Fear of the unknown is selfish, not a response to an epidemic, when an epidemic breaks out locally without obvious reasons [1]. 1977 Edward? Keith used this sentence to describe the public's panic about the new legionnaires' disease at that time. Now we are also facing a new type of atypical pneumonia with unknown pathogen-Severe Acute Respiratory Syndrome (SARS). This sentence seems to be equally applicable to the description of people's psychology at present. Undeniably, some people have a panic about this new epidemic disease, partly because they don't have a comprehensive understanding of this new disease, and they still lack further understanding of its pathogen, transmission mode, pathogenesis, pathogenesis, diagnosis, treatment and prevention. Fortunately, however, human beings have taken positive measures. WHO's international laboratory network jointly carried out research on SARS, and relevant research institutions found the pathogen and deciphered its genome sequence in less than two months, which initially unveiled the mystery of SARS. At present, experts have made some progress and found that SARS is related to a kind of novel coronavirus, but this new virus has not been found in humans or animals before. This discovery has laid a solid foundation for the next work, and experts will continue to uncover more unknowns of this disease. Through global collaboration, the Internet and various research methods, researchers identified the pathogen in just two months. More than 20 years ago, researchers spent more than two years searching for HIV virus, and then it took several years to obtain its nucleotide sequence. In contrast, the rapid response of SARS pathogens was impressive. If we generally review the process of pathogen discovery, we may gain new experience, provide basis for further research and provide experience for another epidemic that may break out in the future.
research progress
Confirm 1. 1 SARS pathogen
On March 6, 2003, WHO organized an international research network, and laboratories from 1 1 countries successively participated in the network.
Joined in early April. The progress of the research network is as follows.
Paramyxovirus was observed from throat swab samples in Germany on March 18, and the Chinese University of Hong Kong obtained the gene amplification product of paramyxovirus, and published the electron microscope picture and gene sequence on the Internet.
On March 9, 2009, paramyxovirus particles were found in respiratory specimens of patients in Singapore, and the amplified products of attenuated virus genes were obtained (according to primers provided by the Chinese University of Hong Kong). The laboratory in Rotterdam, the Netherlands, which was the first to discover paramyxovirus in the world, obtained samples of related patients.
On March 20th, four laboratories around the world began to detect paramyxovirus. The laboratory in Rotterdam, the Netherlands, detected paramyxovirus, but the gene amplification of paramyxovirus was negative. The laboratory sent the test reagents to Singapore and Hong Kong, and the laboratory in Canada sent two serum samples to Rotterdam.
On March 2 1 day, scientists from the Chinese University of Hong Kong obtained the virus isolate through monkey kidney cell culture, and then developed the corresponding serological diagnostic reagent. The research results of many laboratories show that this pathogen has nothing to do with the following pathogens: influenza A and B virus, respiratory syncytial virus, parainfluenza virus 1, 2 and 3, adenovirus, rhinovirus, enterovirus, human interstitial pneumonia virus, mycoplasma pneumoniae and chlamydia pneumoniae; At the same time, the virus particles were observed to be 50-60nm by electron microscope. Serum experiments show that adding patient serum can inhibit the cytopathic effect, but the control serum is ineffective.
On March 2 1, the laboratory in Rotterdam discovered the HMPV virus PCR of respiratory tract samples from three Singaporean patients in Germany.
The test was negative. In addition, Vero cells and monkey kidney cells were inoculated into respiratory specimens of 2 suspicious cases, and lesions were found. Also on this day, British scientists detected the H3N2 influenza virus in the specimens of these two patients. Hong Tao, a virus expert in China, claimed that chlamydia was the main pathogen leading to SARS, but it did not rule out that chlamydia and coronavirus acted at the same time.
On March 2 1, researchers Zhu Qingyu and Qin from the Institute of Microbiology Epidemiology of the Academy of Military Medical Sciences successfully isolated coronavirus-like virus from autopsy samples of SARS patients, inferring that it may be the pathogen of SARS, and reported this important result in writing to the Ministry of Health and the Ministry of Health of the General Logistics Department of the People's Liberation Army.
