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Ron A M Fouchier

Publications and source records attributed to Ron A M Fouchier.

At least 37 records · Page 2Linked to original sources
PubMed2004

Pegylated interferon-alpha protects type 1 pneumocytes against SARS coronavirus infection in macaques.

The primary cause of severe acute respiratory syndrome (SARS) is a newly discovered coronavirus. Replication of this SARS coronavirus (SCV) occurs mainly in the lower respiratory tract, and causes diffuse alveolar damage. Lack of understanding of the pathogenesis of SARS has prevented the rational development of a therapy against this disease. Here we show extensive SCV antigen expression in type 1 pneumocytes of experimentally infected cynomolgus macaques (Macaca fascicularis) at 4 d postinfection (d.p.i.), indicating that this cell type is the primary target for SCV infection early in the disease, and explaining the subsequent pulmonary damage. We also show that prophylactic treatment of SCV-infected macaques with the antiviral agent pegylated interferon-alpha (IFN-alpha) significantly reduces viral replication and excretion, viral antigen expression by type 1 pneumocytes and pulmonary damage, compared with untreated macaques. Postexposure treatment with pegylated IFN-alpha yielded intermediate results. We therefore suggest that pegylated IFN-alpha protects type 1 pneumocytes from SCV infection, and should be considered a candidate drug for SARS therapy.

Animals↗
PubMed2004

Recognition of homo- and heterosubtypic variants of influenza A viruses by human CD8+ T lymphocytes.

In the present study, the recognition of epitope variants of influenza A viruses by human CTL was investigated. To this end, human CD8(+) CTL clones, specific for natural variants of the HLA-B*3501-restricted epitope in the nucleoprotein (NP(418-426)), were generated. As determined in (51)Cr release assays and by flow cytometry with HLA-B*3501-peptide tetrameric complexes, CTL clones were found to be specific for epitopes within one subtype or cross-reactive with heterosubtypic variants of the epitope. Using eight natural variants of the epitope, positions in the 9-mer important for T cell recognition and involved in escape from CTL immunity were identified and visualized using multidimensional scaling. It was shown that positions 4 and 5 in the 9-mer epitope were important determinants of T cell specificity. The in vivo existence of CD8(+) cells cross-reactive with homo- and heterosubtypic variants of the epitope was further confirmed using polyclonal T cell populations obtained after stimulation of PBMC with different influenza A viruses. Based on the observed recognition patterns of the clonal and polyclonal T cell populations and serology, it is hypothesized that consecutive infections with influenza viruses containing different variants of the epitope select for cross-reactive T cells in vivo.

Adult↗
PubMed2004

Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome.

Highly pathogenic avian influenza A viruses of subtypes H5 and H7 are the causative agents of fowl plague in poultry. Influenza A viruses of subtype H5N1 also caused severe respiratory disease in humans in Hong Kong in 1997 and 2003, including at least seven fatal cases, posing a serious human pandemic threat. Between the end of February and the end of May 2003, a fowl plague outbreak occurred in The Netherlands. A highly pathogenic avian influenza A virus of subtype H7N7, closely related to low pathogenic virus isolates obtained from wild ducks, was isolated from chickens. The same virus was detected subsequently in 86 humans who handled affected poultry and in three of their family members. Of these 89 patients, 78 presented with conjunctivitis, 5 presented with conjunctivitis and influenza-like illness, 2 presented with influenza-like illness, and 4 did not fit the case definitions. Influenza-like illnesses were generally mild, but a fatal case of pneumonia in combination with acute respiratory distress syndrome occurred also. Most virus isolates obtained from humans, including probable secondary cases, had not accumulated significant mutations. However, the virus isolated from the fatal case displayed 14 amino acid substitutions, some of which may be associated with enhanced disease in this case. Because H7N7 viruses have caused disease in mammals, including horses, seals, and humans, on several occasions in the past, they may be unusual in their zoonotic potential and, thus, form a pandemic threat to humans.

Amino Acid Sequence↗
PubMed2004

Efficient generation and growth of influenza virus A/PR/8/34 from eight cDNA fragments.

