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Stability of SARS Coronavirus in Human Specimens and Environment and Its Sensitivity to Heating and UV Irradiation
Abstract
The causal agent for SARS is considered as a novel coronavirus that has never been described both in human and animals previously. The stability of SARS coronavirus in human specimens and in environments was studied.
Using a SARS coronavirus strain CoV-P9, which was isolated from pharyngeal swab of a probable SARS case in Beijing, its stability in mimic human specimens and in mimic environment including surfaces of commonly used materials or in household conditions, as well as its resistance to temperature and UV irradiation were analyzed. A total of 10(6) TCID50 viruses were placed in each tested condition, and changes of the viral infectivity in samples after treatments were measured by evaluating cytopathic effect (CPE) in cell line Vero-E6 at 48 h after infection.
The results showed that SARS coronavirus in the testing condition could survive in serum, 1:20 diluted sputum and feces for at least 96 h, whereas it could remain alive in urine for at least 72 h with a low level of infectivity. The survival abilities on the surfaces of eight different materials and in water were quite comparable, revealing reduction of infectivity after 72 to 96 h exposure. Viruses stayed stable at 4 degrees C, at room temperature (20 degrees C) and at 37 degrees C for at least 2 h without remarkable change in the infectious ability in cells, but were converted to be non-infectious after 90-, 60- and 30-min exposure at 56 degrees C, at 67 degrees C and at 75 degrees C, respectively. Irradiation of UV for 60 min on the virus in culture medium resulted in the destruction of viral infectivity at an undetectable level.
The survival ability of SARS coronavirus in human specimens and in environments seems to be relatively strong. Heating and UV irradiation can efficiently eliminate the viral infectivity.
... However, Chin et al. (30) confirm the destruction of SARS-Cov-2 at 56 • C for 30 min. Boiling water might still be an efficient method of decontamination as already shown for SARS (31). ...
... It is also effective on rotavirus, influenza A virus subtype H1N1, and coronavirus. CS at pH 7.2 has an effect on vegetative forms and spores of Bacillus cereus (30)(31)(32). Two cases of raw milk contamination were published while the breast shields were decontaminated with CS (33,34). ...
... To get rid of any contamination from mother to child, a rigorous hand hygiene with liquid soap is required before and after the care of her baby or expressing milk (26). The usual recommendations are a wash of at least 30 s and drying with a disposable wipe, but the most recent data shows that the virus becomes undetectable after 15 min; hydroalcoholic solution appears to be a more efficient and rapid solution but again contact viral load modifies the data (31). If breastfeeding a newborn during the COVID-19 pandemic is not questionable, mothers must follow specific measures and barriers for herself and her environment when she breast pumps milk. ...
To promote breast feeding and breast pumping is essential for the most vulnerable infants even if the current coronavirus disease 2019 (COVID-19) pandemic sanitary crisis imposes more stringent hygienic measures. As recommended by the Centers for Disease Control and Prevention, World Health Organization, and Milk Bank Association, "after each pumping session, all pump part that come into contact with breast milk should be appropriately disinfected." The present study proposed different methods than can be used and focus on the safety analysis of chlorine solution (CS) in terms of residual hypochlorous acid (HCA) and total trihalomethanes (THM). We also performed an efficacy testing of the CS approach to decontaminate the devices used to collect the milk (breast pumps and bottles). The bacteriologic results of 1,982 breast pump milk samples collected in three different settings showed a major decrease of the microbial contamination using either sterile device or decontamination with CS compared to a simple soap washing. The main messages from our study are to propose a guideline for the safe use of CS and to define situations when breast pump decontamination might be necessary: vulnerable babies for which sterile device is recommended; special circumstances, for example the current COVID-19 pandemic; special situations, for example women living in precarious conditions; or women pumping their milk at work but that would have low or no access to boiled water. Overall, cold decontamination reduced losses of milk for bacteriological reasons in human milk banks and may also be interesting to prevent horizontal contamination by virus like COVID-19.