On March 22nd, a coronavirus-like particle (70nm) was found in Hong Kong Virus Laboratory, and a coronavirus-like particle (70- 100nm) was found in cytopathic products obtained from patients in Thailand in the United States, but the same sample obtained a positive result of HMPV virus PCR. At the same time, Canada, France, Singapore and other places began to use PCR and electron microscopy to detect paramyxovirus and HMPV virus. Canada discovered 20nm virus particles and released the genetic phylogenetic tree of HMPV virus.
On March 23rd, Hong Kong Virus Laboratory found two coronavirus RNA positive samples among 8 samples. An American report.
Coronavirus found in Hong Kong samples. At the same time, an immunofluorescence method was established to detect the serum of patients, and the gene amplification primers of coronavirus were published on the Internet. Primate experiments started in Singapore and Hong Kong, and infected bronchi through nasopharyngeal swab samples. Coronavirus was detected by electron microscope in Canada and PCR in France. Viruses were isolated from Vero cells in Germany, Japan and Singapore. Chicken pneumonia virus sequences were detected in respiratory tract and urine samples of suspected cases in British laboratories.
From March 24 to 26, coronavirus was detected by electron microscope and PCR in laboratories in Germany and Hong Kong respectively.
The virus sequence was obtained in the laboratory, and it was confirmed that the amino acid sequence of the new virus was consistent with that of the polymerase of the known coronavirus. More gene amplification sequences were obtained in laboratories in Germany, the Netherlands and Hong Kong, and the phylogenetic tree of the new virus was published on the Internet.
March 27 -3 1, continue to do animal experiments with monkeys. Laboratories in Hong Kong and the United States were confirmed to be positive respectively.
Normal serum is negative for the newly isolated virus. At the same time, more laboratories detected coronavirus: Japan obtained positive results from Singapore specimens; In Hong Kong, 50 patients' serum samples were detected, of which 27 samples showed elevated antibodies to coronavirus. At the same time, 5 samples were found to be virus positive in 10 stool samples, and the virus gene could be detected in the stool of patients 6- 16 days after onset. Human paramyxovirus was found in Canadian patient specimens.
On April 1-8, monkeys co-infected with paramyxovirus and interstitial pneumonia virus showed clinical symptoms in animal experiments.
American researchers use ELISA to detect antibodies in the blood of patients who have been sick for 20 days. Hong Kong researchers detected IgM antibodies from the sera of patients with 10 days of onset by immunofluorescence. The mouse experiment began. More laboratories isolated coronavirus from Vero cultured cells, and some serological experiments confirmed that SARS was a compound infection of paramyxovirus and coronavirus. New sequences of coronavirus were found in Holland, Germany, Hong Kong and the United States, and German scientists found chlamydia in specimens.
On April 9th, China Center for Disease Control and Prevention and Institute of Microbiology and Epidemiology of Academy of Military Medical Sciences were in the Department of Military Medicine.
The college held a symposium on SARS pathogen research, and experts from both sides introduced the research progress of chlamydia and coronavirus respectively.
On the afternoon of April 10, Hong Tao, a famous virologist and academician of China Academy of Engineering, announced to dozens of Chinese and foreign media at the "Introduction Meeting of SARS Prevention and Control Work" that "the research on the pathogen of SARS has achieved initial results, and two main pathogens-chlamydia and coronavirus-like virus have been found".
On April 10, according to Xinhua News Agency, scientific and technical personnel such as Professor Li Dexin, Professor Bi Shengli, Director Duan Shumin and Professor Xu Wenbo from the Institute of Viral Disease Prevention and Control of China Center for Disease Control and Prevention made a major breakthrough in the research of the etiology of atypical pneumonia, successfully isolated a variety of coronaviruses and cloned some genes of the isolated coronaviruses. They detected coronavirus in lung samples of three patients who died of atypical pneumonia and spleen samples of one patient, and purified and amplified coronavirus genes from these samples. The amplified gene was confirmed to be the RNA polymerase gene of coronavirus by nucleotide sequence determination, which confirmed the existence of coronavirus in patients' organs for the first time in the world by molecular biology means. They compared the gene sequence of the new virus with that of other coronaviruses, and found that the coronavirus existing in patients with atypical pneumonia is a variant coronavirus. Novel coronavirus was successfully isolated by using these specimens in various cell cultures. The virus has been subcultured in cultured cells for many times, and all cells have pathological changes, and the genetic detection of the virus continues to be positive. Three strains of coronavirus were isolated from throat swabs of patients with atypical pneumonia in China, and their nucleotide sequences were the same as those of coronavirus isolated from patients' organs. At present, the research results show that coronavirus may be the pathogen of atypical pneumonia to a great extent.
