaks in developed countries, including Europe and the United States, have been associated with significantly higher neuropathology incidence and mortality rate than previously documented. The changing epidemiology, the constant risk of (re-)emergence of more virulent WNV strains, and the lack of effective human antiviral therapy or vaccines makes understanding the pathogenesis of severe disease a priority. Thus, to gain insight into the pathophysiological processes in severe WNV infection, a kinetic analysis of protein expression profiles in the brain of WNV-infected mice was conducted using samples prior to and after the onset of clinical symptoms. Methodology/Principal Findings To this end, 2D-DIGE and gel-free iTRAQ labeling approaches were combined, followed by protein identification by mass spectrometry. Using these quantitative proteomic approaches, a set of 148 proteins with modified abundance was identified. The bioinformatics analysis (Ingenuity Pathway Analysis) of each protein dataset originating from the different time-point comparisons revealed that four major functions were altered during the course of WNV-infection in mouse brain tissue: i) modification of cytoskeleton maintenance associated with virus circulation; ii) deregulation of the protein ubiquitination pathway; iii) modulation of the inflammatory response; and iv) alteration of neurological development and neuronal cell death. The differential regulation of selected host protein candidates as being representative of these biological processes were validated by western blotting using an original fluorescence-based method. Conclusion/Significance This study provides novel insights into the in vivo kinetic host reactions against WNV infection and the pathophysiologic processes involved, according to clinical symptoms. This work offers useful clues for anti-viral research and further evaluation of early biomarkers for the diagnosis and prevention of severe neurological disease caused by WNV.