... But, other biocidal agents such as benzalkonium chloride and chlorhexidine are less effective. [13] A study by Duan et al. [17] revealed that irradiation with ultraviolet light for 60 min on several coronaviruses in a culture medium resulted in undetectable levels of viral infectivity. Household bleach, ethanol (70%), povidone-iodine (7.5%), chloroxylenol (0.05%), chlorhexidine (0.05%), and benzalkonium chloride (0.1%) are also efficient in obtaining not detectable viruses on surfaces. ...
... Once received in the laboratory, these forms can be kept under an ultraviolet chamber, if available, to deactivate coronavirus if any. [17] The intranet server system should be preferred to promote digital workflow. Through this, all requisition forms should be accessible to staff and clinicians online. ...
... In a study, several coronalviruses were inactivated after subjecting to the following time and temperatures: 90 min at 56°C, 60 min at 67°C, and 30 min at 75°C. [17] Paraffin infiltration during the day-to-day histotechnology process uses 60-65°C for 2 h or more. Hence, formalin-fixed paraffin-embedded tissue blocks are of less pathogenic risk. ...
Aim: Histopathology laboratories are assigned laboratories where contagious biopsy samples are regularly received for diagnosis and prognosis. SARS-CoV-2 is a new public health crisis that has created a global alarming. As there is a paradigm shift in the work-flow and specimen management during this COVID-19 pandemic, this review article discusses and summarizes proper sterilization protocols that need to be implemented in histopathological laboratories. This write-up highlights the biosafety level requirement along with step-by-step safety protocol from receiving a specimen till reporting of slides by pathologists. Materials and Methods: A literature search was made to review the published recommendation that is applicable for histopathology laboratories in the light of current knowledge and understanding of COVID-19. A humble effort was also made to review the interim guidelines that are updated by the World Health Organization and Center of Disease Control on day-today basis and are highlighted in this article. Results: Based on keywords used, 29 relevant articles were found useful and were selected for the review. Conclusion: Of all the precautions, proper hand hygiene practice, use of disinfectants, and personal protective equipment are of utmost importance. Also, several studies have shown that coronavirus was inactivated during routine formalin fixation and tissue processing processes. This article focusses on highlighting the guidelines that could help in anti-spread strategies.
... Previous study in Bangladesh have reported significant correlation between average temperature and mutation frequency at ORF1ab and at S-D614G (22). Several research works on influenza virus have reported impact of weather and UV radiation on evolution of substitution mutations (41,42). In similarity with previous studies, we also detected notable correlation between weather and mutation frequency of Japanese isolates, and between hosts factors and mutations (21)(22)(23)(24)(25)(26)(27)(28)(36)(37)(38)(39)(40). ...
... High temperature and UV can reduce the survival period and viability of SARS-CoV-2(21)(22)(23)(24)(25)(26)(36)(37)(38)(39). Temperature above 40 °C can reduce the viability of SARS-CoV, MERS-CoV and SARS-CoV-2 significantly in both laboratory and environmental conditions(40)(41)(42). In this study, the correlation and impact of various regulatory factors on the spread and outcome of COVID-19 in Japan were determined. ...
Though vaccines have become available, emergence and rapid transmission of new variants have added new paradigm in the Coronavirus disease-2019 (COVID-19) pandemic. Weather, population and host immunity have been detected as the regulatory elements of COVID-19. This study aims to investigate the effects of weather, population and host factors on the outcome of COVID-19 and mutation frequency in Japan. Data were collected during January 2020 to February 2021. About 92% isolates were form GR clades. Variants 501Y.V1 (53%) and 452R.V1 (24%) were most prevalent in Japan. The strongest correlation was detected between fatalities and population density (r s = 0.81) followed by total population (r s = 0.72). Relative humidity had the highest correlation (r s =-0.71) with the case fatality rate. Cluster mutations namely N501Y (45%), E484K (30%), N439K (16%), K417N (6%) and T478I (3%) at spike protein have increased during January to February, 2021. Above 90% fatality was detected in patients aged >60 years. The ratio of male to female patients of COVID-19 was 1.35:1. This study will help to understand the seasonality of COVID-19 and impact of weather on the outcome which will add knowledge to reduce the health burden of COVID-19 by the international organizations and policy makers.