On April 1 1, Xinhua News Agency announced the research results of two researchers, Zhu Qingyu and Qin Aide, from the Institute of Microbiology and Epidemiology of the Academy of Military Medical Sciences. At the end of February, the institute isolated and identified coronavirus from an autopsy specimen of a SARS patient. By March 2 1 day, further proof of coronavirus was obtained through serology, immunology and molecular biology research. On April 9th, the sequences of four coronaviruses were determined. These results were published on April 65438, 2006.
On April 12, Dr. Holt of the British Columbia Cancer Institute in Vancouver, Canada and his research team announced SARS.
Genome sequence of suspected pathogen.
On April 14, the research team of Dr. Anderson from CDC in Atlanta, USA also completed the genome sequencing and posted it online.
Published, the sequencing results of the two research groups are basically the same.
April 16, David, Executive Director of Infectious Diseases of WHO? Hyman announced that through the joint efforts of researchers around the world, it was officially confirmed that a mutated coronavirus caused SARS.
On April 17, WHO announced at a press conference in Geneva that the last step of pathogen identification had been completed.
Which is the fourth step of Koch's presumption. The research team led by Dr. Albert Osterhaus of Erasmus University in the Netherlands successfully infected experimental monkeys with novel coronavirus, and then the research team isolated the virus from the infected monkeys and cultured it in the laboratory. It shows that after a month of joint efforts, scientists around the world have initially identified the pathogen of SARS [2-9].
On April 22nd, China released the coronavirus map of the culprit of SARS [3][7][ 10].
Research progress on genome decoding of 1.2 SARS virus
1.2. 1 Canadian Smith Gene Science Center
On April 12, Canadian scientists cracked the gene of suspected SARS pathogen, and developed the diagnosis method and research of SARS.
Take the first step in distributing SARS vaccines and drugs.
The Smith Gene Science Center in Vancouver has joined the global fight against SARS. Mara, director of the center, said that genes
Coding is the basic data for scientists to develop diagnostic and testing methods. Smith Genetic Science Center cracked this genetic code and immediately published it on the Internet (http://www.bcgsc.bc.ca) for other scientists around the world to use.
1.2.2 University of Hong Kong
Yuan, director of the Department of Microbiology at the University of Hong Kong, said that the Medical College of the University of Hong Kong had completed the sequencing of the coronavirus that caused SARS, and it was proved to be a brand-new virus, which was suspected to be transmitted from animals to people. As for what animals, it remains to be studied. Yuan believes that this discovery will help to improve the current rapid detection methods. Previously, researchers from the Chinese University of Hong Kong handed over part of their decoded novel coronavirus gene sequence to the SARS working group of the World Health Organization on the evening of 13.
1.2.3 CDC, USA
14 On April 4th, the US Centers for Disease Control and Prevention announced that it had completed the research on the SARS epidemic.
Genome sequencing of novel coronavirus. The results of gene sequencing are basically consistent with those of a laboratory in Canada. Comparing the sequencing results of the two institutions, it is found that the difference is that their sequencing results are 15 nucleotides, which will be a major start to continue sequencing work. The sequencing results were obtained after 10 scientists and many technicians 12 days. The researchers cultured the throat secretions of 1 SARS patients in the kidney cells of African green monkeys for passage, purified the nucleic acid sequence of coronavirus causing the disease, and then amplified and sequenced it. The new gene sequence * * * has 29727 nucleosides. Within the ribonucleic acid boundary of a typical coronavirus family, members of the coronavirus family generally have 29,000 to 365,438+0,000 nucleosides. Dr Julie Gerbertin, director of the US Centers for Disease Control and Prevention, said that it is very important to determine the gene sequence of a new virus for the treatment and prevention of diseases. Using the information about the gene sequence, we can start the laboratory research of antiviral drugs, which can be used as the basis for developing vaccines and developing diagnostic tests for early detection of cases. It is very important that the research results in the United States and Canada are almost the same, indicating that the virus may have the same source, because these samples are collected from different individuals infected in different countries. However, officials of the US Centers for Disease Control and Prevention stressed that the analysis of the virus is far from complete, and coronavirus can
Rapid mutation, researchers need to compare the virus isolated from cell culture with the virus obtained from the diseased tissues of SARS patients, and gene sequencing will speed up the comparison.
