Crouching tiger, hidden dragon: the laboratory diagnosis of severe acute respiratory syndrome.
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Genome, Viral↗
Biomedical subjects
J S M Peiris
Publications and source records attributed to J S M Peiris.
Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China.
A novel coronavirus (SCoV) is the etiological agent of severe acute respiratory syndrome (SARS). SCoV-like viruses were isolated from Himalayan palm civets found in a live-animal market in Guangdong, China. Evidence of virus infection was also detected in other animals (including a raccoon dog, Nyctereutes procyonoides) and in humans working at the same market. All the animal isolates retain a 29-nucleotide sequence that is not found in most human isolates. The detection of SCoV-like viruses in small, live wild mammals in a retail market indicates a route of interspecies transmission, although the natural reservoir is not known.
Amino Acid Sequence↗
Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study.
BACKGROUND: We investigated the temporal progression of the clinical, radiological, and virological changes in a community outbreak of severe acute respiratory syndrome (SARS). METHODS: We followed up 75 patients for 3 weeks managed with a standard treatment protocol of ribavirin and corticosteroids, and assessed the pattern of clinical disease, viral load, risk factors for poor clinical outcome, and the usefulness of virological diagnostic methods. FINDINGS: Fever and pneumonia initially improved but 64 (85%) patients developed recurrent fever after a mean of 8.9 (SD 3.1) days, 55 (73%) had watery diarrhoea after 7.5 (2.3) days, 60 (80%) had radiological worsening after 7.4 (2.2) days, and respiratory symptoms worsened in 34 (45%) after 8.6 (3.0) days. In 34 (45%) patients, improvement of initial pulmonary lesions was associated with appearance of new radiological lesions at other sites. Nine (12%) patients developed spontaneous pneumomediastinum and 15 (20%) developed acute respiratory distress syndrome (ARDS) in week 3. Quantitative reverse-transcriptase (RT) PCR of nasopharyngeal aspirates in 14 patients (four with ARDS) showed peak viral load at day 10, and at day 15 a load lower than at admission. Age and chronic hepatitis B virus infection treated with lamivudine were independent significant risk factors for progression to ARDS (p=0.001). SARS-associated coronavirus in faeces was seen on RT-PCR in 65 (97%) of 67 patients at day 14. The mean time to seroconversion was 20 days. INTERPRETATION: The consistent clinical progression, shifting radiological infiltrates, and an inverted V viral-load profile suggest that worsening in week 2 is unrelated to uncontrolled viral replication but may be related to immunopathological damage.
Administration, Oral↗
Effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (SARS).
We did a case-control study in five Hong Kong hospitals, with 241 non-infected and 13 infected staff with documented exposures to 11 index patients with severe acute respiratory syndrome (SARS) during patient care. All participants were surveyed about use of mask, gloves, gowns, and hand-washing, as recommended under droplets and contact precautions when caring for index patients with SARS. 69 staff who reported use of all four measures were not infected, whereas all infected staff had omitted at least one measure (p=0.0224). Fewer staff who wore masks (p=0.0001), gowns (p=0.006), and washed their hands (p=0.047) became infected compared with those who didn't, but stepwise logistic regression was significant only for masks (p=0.011). Practice of droplets precaution and contact precaution is adequate in significantly reducing the risk of infection after exposures to patients with SARS. The protective role of the mask suggests that in hospitals, infection is transmitted by droplets.
Case-Control Studies↗
Coronavirus as a possible cause of severe acute respiratory syndrome.
BACKGROUND: An outbreak of severe acute respiratory syndrome (SARS) has been reported in Hong Kong. We investigated the viral cause and clinical presentation among 50 patients. METHODS: We analysed case notes and microbiological findings for 50 patients with SARS, representing more than five separate epidemiologically linked transmission clusters. We defined the clinical presentation and risk factors associated with severe disease and investigated the causal agents by chest radiography and laboratory testing of nasopharyngeal aspirates and sera samples. We compared the laboratory findings with those submitted for microbiological investigation of other diseases from patients whose identity was masked. FINDINGS: Patients' age ranged from 23 to 74 years. Fever, chills, myalgia, and cough were the most frequent complaints. When compared with chest radiographic changes, respiratory symptoms and auscultatory findings were disproportionally mild. Patients who were household contacts of other infected people and had older age, lymphopenia, and liver dysfunction were associated with severe disease. A virus belonging to the family Coronaviridae was isolated from two patients. By use of serological and reverse-transcriptase PCR specific for this virus, 45 of 50 patients with SARS, but no controls, had evidence of infection with this virus. INTERPRETATION: A coronavirus was isolated from patients with SARS that might be the primary agent associated with this disease. Serological and molecular tests specific for the virus permitted a definitive laboratory diagnosis to be made and allowed further investigation to define whether other cofactors play a part in disease progression.