... UV index is another major environmental factor that has antiviral effect. In several previous studies UV was found to be effective against SARS-CoV and MERS-CoV in laboratory set up [36,37]. In the environment, the intensity and exposure time of UV index vary greatly that might be regulatory for the viability and transmission of SARS-CoV-2. ...
Background
Coronavirus disease-2019 (COVID-19) has caused worldwide health emergencies during the last 6 months of 2020. Within very short time, severe acute respiratory coronavirus-2 (SARS-CoV-2) has infected over 64,516,333 people with 1,493,264 fatalities in 210 countries and regions. Previous studies have reported that environmental factors can affect the viability and transmission of SARS-CoV-2. This study aimed to determine the correlation of environmental factors with COVID-19 pandemic and epidemiology of COVID-19 across nine countries in five continents.
Methods
Both environmental and health data were retrieved from various databases during January 1, 2020 to June 30, 2020. Mean value of environmental factors were calculated for weekly and daily cases and fatalities. Spearman correlation test was conducted.
Results
In this study, most of the COVID-19 cases and fatalities were detected from regions (New York, Madrid, Lombardy, London and Sau Paulo) with 7 °C–25 °C mean temperature per day, 3 to 6 mean UV index per day and 14 km/h to 22 km/h mean wind velocity per day. Both cases and fatalities increased significantly after removing lockdown in Bangladesh, India, Brazil and South Africa. Over 50% COVID-19 patients were asymptomatic in every country except Brazil and Australia. Fever (>50%) was the most common symptom followed by cough (45%), tiredness (38%) and sore throat (30%), respectively. In India and Bangladesh over 70% of cases were reported in male. Significant correlation of COVID-19 cases with temperature and UV were detected in London, Lombardy, Madrid, New York and Dhaka.
Conclusion
This is one of the first cross-country epidemiologic and correlation studies between environmental factors and COVID-19 pandemics. This study will help both local and international health organizations and policy makers to face the COVID-19 challenge.
... Arbaeen walk participants can reach into big buckets of iced water to take sealed packages or bottles of water as they pass by. As viruses (including coronaviruses) can survive well at low temperatures [42,43], there have been concerns that this practice may cause disease transmission. However, the sealed water containers may convey less risk than available alternatives, such as water from a reuseable cup. ...
COVID-19 poses grave challenges for mass gatherings. One of the world’s largest annual gatherings, Arbaeen, occurs in Iraq. We studied respiratory symptoms and risk and protective factors using representative sampling of Arbaeen pilgrims in 2019 to inform prevention of COVID-19 transmission. Structured sampling was used to recruit walking pilgrims. A questionnaire asked about respiratory symptoms, risk, and preventive factors, including hygiene-related resources of toilet facilities. The commonest symptom reported by the 1842 participants (63.3% male, 36.7% female) was cough (25.6%). Eating in mawkibs (rest areas) with indoor kitchens and drinking only packaged water were associated with lower risk of cough (AOR = 0.72, CI = 0.56–0.94; AOR = 0.60; CI = 0.45–0.78, p < 0.05). Facemask use was associated with increased risk of cough (AOR = 2.71, CI = 2.08–3.53, p < 0.05). Handwashing was not protective against cough, or against (one or more of) cough, fever, or breathlessness in multivariate analysis. Toilet facilities often lacked running water (32.1%) and soap (26.1%), and had shared hand towels (17%). To reduce risk of respiratory infections including COVID-19 during Arbaeen or other mass gatherings, needs include running water, soap, and hygienic hand drying options or hand sanitiser. Education on proper handwashing and facemask approaches and monitoring around food preparation and eating spaces are needed.
... Some authors evaluated the stability of SARS-CoV-2 at different temperatures, showing that the virus is highly stable at +4 • C but that it is sensitive to heat. Further studies confirmed that SARS-CoV-2 lost its infectivity after 90-, 60-, and 30-min exposures to temperatures of +56 • C, +67 • C, and +75 • C, respectively [21]. ...