1.2.4 Beijing Institute of Genomics, Chinese Academy of Sciences and Institute of Microbial Epidemiology, Academy of Military Medical Sciences
On April 6th, 2003, Beijing Genome Institute of Chinese Academy of Sciences cooperated with Institute of Microbiology and Epidemiology of Academy of Military Medical Sciences to decipher the genomes of four coronaviruses isolated from different SARS cases. The results showed that the virus was about 30,000 base pairs in length, which was basically consistent with the sequences reported in Canada and the United States, and belonged to a novel coronavirus. This achievement is only two days later than the time when Canadian scientists announced the decoding of coronavirus genes. The successful determination of the whole genome sequence of coronavirus has laid a solid foundation for tracing the origin of coronavirus and developing diagnostic preparations, vaccines and therapeutic drugs for atypical pneumonia, which has made an important step forward in the prevention and treatment of atypical pneumonia in China [2][3][4][5][8][ 10].
The researchers isolated and identified pathogens.
In this global joint search for pathogens by related laboratories, Hong Kong researchers took the lead in making a breakthrough. They used traditional virus culture, serological detection technology and modern molecular genetics methods to identify the pathogen-a kind of novel coronavirus-in 50 SARS patients. In addition, the analysis of the control group samples further supports their argument about pathogens: none of the 40 respiratory samples from other respiratory diseases patients contained RNA novel coronavirus; None of the 200 serum samples from blood donors contained antibodies to this virus.
These findings also support the views put forward by two other research institutions. Novel coronavirus was also isolated from SARS patients by the Centers for Disease Control and Prevention (CDC) and related research institutions in Toronto, Canada, and it was believed that the virus was related to the outbreak of SARS. German researchers found novel coronavirus in the first three patients, and further collected samples of patients from Hanoi for inspection and analysis. The results also support this conclusion.
2. 1 international laboratory network research results
2. 1. 1 hongkong, China
The research team led by Peiris, a professor of microbiology at the University of Hong Kong, studied patients in Hong Kong to find pathogens.
Body.
The team selected 50 SARS patients who met the revised definition of WHO from three emergency hospitals in Hong Kong and collected their data.
Swallow swab samples and serum samples were collected from all patients, and serum and excreta were collected from some patients in severe stage and rehabilitation stage respectively.
Material samples. In addition, they obtained a lung tissue sample from a patient, and carried out virus culture and isolation, reverse transcription PCR(RT-PCR).
Routine tissue autoradiography and electron microscope observation. Microorganisms in throat swabs, excreta and serum samples of other patients.
The test results are used as a control.
The researchers initially carried out routine blood tests, biochemical tests and microbial tests, as well as blood samples and throat swabs.
Bacterial culture and serological detection were carried out respectively, and rapid fluorescent antigen detection was carried out on throat swab samples to determine the pathogen.
Whether it is a common respiratory infection virus, and isolate the pathogen with various cell cultures; Direct use of clinical samples
RT-PCR was used to detect whether it was influenza A virus and human paramyxovirus infection. In addition, ELISA is used to culture cells.
Methods for detecting the presence of chlamydia.
The breakthrough of this study was the observation of coronavirus-like particles in the samples of two patients. One of the samples comes from a
A 53-year-old male patient detected coronavirus RNA in his throat swab and lung biopsy.
The antibody titer of coronavirus increased significantly (1/200 ~11600). Another sample was taken from a 42-year-old female patient for PCR detection.
She was positive for coronavirus, and her autoantibodies changed (1150 ~11600).
The researchers inoculated the cultured cells into two samples respectively, and after 2-4 days, the diseased cells with circular refraction appeared, indicating that
The pathogen is separated, and the separated pathogen does not react with the reagent plate for identifying common viruses. Extracting the cell culture
Forty two.