A reverse genetics system for the generation of influenza virus A/PR/8/34 (NIBSC vaccine strain) from plasmid DNA was developed. Upon transfection of eight bidirectional transcription plasmids encoding the gene segments of A/PR/8/34 into 293T cells, virus titers in the supernatant were about 10(4) TCID50/ml. The production of A/PR/8/34 in 293T cells was compared to that of A/WSN/33, for which virus titers in the supernatant were 10(7)-10(8) TCID50/ml. Time-course analysis of virus production indicated that the differences in virus titers were due to reinfection of 293T cells by A/WSN/33 but not A/PR/8/34. Indeed, virus titers of A/PR/8/34 comparable to those of A/WSN/33 were achieved upon addition of trypsin to the culture medium of transfected cells. The production of chimeric viruses revealed that the difference in virus titers between A/PR/8/34 and A/WSN/33 are determined primarily by differences in the surface glycoproteins hemagglutinin and neuraminidase and the polymerase protein PB1. In conclusion, high-titer virus stocks of recombinant influenza A/PR/8/34 virus can be produced as well as virus stocks with much lower titers, but without the requirement of virus amplification through replication.

Animals↗
PubMed2004

Experimental human metapneumovirus infection of cynomolgus macaques (Macaca fascicularis) results in virus replication in ciliated epithelial cells and pneumocytes with associated lesions throughout the respiratory tract.

A substantial proportion of hitherto unexplained respiratory tract illnesses is associated with human metapneumovirus (hMPV) infection. This virus also was found in patients with severe acute respiratory syndrome (SARS). To determine the dynamics and associated lesions of hMPV infection, six cynomolgus macaques (Macaca fascicularis) were inoculated with hMPV and examined by pathological and virological assays. They were euthanized at 5 (n = 2) or 9 (n = 2) days post-infection (dpi), or monitored until 14 dpi (n = 2). Viral excretion peaked at 4 dpi and decreased to zero by 10 dpi. Viral replication was restricted to the respiratory tract and associated with minimal to mild, multi-focal erosive and inflammatory changes in conducting airways, and increased numbers of macrophages in alveoli. Viral expression was seen mainly at the apical surface of ciliated epithelial cells throughout the respiratory tract, and less frequently in type 1 pneumocytes and alveolar macrophages. Both cell tropism and respiratory lesions were distinct from those of SARS-associated coronavirus infection, excluding hMPV as the primary cause of SARS. This study demonstrates that hMPV is a respiratory pathogen and indicates that viral replication is short-lived, polarized to the apical surface, and occurs primarily in ciliated respiratory epithelial cells.

Animals↗
PubMed2004

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.

Acute Disease↗
PubMed2004

Identification of small-animal and primate models for evaluation of vaccine candidates for human metapneumovirus (hMPV) and implications for hMPV vaccine design.

Human metapneumovirus (hMPV), a recently identified paramyxovirus, is the causative agent of respiratory tract disease in young children. Epidemiological studies have established the presence of hMPV in retrospective as well as current clinical samples in Europe, USA, Canada, Hong Kong and Australia. The hMPV disease incidence rate varied from 7 to 12 %. This rate of disease attack places hMPV in severity between respiratory syncytial virus and human parainfluenza virus type 3, two common respiratory pathogens of young children, the elderly and immunosuppressed individuals. To evaluate the effectiveness and safety of future hMPV antiviral drugs, therapeutic and prophylactic monoclonal antibodies (mAbs), and vaccine candidates, it was necessary to identify small-animal and primate models that efficiently supported hMPV replication in the respiratory tract and produced neutralizing serum antibodies, commonly a clinical correlate of protection in humans. In this study, various rodents (mice, cotton rats, hamsters and ferrets) and two primate species, rhesus macaques and African green monkeys (AGMs), were evaluated for hMPV replication in the respiratory tract. The results showed that hamsters, ferrets and AGMs supported hMPV replication efficiently and produced high levels of hMPV-neutralizing antibody titres. Hamsters vaccinated with subgroup A hMPV were protected from challenge with subgroup A or subgroup B hMPV, which has implications for hMPV vaccine design. Although these animal models do not mimic human hMPV disease signs, they will nevertheless be invaluable for the future evaluation of hMPV antivirals, mAbs and vaccines.