Adult↗
Severe Acute Respiratory Syndrome (SARS).
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Animals↗
The next influenza pandemic: lessons from Hong Kong.
Pandemic influenza is a zoonosis. Studies on influenza ecology conducted in Hong Kong since the 1970s in which Hong Kong essentially functioned as an influenza sentinel post indicated that it might be possible, for the first time, to have influenza preparedness at the baseline avian level. This appreciation of influenza ecology facilitated recognition of the H5N1 'bird flu' incident in Hong Kong in 1997 in what was considered to be an incipient pandemic situation, the chicken being the source of virus for humans and, if so, was the first instance where a pandemic may have been averted. The 2001 and 2002 H5N1 incidents demonstrated that it was possible to have an even higher order of baseline preparedness with the recognition in chicken of a range of genotypes of H5N1-like viruses before they had the opportunity to infect humans. Investigations of these incidents revealed a complex ecology involving variously precursor avian H5N1 virus in geese and ducks, and H9N2 and H6N1 viruses in quail, the quail possibly functioning as an avian 'mixing vessel' for key genetic reassortment events for onward transmission of H5N1 viruses highly pathogenic for chicken and humans. These findings highlight the importance of systematic virus surveillance of domestic poultry in recognizing changes in virus occurrence, host range and pathogenicity as signals at the avian level that could presage a pandemic. For example, there is now an increasing prevalence of avian influenza viruses in terrestrial (in contrast to aquatic) poultry. Prior to 1997, no particular virus subtype other than H4N6 would have been considered a candidate for pandemicity and this was based, in the absence of any other data, on its high frequency of occurrence in ducks in southern China. Now,with the isolation of H5N1 and H9N2 viruses from humans supported by genetic, molecular and biological studies on these and other avian isolates, there is credible evidence for the candidacy, in order, of H5N1, H9N2 and H6N1 viruses. These viruses have been made available for the production of diagnostic reagents and exploratory vaccines. The 1997 incident upheld the hypothesis that southern China is an epicentre for the emergence of pandemic influenza viruses. However, the intensification of the poultry (chicken) industry worldwide coupled with the spread of viruses such as the Eurasian lineage of H9N2 suggest that the genesis of a pandemic could take place elsewhere in the world. This re-emphasizes the importance of systematic virus surveillance of poultry globally for international public health and for economic and food concerns. Faced with an incipient pandemic in 1997, Hong Kong brought in international experts to join the investigative effort. Good teamwork at all levels is essential in dealing with the many facets. The threat of a pandemic should not be minimized, nor should governments be lulled into a sense of false security. The media is a powerful channel and has the responsibility and the avenues to convey and influence public perception of events. Close liaison between the media and those on the operational side ensures effective, accurate and timely dissemination of information. This will enhance public confidence in the investigative process and in steps taken for its safety and health.
Animals↗
A newly discovered verotoxin variant, VT2g, produced by bovine verocytotoxigenic Escherichia coli.
A new verotoxin (VT) variant, designated vt2g, was identified from a bovine strain of verocytotoxigenic Escherichia coli (VTEC) serotype O2:H25. When vt2g was aligned with published sequences of vt2 and vt variants, it exhibited the highest DNA sequence homology with vt2 and vt2c. However, vt2g was not detected by vt2-specific primers and probes, although it was partially neutralized by an antiserum to the VT2A subunit. VT2g was cytotoxic for Vero and HeLa cells and was not activated by mouse intestinal mucus. The vt2g gene was detected in 3 of 409 (0.7%) bovine VTEC strains, including serotypes O2:H25, O2:H45 and Ont:H-.