After high-speed centrifugation, the irregular enveloped virus with a diameter of about 80-90nm was found by negative staining with electron microscope. Table.
Facial features are similar to coronavirus. The ultrathin section of infected cells was observed by electron microscope, and it was found that the cytoplasm and cell membrane surface were covered by
There are similar virus particles, and the virus isolated from two patients is similar in size and morphological characteristics.
In order to obtain the gene sequence information of the newly isolated virus, the researchers conducted random RT-PCR and cloned and sequenced the virus sensation.
The characteristic chromosome bands of stained cells were compared in GenBank and found in 30 clones.
Unknown sequence found. By analyzing this DNA sequence, it is found that it has low homology with coronavirus, but it is inferred from this.
Its amino acid sequence has a high homology of 57% with bovine coronavirus and murine pneumonia virus RNA polymerase.
Phylogenetic study of protein sequence showed that the new virus was highly correlated with Coronavirus Group 2. Serological reaction to virus
Indirect immunofluorescence detection was used, and the results showed that 32 patients had serum in severe stage and rehabilitation stage.
Transformation, there is a phenomenon that the antibody titer of coronavirus increases.
The research team also tested the human paramyxovirus by RT-PCR and serum antibody titer, and the results were negative, no.
Other pathogens were detected. Therefore, the research team believes that the isolated coronavirus is the pathogen of SARS or a necessity.
However, it remains to be seen whether there are other microbial or non-microbial cofactors [1 1][ 12].
2. 1.2 Germany
SARS first broke out in Asia. Due to intercontinental travel, the disease spread from Asia to other continents. In view of SARS disease
It is a new disease, and human beings know nothing about its pathogen. The initial research work focused on the identification of pathogens.
WHO has organized an international laboratory network to pool the research efforts of relevant countries to find out the pathogen of SARS. German research institute
As a part of the network, the institute also carried out pathogen identification research.
The sample originally selected by the research team comes from three people in the same family: a 32-year-old male patient whose
His wife and mother-in-law are a doctor in Singapore. He once treated a SARS patient and was infected.
And he infected his wife and mother-in-law, who came to the United States from Singapore for some reason, and the doctor developed symptoms during his stay in the United States, he told me.
He met his colleagues in Singapore and reported to WHO that WHO was a transit point for the three people to return to Singapore-
Frankfurt isolated them. German researchers obtained their respiratory tract samples and blood samples, and then
The team also obtained 18 samples of other suspected or possible SARS cases and 2 1 samples of people who had been exposed to SARS from Asia.
Samples of patients but uninfected people.
The researchers first tested the samples of the above three patients by PCR to determine whether there was pneumococcus.
Chlamydia pneumoniae, human cytomegalovirus, parainfluenza virus, influenza virus, human paramyxovirus, rhinovirus and human coronavirus.
Known respiratory pathogens, such as OC43 and 229E, perform antigen ELISA on respiratory samples to detect the presence of lung.
Serological detection of inflammatory cocci, influenza virus and respiratory syncytial virus, as well as blood samples; In addition, researchers
Respiratory tract samples and blood samples were observed by negative staining electron microscope, and the samples were inoculated with cultured cells.
The researchers extracted RNA from sputum samples and analyzed some designed PCR by random RT-PCR technology.
Primers contain degenerate sites, and most of the 3' end is T base, so DNA polymerase has no base at the end of primers.
It can only take effect if it is a perfect match. The homology of cloned products was compared by BLAST tool.
The research team conducted several tests on the samples of three patients, aiming at the test results of known respiratory pathogens.
Most of them are negative. Rare paramyxovirus-like particles were found in respiratory specimens under electron microscope, but they were repeatedly targeted at paramyxosis.
PCR detection of virus family showed negative results. Six days after the sputum samples were inoculated with cultured cells, the researchers found that
In the diseased cells, RNA was extracted immediately, and the extracted RNA was amplified and cloned by RT-PCR, and about 20 non-RNA were cloned.
The same DNA fragments were sequenced and searched by BLAST, and three new fragments were found, which were different from the original DNA fragments.
The sequences in the database match, but the amino acid sequence deduced from the new fragment shows homology with the coronavirus family, indicating that
A new coronavirus was isolated. The researchers compared the new fragment with that measured by the Centers for Disease Control and Prevention (CDC).