Animals↗
PubMed2004

Real-time reverse transcriptase PCR assay for detection of human metapneumoviruses from all known genetic lineages.

The discovery of human metapneumovirus and its implications for respiratory tract disease have emphasized the need for a sensitive, specific, and rapid assay to detect this virus in a clinical setting. It recently became clear that human metapneumovirus can be grouped into at least four genetic lineages. Previously described assays for the detection of human metapneumovirus were developed by using limited sequence information and failed to detect viruses from all four genetic lineages with comparable sensitivities. Here we describe the development and evaluation of a real-time reverse transcriptase PCR assay that detects human metapneumovirus from the four known genetic lineages with equal specificities and sensitivities.

Base Sequence↗
PubMed2004

Recovery of human metapneumovirus genetic lineages a and B from cloned cDNA.

Human metapneumovirus (hMPV) is a newly discovered pathogen associated with respiratory tract illness, primarily in young children, immunocompromised individuals, and the elderly. The genomic sequence of the prototype hMPV isolate NL/1/00 without the terminal leader and trailer sequences has been reported previously. Here we describe the leader and trailer sequences of two hMPV isolates, NL/1/00 and NL/1/99, representing the two main genetic lineages of hMPV. Minigenome constructs in which the green fluorescent protein or chloramphenicol acetyltransferase genes are flanked by the viral genomic ends derived from both hMPV lineages and transcribed using a T7 RNA polymerase promoter-terminator cassette were generated. Cotransfection of minigenome constructs with plasmids expressing the polymerase complex components L, P, N, and M2.1 in 293T or baby hamster kidney cells resulted in expression of the reporter genes. When the minigenome was replaced by a sense or antisense full-length cDNA copy of the NL/1/00 or NL/1/99 viral genomes, recombinant virus was recovered from transfected cells. Viral titers up to 10(7.2) and 10(5.7) 50% tissue culture infective dose/ml were achieved with the sense and antisense plasmids, respectively. The recombinant viruses replicated with kinetics similar to those of the parental viruses in Vero cells. This reverse genetics system provides an important new tool for applied and fundamental research.

5' Untranslated Regions↗
PubMed2004

Antigenic and genetic variability of human metapneumoviruses.

Human metapneumovirus (HMPV) is a member of the subfamily Pneumovirinae within the family Paramyxo- viridae. Other members of this subfamily, respiratory syncytial virus and avian pneumovirus, can be divided into subgroups on the basis of genetic or antigenic differences or both. For HMPV, the existence of different genetic lineages has been described on the basis of variation in a limited set of available sequences. We address the antigenic relationship between genetic lineages in virus neutralization assays. In addition, we analyzed the genetic diversity of HMPV by phylogenetic analysis of sequences obtained for part of the fusion protein (n = 84) and the complete attachment protein open reading frames (n = 35). On the basis of sequence diversity between attachment protein genes and the differences in virus neutralization titers, two HMPV serotypes were defined. Each serotype could be divided into two genetic lineages, but these did not reflect major antigenic differences.

Amino Acid Sequence↗
PubMed2004

Avian influenza H5N1 in tigers and leopards.

Influenza virus is not known to affect wild felids. We demonstrate that avian influenza A (H5N1) virus caused severe pneumonia in tigers and leopards that fed on infected poultry carcasses. This finding extends the host range of influenza virus and has implications for influenza virus epidemiology and wildlife conservation.

Animals↗
PubMed2003

Prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients.

