“When an epidemic of unknown cause attacks mankind, people’s reaction to it shows mankind’s fear of the unknown

” (The terror of the unknown is seldom better displayed than by the response of an epidemic, particularly when the epidemic strikes without apparent cause) [1]. In 1977, Edward Keyes used this sentence to describe the public's panic about the newly emerged Legionnaires' disease. Now we are also facing a new type of atypical pneumonia with an unknown pathogen - Severe Acute Respiratory Syndrome (SARS). This sentence seems to be equally applicable to the description of current people's psychology. It is undeniable that some members of the public are panic-stricken about this new epidemic. Part of this panic is due to incomplete understanding of the new disease, including its pathogens, mode of transmission, pathogenesis, pathogenesis, and diagnosis. There is still a lack of further understanding of treatment and prevention. Fortunately, humans have taken active countermeasures. The WHO organized an international laboratory network to jointly carry out research on SARS. In less than two months, relevant research institutions found the pathogen and deciphered its genome sequence, initially revealing the The mystery of SARS was opened. At present, experts have made some progress. Research has found that SARS disease is related to a new coronavirus, and this new virus has not been found in humans or animals before. This discovery has laid a solid foundation for the next step of work. Experts continue to uncover more unknowns about this disease. Through global collaboration, researchers used the Internet and a variety of research methods to identify the pathogen in just two months. More than 20 years ago, researchers spent more than two years searching for the HIV virus, and then several more years to obtain its nucleotide sequence. In contrast, the rapid response to the search for the SARS pathogen was impressive. If we roughly review the process of discovering pathogens, we may be able to gain new experience, provide a basis for further research, and provide experience for another epidemic that may break out in the future.

Research Progress

1.1 Confirmation of the SARS pathogen

On March 15, 2003, WHO organized an international research network, with laboratories from 11 countries participating. , my country joined in

early April. The progression of the research network is roughly as follows.

On March 18, Germany observed paramyxovirus from throat swab specimens through electron microscopy. At the same time, the Chinese University of Hong Kong obtained the gene amplification product of paramyxovirus and published the electron microscopy pictures and genes on the Internet. sequence.

On March 19, Singapore found paramyxovirus particles in patient respiratory specimens and obtained weaker viral gene amplification products (based on primers provided by the Chinese University of Hong Kong). The Rotterdam laboratory in the Netherlands, which first discovered paramyxovirus in the world, obtained samples from relevant patients.

On March 20, four international laboratories began to conduct paramyxovirus testing. The Rotterdam laboratory in the Netherlands detected paramyxovirus, but the paramyxovirus gene amplification was negative. The laboratory sent detection reagents to Singapore and Hong Kong, and the Canadian laboratory sent double serum to Rotterdam.

On March 21, scientists from the Chinese University of Hong Kong obtained virus isolates using monkey kidney cell culture methods, and then developed corresponding serological diagnostic reagents. Research results from many laboratories indicate that the pathogen is not related to the following pathogens: influenza A and B viruses, respiratory syncytial virus, parainfluenza virus types 1, 2, and 3, adenovirus, rhinovirus, enterovirus, human pneumoniae virus, Mycoplasma pneumoniae, and Chlamydia pneumoniae; at the same time, electron microscopy observed that the virus particles were 50-60nm. Serum experiments proved that adding patient serum can inhibit cytopathic effects while control serum is ineffective.

On March 21, the Rotterdam laboratory found that the HMPV virus PCR test of the respiratory specimens of three Singaporean patients in Germany was negative. Moreover, lesions were found in the respiratory tract specimens of the two suspicious cases inoculated with Vero cells and monkey kidney cells. Also on this day, British scientists tested the H3N2 influenza virus in the specimens of the two patients; Chinese virus expert Hong Tao announced that chlamydia was the main pathogen causing SARS, but he did not rule out the simultaneous action of chlamydia and coronavirus.

On March 21, researchers Zhu Qingyu and Qin Ede of the Institute of Microbiology and Epidemiology of the Academy of Military Medical Sciences successfully isolated a coronavirus-like virus from autopsy specimens of SARS patients through various studies, inferring that it may be SARS. pathogens, and reported this important result in writing to the Ministry of Health of the General Logistics Department of the People's Liberation Army and the National Ministry of Health.