The nucleic acid sequences of the virus were compared and found to have homology. In the serum samples and infection cultures of these three patients.
Cells were detected by immunofluorescence to determine whether antibodies existed. Elevated IgG antibodies were detected in the sera of two patients.
Indicating the existence of novel coronavirus's serum reaction.
43
In order to test whether this new virus is related to SARS, the researchers further collected information about possible or suspected SARS patients.
And samples of people who had contact with SARS patients but were not infected (further samples were collected from Hanoi, Vietnam), right?
Nested PCR analysis of this batch of samples showed that the proportion of virus found in possible SARS cases was 100%, which was 100%.
23% of suspected cases, but no virus was found in healthy contacts. These data may further prove that novel coronavirus and
The connection of SARS.
The research team also detected paramyxovirus and pneumococcus, but the results of subsequent targeted PCR experiments were negative. exist
Chlamydia infection was found in several patients, but not in other SARS patients. So the researchers didn't
It is clear whether these pathogens are pathogenic factors of SARS or joint pathogenic factors [13].
2. 1.3 USA
The Centers for Disease Control and Prevention is one of the research institutions of the international laboratory network organized by WHO. it
Scientists have also begun to identify the pathogen of SARS. CDC samples come from Vietnam, Singapore, Thailand and Canada.
The United States, China, Hongkong, Taiwan Province Province and the United States have tried to find out the causes of this situation from a series of known pathogens.
The source of the outbreak of SARS.
Because the clinical symptoms of SARS patients are not specific, the initial research focused on the discharge of known respiratory pathogens.
Inspection, comprehensive use of a variety of detection methods. The researchers collected blood, serum, nasopharyngeal swab, mouthwash and organs.
A variety of samples including tissues are cultured in a variety of cells, and suckling mice are inoculated to isolate pathogens;
Observe the cultured cells and suckling mice, and prepare sections of diseased cells or individuals for electron microscope observation; Perform serum samples
Carry out serological experiments to detect antibodies; Common and special bacterial cultures are carried out, and molecular biotechnology such as PCR is also used.
、RT-PCR； A variety of respiratory pathogens were screened, such as Yersinia, mycoplasma, chlamydia, rickettsia and so on.
Legionella, influenza A and B viruses, parainfluenza virus family, etc.
The breakthrough of this study is to observe coronavirus-like particles through electron microscope. Respiratory tract specimens of patients inoculated with cultured bacteria
Cell lesions appeared in cells, and the diseased cells were made into ultrathin sections and observed by electron microscope.
Coronavirus-like particles were found in the membrane: the diameter was about 80- 140nm, and the virus surface had complex protrusions of 20-40nm. profit
Observing the bronchial lavage fluid samples of patients with electron microscope, it was also found that many infected cells had coronavirus.
Researchers performed RNA extraction and RT-PCR on diseased cells to amplify new virus sequences, and primers were based on GenBank.
Based on the sequence information of known coronavirus. The amplified purified products were sequenced and analyzed, and compared with crown published in public.
The sequence of the virus was compared and analyzed by bioinformatics technology, and the evolutionary tree of the virus was obtained. New viruses and others
Compared with the sequence of coronavirus and the amino acid sequence deduced from the sequence, it was found that the new virus was different from the group2 of coronavirus family.
Have high homology. However, the phylogenetic tree shows that this virus is genetically different from other coronaviruses, which indicates that
The isolate is a novel coronavirus. Serological detection of the samples showed that the infected cells were in recovery period.
The sera of suspected SARS patients from Hong Kong, Bangkok and the United States showed specific anti-reaction.
It should be that it shows a transition from negative to positive or an increase in reactivity in indirect fluorescent antibody experiments; For the same batch of serum samples
ELISA antigen detection showed that the reaction of the recovered samples was highly specific, and the antibody titer gradually increased.
The pathogen was amplified by inoculating cultured cells with samples, and it was found by electron microscope that the pathogen was coronavirus.
In order to further determine the essential characteristics of this new virus, serological experiments have determined its relationship with diseases, so researchers believe that separation
Novel coronavirus may be the pathogen of SARS. However, the research team also pointed out that the patient's focus tissue cells
No new coronavirus antigen or viral RNA was directly detected in the pathological process. At the same time, the new virus cannot be confirmed.