Amino Acid Sequence↗
Evaluation of reverse transcription-PCR assays for rapid diagnosis of severe acute respiratory syndrome associated with a novel coronavirus.
The reverse transcription (RT)-PCR protocols of two World Health Organization (WHO) severe acute respiratory syndrome (SARS) network laboratories (WHO SARS network laboratories at The University of Hong Kong [WHO-HKU] and at the Bernhard-Nocht Institute in Hamburg, Germany [WHO-Hamburg]) were evaluated for rapid diagnosis of a novel coronavirus (CoV) associated with SARS in Hong Kong. A total of 303 clinical specimens were collected from 163 patients suspected to have SARS. The end point of both WHO-HKU and WHO-Hamburg RT-PCR assays was determined to be 0.1 50% tissue culture infective dose. Using seroconversion to CoV as the "gold standard" for SARS CoV diagnosis, WHO-HKU and WHO-Hamburg RT-PCR assays exhibited diagnostic sensitivities of 61 and 68% (nasopharyngeal aspirate specimens), 65 and 72% (throat swab specimens), 50 and 54% (urine specimens), and 58 and 63% (stool specimens), respectively, with an overall specificity of 100%. For patients confirmed to have SARS CoV and from whom two or more respiratory specimens were collected, testing the second specimen increased the sensitivity from 64 and 71% to 75 and 79% for the WHO-HKU and WHO-Hamburg RT-PCR assays, respectively. Testing more than one respiratory specimen will maximize the sensitivity of PCR assays for SARS CoV.
Humans↗
Characterization of H9 subtype influenza viruses from the ducks of southern China: a candidate for the next influenza pandemic in humans?
A current view of the emergence of pandemic influenza viruses envisages a gene flow from the aquatic avian reservoir to humans via reassortment in pigs, the hypothetical "mixing vessel." Understanding arising from recent H5N1 influenza outbreaks in Hong Kong since 1997 and the isolation of avian H9N2 virus from humans raises alternative options for the emergence of a new pandemic virus. Here we report that H9N2 influenza viruses established in terrestrial poultry in southern China are transmitted back to domestic ducks, in which the viruses generate multiple reassortants. These novel H9N2 viruses are double or even triple reassortants that have amino acid signatures in their hemagglutinin, indicating their potential to directly infect humans. Some of them contain gene segments that are closely related to those of A/Hong Kong/156/97 (H5N1/97, H5N1) or A/Quail/Hong Kong/G1/97 (G1-like, H9N2). More importantly, some of their internal genes are closely related to those of novel H5N1 viruses isolated during the outbreak in Hong Kong in 2001. This study reveals a two-way transmission of influenza virus between terrestrial and aquatic birds that facilitates the generation of novel reassortant H9N2 influenza viruses. Such reassortants may directly or indirectly play a role in the emergence of the next pandemic virus.
Animals↗
The complete genome sequence of severe acute respiratory syndrome coronavirus strain HKU-39849 (HK-39).
The complete genomic nucleotide sequence (29.7kb) of a Hong Kong severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) strain HK-39 is determined. Phylogenetic analysis of the genomic sequence reveals it to be a distinct member of the Coronaviridae family. 5' RACE assay confirms the presence of at least six subgenomic transcripts all containing the predicted intergenic sequences. Five open reading frames (ORFs), namely ORF1a, 1b, S, M, and N, are found to be homologues to other CoV members, and three more unknown ORFs (X1, X2, and X3) are unparalleled in all other known CoV species. Optimal alignment and computer analysis of the homologous ORFs has predicted the characteristic structural and functional domains on the putative genes. The overall nucleotides conservation of the homologous ORFs is low (<5%) compared with other known CoVs, implying that HK-39 is a newly emergent SARS-CoV phylogenetically distant from other known members. SimPlot analysis supports this finding, and also suggests that this novel virus is not a product of a recent recombinant from any of the known characterized CoVs. Together, these results confirm that HK-39 is a novel and distinct member of the Coronaviridae family, with unknown origin. The completion of the genomic sequence of the virus will assist in tracing its origin.
3' Untranslated Regions↗
The impact of a monthly rest day on avian influenza virus isolation rates in retail live poultry markets in Hong Kong.