A Coronavirus disease (COVID-19), caused by a new virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spreads via direct contact through droplets produced by infected individuals. The transmission of this virus can also occur via indirect contact if objects and surfaces are contaminated by secretions from individuals with COVID-19 or asymptomatic carriers. Environmental contamination with SARS-CoV-2 is high in hospital settings; on the contrary, surface contamination in non-healthcare settings is still poorly studied. In this study, the presence of SARS-CoV-2 on the surfaces of 20 tourist-recreational facilities was investigated by performing a total of 100 swabs on surfaces, including refrigerator handles, handrails, counters, tables, and bathroom access doors. Six (6%) swabs from four (20%) tourist-recreational facilities tested positive for SARS-CoV-2; the surfaces that were involved were toilet door handles, refrigerator handles, handrails, and bar counters. This study highlights that SARS-CoV-2 is also present in non-healthcare environments; therefore, in order to limit this worrying pandemic, compliance with behavioral rules and the adoption of preventive and protective measures are of fundamental importance not only in healthcare or work environments but also in life environments.
... In vitro exposure in cell cultures of the virus for 15 min reduced severely the infectivity potential. After 30 min exposure no infectivity could be observed (Duan et al., 2003). The geographic location and the elevation influence are important factors, when it comes to solar light inactivation of viruses. ...
The disinfecting properties of sun (heat and UV radiation) are adequate in warm sunny regions to rid beach sand of coronavirus particles, if present. Here we detail the mechanism of natural disinfection offered by the sun on coronaviral particles that may find their way onto beach sand. We conclude that heat and UV radiation generated by the sun destroy the virus infection ability.
The rapid increase in global cases of COVID-19 illness and death requires the implementation of appropriate and efficient engineering controls to improve indoor air quality. This manuscript focuses on the use of the ultraviolet germicidal irradiation (UVGI) air purification technology in HVAC ducts, which is particularly applicable to buildings where fully shutting down air recirculation is not feasible. Given the poor understanding of the in-duct UVGI system regarding its working mechanisms, designs, and applications, this review has the following key research objectives:
Cisplatin is an efcient anticancer drug against various types of cancers however, its usage involves
side efects. We investigated the mechanisms of action of indole derivative, 2-(5-methoxy-2-methyl1H-indol-3-yl)-N’-[(E)-(3-nitrophenyl) methylidene] acetohydrazide (MMINA) against anticancer drug
(cisplatin) induced organ damage using a rodent model. MMINA treatment reversed Cisplatin-induced
NO and malondialdehyde (MDA) augmentation while boosted the activity of glutathione peroxidase
(GPx), and superoxide dismutase (SOD). The animals were divided into fve groups (n= 7). Group1:
Control (Normal) group, Group 2: DMSO group, Group 3: cisplatin group, Group 4: cisplatin+MMINA
group, Group 5: MMINA group. MMINA treatment normalized plasma levels of biochemical enzymes.
We observed a signifcant decrease in CD4+
COX-2, STAT3, and TNF-α cell population in whole blood
after MMINA dosage. MMINA downregulated the expression of various signal transduction pathways
regulating the genes involved in infammation i.e. NF-κB, STAT-3, IL-1, COX-2, iNOS, and TNF-α. The
protein expression of these regulatory factors was also downregulated in the liver, kidney, heart,
and brain. In silico docking and dynamic simulation data were in agreement with the experimental findings. The physicochemical properties of MMINA predicted it as a good drug-like molecule and its
mechanism of action is predictably through inhibition of ROS and inflammation.
RESUMO O presente trabalho apresenta o projeto de um dispositivo e os testes realizados em um sistema de auxílio na prevenção de contaminações pelo Sars-Cov2 com a desinfecção do ar pela utilização de UVC como ação germicida. O sistema foi testado com E.colis e proporcionou no ar tratado uma visível taxa de inativação das colônias de bactérias aspergidas em sua entrada.
In March 2003, a novel coronavirus (SARS-CoV) was discovered in association with cases of severe acute respiratory syndrome (SARS). The sequence of the complete genome of SARS-CoV was determined, and the initial characterization of the viral genome is presented in this report. The genome of SARS-CoV is 29,727 nucleotides in length and has 11 open reading frames, and its genome organization is similar to that of other coronaviruses. Phylogenetic analyses and sequence comparisons showed that SARS-CoV is not closely related to any of the previously characterized coronaviruses.