During a 17-month period, we performed retrospective analyses of the prevalence of and clinical symptoms associated with human metapneumovirus (hMPV) infection, among patients in a university hospital in The Netherlands. All available nasal-aspirate, throat-swab, sputum, and bronchoalveolar-lavage samples (N=1515) were tested for hMPV RNA by reverse-transcriptase polymerase chain reaction. hMPV RNA was detected in 7% of samples from patients with respiratory tract illnesses (RTIs) and was the second-most-detected viral pathogen in these patients during the last 2 winter seasons. hMPV was detected primarily in very young children and in immunocompromised individuals. In young children, clinical symptoms associated with hMPV infection were similar to those associated with human respiratory syncytial virus (hRSV) infection, but dyspnea, feeding difficulties, and hypoxemia were reported more frequently in hRSV-infected children. Treatment with antibiotics and corticosteroids was reported more frequently in hMPV-infected children. From these data, we conclude that hMPV is an important pathogen associated with RTI.

Adolescent↗
PubMed2003

Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome.

BACKGROUND: The worldwide outbreak of severe acute respiratory syndrome (SARS) is associated with a newly discovered coronavirus, SARS-associated coronavirus (SARS-CoV). We did clinical and experimental studies to assess the role of this virus in the cause of SARS. METHODS: We tested clinical and postmortem samples from 436 SARS patients in six countries for infection with SARS-CoV, human metapneumovirus, and other respiratory pathogens. We infected four cynomolgus macaques (Macaca fascicularis) with SARS-CoV in an attempt to replicate SARS and did necropsies on day 6 after infection. FINDINGS: SARS-CoV infection was diagnosed in 329 (75%) of 436 patients fitting the case definition of SARS; human metapneumovirus was diagnosed in 41 (12%) of 335, and other respiratory pathogens were diagnosed only sporadically. SARS-CoV was, therefore, the most likely causal agent of SARS. The four SARS-CoV-infected macaques excreted SARS-CoV from nose, mouth, and pharynx from 2 days after infection. Three of four macaques developed diffuse alveolar damage, similar to that in SARS patients, and characterised by epithelial necrosis, serosanguineous exudate, formation of hyaline membranes, type 2 pneumocyte hyperplasia, and the presence of syncytia. SARS-CoV was detected in pneumonic areas by virus isolation and RT-PCR, and was localised to alveolar epithelial cells and syncytia by immunohistochemistry and transmission electron microscopy. INTERPRETATION: Replication in SARS-CoV-infected macaques of pneumonia similar to that in human beings with SARS, combined with the high prevalence of SARS-CoV infection in SARS patients, fulfill the criteria required to prove that SARS-CoV is the primary cause of SARS.

Animals↗
PubMed2003

Animal influenza virus surveillance.

Influenza surveillance studies in humans and animals performed for many years have provided a wealth of data on the ecology and evolution of influenza viruses and their public health related and economical impact. Here we provide a brief overview of some of the data from the past and give our view on how the continued and intensified animal influenza virus surveillance may aid in the early recognition and management of outbreaks in the future.

Animals↗
PubMed2003

Identification of a novel coronavirus in patients with severe acute respiratory syndrome.

BACKGROUND: The severe acute respiratory syndrome (SARS) has recently been identified as a new clinical entity. SARS is thought to be caused by an unknown infectious agent. METHODS: Clinical specimens from patients with SARS were searched for unknown viruses with the use of cell cultures and molecular techniques. RESULTS: A novel coronavirus was identified in patients with SARS. The virus was isolated in cell culture, and a sequence 300 nucleotides in length was obtained by a polymerase-chain-reaction (PCR)-based random-amplification procedure. Genetic characterization indicated that the virus is only distantly related to known coronaviruses (identical in 50 to 60 percent of the nucleotide sequence). On the basis of the obtained sequence, conventional and real-time PCR assays for specific and sensitive detection of the novel virus were established. Virus was detected in a variety of clinical specimens from patients with SARS but not in controls. High concentrations of viral RNA of up to 100 million molecules per milliliter were found in sputum. Viral RNA was also detected at extremely low concentrations in plasma during the acute phase and in feces during the late convalescent phase. Infected patients showed seroconversion on the Vero cells in which the virus was isolated. CONCLUSIONS: The novel coronavirus might have a role in causing SARS.

Adult↗