Retail live poultry markets (LPMs) may act as a reservoir of avian influenza viruses (AIV). In this study we test the hypothesis that a rest day in the LPMs where the stalls are completely emptied of poultry, cleansed, and restocked will reduce the isolation rates of avian influenza viruses. The isolation rate of H9N2 subtype viruses from chicken was significantly lower after the rest day than prior to it, indicating its impact in reducing transmission. In contrast, Newcastle disease virus (NDV) isolation rates appear unaffected by this intervention, possibly reflecting differences in herd immunity or virus transmission dynamics.
Animals↗
Reassortants of H5N1 influenza viruses recently isolated from aquatic poultry in Hong Kong SAR.
The H5N1 virus (H5N1/97) that caused the bird flu incident in Hong Kong in 1997 has not been isolated since the poultry slaughter in late 1997. But the donor of its H5 hemagglutinin gene, Goose/Guangdong/1/96-like (Gs/Gd/96-like) virus, established a distinct lineage and continued to circulate in geese in the area. In 2000, a virus from the Goose/Guangdong/1/96 lineage was isolated for the first time from domestic ducks. Subsequently, it has undergone reassortment, and these novel reassortants now appear to have replaced Gs/Gd/96-like viruses from its reservoir in geese and from ducks. The internal gene constellation is also different from H5N1/97, but these variants have the potential for further reassortment events that may allow the interspecies transmission of the virus.
Animals↗
Treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicentre retrospective matched cohort study.
OBJECTIVES: To investigate the possible benefits and adverse effects of the addition of lopinavir/ritonavir to a standard treatment protocol for the treatment of severe acute respiratory syndrome. DESIGN: Retrospective matched cohort study. SETTING: Four acute regional hospitals in Hong Kong. PATIENTS AND METHODS: Seventy-five patients with severe acute respiratory syndrome treated with lopinavir/ritonavir in addition to a standard treatment protocol adopted by the Hospital Authority were matched with controls retrieved from the Hospital Authority severe acute respiratory syndrome central database. Matching was done with respect to age, sex, the presence of co-morbidities, lactate dehydrogenase level and the use of pulse steroid therapy. The 75 patients treated with lopinavir/ritonavir were divided into two subgroups for analysis: lopinavir/ritonavir as initial treatment, and lopinavir/ritonavir as rescue therapy. These groups were compared with matched cohorts of 634 and 343 patients, respectively. Outcomes including overall death rate, oxygen desaturation, intubation rate, and use of pulse methylprednisolone were reviewed. RESULTS: The addition of lopinavir/ritonavir as initial treatment was associated with a reduction in the overall death rate (2.3%) and intubation rate (0%), when compared with a matched cohort who received standard treatment (15.6% and 11.0% respectively, P<0.05) and a lower rate of use of methylprednisolone at a lower mean dose. The subgroup who had received lopinavir/ritonavir as rescue therapy, showed no difference in overall death rate and rates of oxygen desaturation and intubation compared with the matched cohort, and received a higher mean dose of methylprednisolone. CONCLUSION: The addition of lopinavir/ritonavir to a standard treatment protocol as an initial treatment for severe acute respiratory syndrome appeared to be associated with improved clinical outcome. A randomised double-blind placebo-controlled trial is recommended during future epidemics to further evaluate this treatment.
Adolescent↗
Induction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human disease?
BACKGROUND: In 1997, the first documented instance of human respiratory disease and death associated with a purely avian H5N1 influenza virus resulted in an overall case-fatality rate of 33%. The biological basis for the severity of human H5N1 disease has remained unclear. We tested the hypothesis that virus-induced cytokine dysregulation has a role. METHODS: We used cDNA arrays and quantitative RT-PCR to compare the profile of cytokine gene expression induced by viruses A/HK/486/97 and A/HK/483/97 (both H5N1/97) with that of human H3N2 and H1N1 viruses in human primary monocyte-derived macrophages in vitro. Secretion of tumour necrosis factor alpha (TNF alpha) from macrophages infected with the viruses was compared by ELISA. By use of naturally occurring viral reassortants and recombinant viruses generated by reverse genetic techniques, we investigated the viral genes associated with the TNF-alpha response. FINDINGS: The H5N1/97 viruses induced much higher gene transcription of proinflammatory cytokines than did H3N2 or H1N1 viruses, particularly TNF alpha and interferon beta. The concentration of TNF-alpha protein in culture supernatants of macrophages infected with these viruses was similar to that induced by stimulation with Escherichia coli lipopolysaccharide. The non-structural (NS) gene-segment of H5N1/97 viruses contributed to the increase in TNF alpha induced by the virus. INTERPRETATION: The H5N1/97 viruses are potent inducers of proinflammatory cytokines in macrophages, the most notable being TNF alpha. This characteristic may contribute to the unusual severity of human H5N1 disease.