Information on the clinical features of the severe acute respiratory syndrome (SARS) will be of value to physicians caring for patients suspected of having this disorder.
We abstracted data on the clinical presentation and course of disease in 10 epidemiologically linked Chinese patients (5 men and 5 women 38 to 72 years old) in whom SARS was diagnosed between February 22, 2003, and March 22, 2003, at our hospitals in Hong Kong, China.
Exposure between the source patient and subsequent patients ranged from minimal to that between patient and health care provider. The incubation period ranged from 2 to 11 days. All patients presented with fever (temperature, >38 degrees C for over 24 hours), and most presented with rigor, dry cough, dyspnea, malaise, headache, and hypoxemia. Physical examination of the chest revealed crackles and percussion dullness. Lymphopenia was observed in nine patients, and most patients had mildly elevated aminotransferase levels but normal serum creatinine levels. Serial chest radiographs showed progressive air-space disease. Two patients died of progressive respiratory failure; histologic analysis of their lungs showed diffuse alveolar damage. There was no evidence of infection by Mycoplasma pneumoniae, Chlamydia pneumoniae, or Legionella pneumophila. All patients received corticosteroid and ribavirin therapy a mean (+/-SD) of 9.6+/-5.42 days after the onset of symptoms, and eight were treated earlier with a combination of beta-lactams and macrolide for 4+/-1.9 days, with no clinical or radiologic efficacy.
SARS appears to be infectious in origin. Fever followed by rapidly progressive respiratory compromise is the key complex of signs and symptoms from which the syndrome derives its name. The microbiologic origins of SARS remain unclear.
There has been an outbreak of the severe acute respiratory syndrome (SARS) worldwide. We report the clinical, laboratory, and radiologic features of 138 cases of suspected SARS during a hospital outbreak in Hong Kong.
From March 11 to 25, 2003, all patients with suspected SARS after exposure to an index patient or ward were admitted to the isolation wards of the Prince of Wales Hospital. Their demographic, clinical, laboratory, and radiologic characteristics were analyzed. Clinical end points included the need for intensive care and death. Univariate and multivariate analyses were performed.
There were 66 male patients and 72 female patients in this cohort, 69 of whom were health care workers. The most common symptoms included fever (in 100 percent of the patients); chills, rigors, or both (73.2 percent); and myalgia (60.9 percent). Cough and headache were also reported in more than 50 percent of the patients. Other common findings were lymphopenia (in 69.6 percent), thrombocytopenia (44.8 percent), and elevated lactate dehydrogenase and creatine kinase levels (71.0 percent and 32.1 percent, respectively). Peripheral air-space consolidation was commonly observed on thoracic computed tomographic scanning. A total of 32 patients (23.2 percent) were admitted to the intensive care unit; 5 patients died, all of whom had coexisting conditions. In a multivariate analysis, the independent predictors of an adverse outcome were advanced age (odds ratio per decade of life, 1.80; 95 percent confidence interval, 1.16 to 2.81; P=0.009), a high peak lactate dehydrogenase level (odds ratio per 100 U per liter, 2.09; 95 percent confidence interval, 1.28 to 3.42; P=0.003), and an absolute neutrophil count that exceeded the upper limit of the normal range on presentation (odds ratio, 1.60; 95 percent confidence interval, 1.03 to 2.50; P=0.04).
SARS is a serious respiratory illness that led to significant morbidity and mortality in our cohort.
A worldwide outbreak of severe acute respiratory syndrome (SARS) has been associated with exposures originating from a single ill health care worker from Guangdong Province, China. We conducted studies to identify the etiologic agent of this outbreak.
We received clinical specimens from patients in seven countries and tested them, using virus-isolation techniques, electron-microscopical and histologic studies, and molecular and serologic assays, in an attempt to identify a wide range of potential pathogens.