It exists in all SARS patients, and some SARS patients have not detected coronavirus, so further related research is needed [14][ 15].
2. 1.4 Canada
Canada's National Microbiology Laboratory and related research institutions organized a SARS research group to conduct research on SARS.
The study of disease.
After follow-up investigation, the researchers confirmed that all the original SARS patients in the country had been to Hong Kong and were infected there.
Forty-four
The patient returned to Canada and spread SARS to that country. The sample obtained by the researchers comes from this patient (already
Death) directly or indirectly infected 9 patients. The researcher made a histopathological examination of 1 autopsy tissue sample,
All the specimens of 9 patients were detected by microbiology. Immunohistochemical examination of autopsy tissue samples was carried out to
Detect whether there are influenza A and B viruses, respiratory syncytial virus, adenovirus, hantavirus, measles virus, enterovirus,
Flavivirus and other known common viruses, as well as Rickettsia, Mycoplasma pneumoniae, Chlamydia pneumoniae, Yersinia, etc. , have been tested.
The test results are all negative. Blood samples, respiratory tract samples and urine samples of 9 patients were subjected to routine bacteria and bacteria.
Bacterial detection, and culture of these samples, the results showed negative, and the special detection of Legionella also showed negative.
The researchers detected the common bacteria in respiratory tract samples by molecular biology, extracted DNA and studied the common bacteria.
The RT-PCR results of pneumonia, Yersinia, Chlamydia and other microorganisms were also negative.
The researchers also carried out the detection of common viruses, and all the respiratory tract samples and stool samples of 9 patients were examined.
Direct virus detection, including electron microscope observation and direct fluorescent antibody detection, did not find influenza A and B viruses.
Parainfluenza virus 1, 2, 3, adenovirus, respiratory syncytial virus, etc. Similarly, for common respiratory viruses, researchers
Molecular biological detection of the virus was also carried out, and most of the results were negative, but the samples of bronchial lavage fluid were similar to those of the virus.
Human paramyxovirus was found in throat swab samples by nested RT-PCR, which ruled out the possibility of cross-infection in laboratory.
In addition, a novel coronavirus was isolated from cultured cells inoculated with respiratory specimens, accounting for 5.5% of 9 SARS patients.
Viruses were isolated from samples of 10 patients, and paramyxovirus was isolated from samples of 4 patients. At this point, research
Institutions, Hong Kong research groups and CDC researchers in the United States all reported that a new type of coronary heart disease was isolated from SARS patients.
Poison. The researchers then carried out RT-PCR on this new virus, amplified and cloned the nucleotide sequence of the virus, and measured it.
The sequencing results were analyzed by bioinformatics technology: the sequence of the new virus was different from that of the known coronavirus,
However, the amino acid sequence deduced from this sequence has high homology (78%) with that of several known coronaviruses.
Phylogenetic tree analysis showed that the new virus was the same as the known coronavirus family (group 1, group2 and group3).
Not very close.
Based on the existing findings, the researchers concluded that the isolated human paramyxovirus and neocoronavirus may be related to
SARS is related to the disease, but it is not clear whether the two viruses work together, or whether the two viruses work separately, or even others.
Undetected viruses are working, and further research is needed to confirm [16].
2.2 China
The research team led by Academician Hong Tao from the Institute of Virus Prevention and Control of China Center for Disease Control and Prevention showed the pathogen of SARS.
A study was conducted, and chlamydia and coronavirus particles were found in autopsy samples of SARS patients by electron microscope, which was published in China.
Their research literature was published in medical journals.
The research team selected samples from seven patients who died of SARS, and the researchers collected the organs of the deceased.
Tissue specimens, electron microscope specimens and pathological specimens were made. Of the seven dead, four were from Guangdong, 1 from Shanxi, and 1 came.
From Beijing, 1 case is a representative Hong Kong patient who died in Chengdu. In order to isolate pathogens, lung tissue extracts of patients were extracted.
Inoculated into 293 cells (human embryonic kidney transformed cell line), the tissues of patients and their isolated pathogens were identified by immunology, and samples were collected.
Indirect immunofluorescence and immunohistochemistry were used.