Cells, Cultured↗
Emergence of multiple genotypes of H5N1 avian influenza viruses in Hong Kong SAR.
Although A/Hong Kong/156/97 (H5N1/97)-like viruses associated with the "bird flu" incident in Hong Kong SAR have not been detected since the slaughter of poultry in 1997, its putative precursors continue to persist in the region. One of these, Goose/Guangdong/1/96 (H5N1 Gs/Gd)-like viruses, reassorted with other avian viruses to generate multiple genotypes of H5N1 viruses that crossed to chickens and other terrestrial poultry from its reservoir in geese. Whereas none of these recent reassortants had acquired the gene constellation of H5N1/97, these events provide insight into how such a virus may have been generated. The recent H5N1 reassortants readily infect and kill chicken and quail after experimental infection, and some were associated with significant mortality of chickens within the poultry retail markets in Hong Kong. Some genotypes are lethal for mice after intra-nasal inoculation and spread to the brain. On this occasion, the early detection of H5N1 viruses in the retail, live poultry markets led to preemptive intervention before the occurrence of human disease, but these newly emerging, highly pathogenic H5N1 viruses provide cause for pandemic concern.
Animals↗
Methods in virus diagnosis: immunofluorescence revisited.
BACKGROUND: Immunofluorescence (IF) has been used in many laboratories for virus diagnosis but has begun to fall inappropriately out of favour as a diagnostic method as pressure on budgets and for objective quality control increases. OBJECTIVES: To review the status, value and benefits of IF. CONCLUSIONS: IF has, we believe, still a valuable role to play in routine virus diagnosis because it is rapid, accurate (with properly validated reagents), flexible and, by giving feedback on the quality of the specimens collected, promotes dialogue with the customer clinicians to their benefit and to that of the diagnostic laboratory. These benefits are not easily duplicated by other methods or techniques. While such rapid diagnosis primarily benefits the individual patient, providing results within a clinically relevant time has a secondary effect of increasing use of the service. It is our experience that the availability of rapid IF diagnosis (as opposed to culture or serology) for respiratory viral infections leads to a substantial increase in its use, thereby enhancing the amount and breadth of the resultant epidemiological data.
Child↗
Polyclonal antibodies to glutathione S-transferase--verotoxin subunit a fusion proteins neutralize verotoxins.
The A1 subunits of verotoxin-1 (VT1) and VT2 genes were cloned into pGEX-4T-2 for the expression of glutathione S-transferase (GST) fusion proteins. The N-terminal and the transmembrane regions of the A1 subunits were excluded from the constructs in order to increase the product yields. Polyclonal anti-VT1A1 and anti-VT2A1 antibodies were produced by immunizing rabbits with GST-VT1A1 and GST-VT2A1 fusion proteins, respectively. The antibodies were tested for their ability to neutralize active toxins from 45 VT-producing Escherichia coli (VTEC) strains. The antibodies had significantly high neutralizing activities against their homologous toxins. The average percentages of neutralization of VT1 by anti-GST-VT1A1 and anti-GST-VT2A1 were 76.7% +/- 7.9% and 3.6% +/- 2.3%, respectively, and those of VT2 were 1.7% +/- 2.3% and 82.5% +/- 13.9%, respectively. VT2 variant toxin was neutralized by anti-GST-VT2A1, with cross neutralization being a possible consequence of sequence homology between VT2 and a VT2 variant. To our knowledge, this is the first report on the production of polyclonal antibodies from GST-VT fusion proteins. The antibodies were shown to exhibit specific toxin neutralizing activities and may be useful for immunological diagnosis of VTEC infections.
Animals↗