None of the previously described respiratory pathogens were consistently identified. However, a novel coronavirus was isolated from patients who met the case definition of SARS. Cytopathological features were noted in Vero E6 cells inoculated with a throat-swab specimen. Electron-microscopical examination revealed ultrastructural features characteristic of coronaviruses. Immunohistochemical and immunofluorescence staining revealed reactivity with group I coronavirus polyclonal antibodies. Consensus coronavirus primers designed to amplify a fragment of the polymerase gene by reverse transcription-polymerase chain reaction (RT-PCR) were used to obtain a sequence that clearly identified the isolate as a unique coronavirus only distantly related to previously sequenced coronaviruses. With specific diagnostic RT-PCR primers we identified several identical nucleotide sequences in 12 patients from several locations, a finding consistent with a point-source outbreak. Indirect fluorescence antibody tests and enzyme-linked immunosorbent assays made with the new isolate have been used to demonstrate a virus-specific serologic response. This virus may never before have circulated in the U.S. population.
A novel coronavirus is associated with this outbreak, and the evidence indicates that this virus has an etiologic role in SARS. Because of the death of Dr. Carlo Urbani, we propose that our first isolate be named the Urbani strain of SARS-associated coronavirus.
We sequenced the 29,751-base genome of the severe acute respiratory syndrome (SARS)-associated coronavirus known as the Tor2 isolate. The genome sequence reveals that this coronavirus is only moderately related to other known coronaviruses, including two human coronaviruses, HCoV-OC43 and HCoV-229E. Phylogenetic analysis of the predicted viral proteins indicates that the virus does not closely resemble any of the three previously known groups of coronaviruses. The genome sequence will aid in the diagnosis of SARS virus infection in humans and potential animal hosts (using polymerase chain reaction and immunological tests), in the development of antivirals (including neutralizing antibodies), and in the identification of putative epitopes for vaccine development.
The susceptibility of laboratory mice to intranasal and contact infection with mouse hepatitis virus (MHV)-related coronaviruses was tested in infant CD1 mice. One day old mouse pups were inoculated intranasally with respiratory MHV-S, enteric MHV-Y, rat sialodacryoadenitis virus (SDAV), human coronavirus OC43 (HCV-OC43) or bovine coronavirus (BCV). Twenty-four hours later, they were placed in direct contact with age matched sham inoculated pups. Indices of infection in virus inoculated mice included lesions by histopathology and viral antigen by immunoperoxidase histochemistry in brain, lung, liver and intestine at 3 days after inoculation. Indices of infection in contact mice included mortality or seroconversion by 21 days after exposure. Infant mice were susceptible to infection with all five viruses. Transmission by direct contact exposure occurred with MHV and SDAV, but not HCV or BCV. Furthermore, adult mice were not susceptible to infection with HCV. Tissue distribution of lesions and antigen varied markedly among viruses, indicating that they do not induce the same disease as MHV. This study demonstrates that although these coronaviruses are antigenically closely related, they are biologically different viruses and disease patterns in susceptible infant mice can be used to differentiate viruses.
Rhinoviruses are picornaviruses that cause colds. They are naturally temperature sensitive and highly adapted to grow in respiratory epithelium. Coronaviruses are structurally quite different, being enveloped viruses but are also adapted to grow in respiratory epithelium and also cause colds.
The quasispecies nature of three animal pathogenic RNA viruses of field origin was examined by testing variants of classical swine fever virus (CSFV) originating from geographically different areas, feline coronavirus (FCoV) detected from the same animal by successive sampling, and rabbit haemorrhagic disease virus (RHDV) originating from successive outbreaks in the same geographic area. Clinical samples were investigated using reverse transcriptase polymerase chain reaction (RT-PCR) and ensuing single strand conformational polymorphism (SSCP) assay. By the combination of these methods even subtle differences could be detected among the amplified fragments of the same virus species of different origin. FCoV proved to comprise the most and CSFV the less heterogeneous virus quasispecies. The results show that the combination of RT-PCR and SSCP provides novel and highly sensitive means for the characterisation of RNA viruses, with special regard to genome composition, evolution, features of pathogenicity and molecular epizootiology.
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.
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.
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.
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.