On March 22, coronavirus-like particles (70nm) were found under an electron microscope at the Hong Kong Virology Laboratory, and coronavirus-like particles (70-100nm) were found in cytopathic products obtained from Thai patient specimens in the United States, but the same Positive PCR results for HMPV virus were obtained from 2 samples.

At the same time, Canada, France, Singapore and other places began to use PCR and electron microscopy methods to detect paramyxovirus and HMPV viruses. Canada discovered 20nm virus particles and released the genetic evolutionary tree of the HMPV virus.

On March 23, the Hong Kong Virus Laboratory found that 2 of 8 specimens were positive for coronavirus RNA. The United States reported that coronavirus was found in Hong Kong specimens. At the same time, it established an immunofluorescence method to detect patient serum and published the coronavirus gene amplification primers on the Internet. Primate experiments using nasopharyngeal swab samples to infect bronchial tubes began in Singapore and Hong Kong. Canada and France have detected coronavirus using electron microscopy and PCR technology respectively. Viruses were isolated from Vero cells in Germany, Japan, and Singapore. British laboratories have detected chicken pneumonia virus sequences in respiratory and urine specimens from suspected cases.

On March 24-26, laboratories in Germany and Hong Kong detected the coronavirus using electron microscopy and PCR technology respectively. The German laboratory obtained the virus sequence and confirmed the identity of the new virus. The amino acid sequence is consistent with the amino acid sequence of the polymerase of known coronaviruses. Laboratories in Germany, the Netherlands, Hong Kong and other places have obtained more gene amplification sequences and published the gene evolution tree of the new virus on the Internet.

From March 27th to 31st, animal experiments using monkeys as experimental subjects continued. Laboratories in Hong Kong and the United States respectively confirmed that normal human serum and the newly isolated virus were negative. Meanwhile, more laboratories have detected the coronavirus: Japan obtained positive results from specimens from Singapore; Hong Kong tested 50 patient sera, 27 of which had elevated coronavirus antibodies, while 10 patient stool samples were also tested 5 samples were found to be positive for the virus, and viral genes were found in the patient's feces 6-16 days after onset of illness; in Canada, human paramyxovirus was found in patient specimens.

On April 1-8, clinical symptoms appeared in monkeys infected with paramyxovirus and metapneumovirus in animal experiments.

American researchers used ELISA to detect antibodies in the blood of patients 20 days after the onset of the disease. Hong Kong researchers used immunofluorescence method to detect IgM antibodies in the serum of patients 10 days after the onset of the disease. The mouse experiment begins. More laboratories have isolated coronaviruses from Vero cultured cells, and some serological experiments have confirmed that SARS disease is a composite infection of paramyxovirus and coronavirus. The Netherlands, Germany, Hong Kong and the United States have successively discovered new sequences of the coronavirus, and German scientists have discovered chlamydia in specimens.

On April 9, the Chinese Center for Disease Control and Prevention and the Institute of Microbiology and Epidemiology of the Academy of Military Medical Sciences held a symposium on SARS pathogen research at the Academy of Military Medical Sciences. Relevant experts from both sides Research progress on chlamydia and coronaviruses were introduced respectively.

On the afternoon of April 10, Hong Tao, a well-known virology expert and academician of the Chinese Academy of Engineering, announced to dozens of Chinese and foreign media at the "Knowledge Introduction Conference on SARS Prevention and Control": "The research on SARS pathogens The research has initially yielded results, and two main pathogens have been found - chlamydia and coronavirus-like viruses."

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 of the Institute of Viral Disease Prevention and Control of the Chinese Center for Disease Control and Prevention reported on the pathogenic factors of SARS. A major breakthrough has been made in scientific research, several strains of coronavirus have been successfully isolated, and part of the isolated coronavirus genes have been cloned. They tested the coronavirus in the lung samples of three patients who died of SARS and the spleen sample of one of them, purified and amplified the coronavirus gene from these samples, and confirmed the amplified gene through nucleotide sequence determination. Because of the RNA polymerase gene of coronavirus, it was the first time in the world that molecular biology methods were used to confirm the presence of coronavirus in the patient's organs. They compared the genetic sequences of the new virus with other coronaviruses and found that the coronavirus present in SARS patients was a mutated coronavirus. These specimens were used to culture a variety of cells, and the new coronavirus was successfully isolated. The virus was passaged several times in the cultured cells, and cytopathic effects were stable in all cases. The genetic test for the virus continued to be positive. Three strains of coronavirus were isolated from throat swabs of domestic SARS patients. The nucleotide sequences of these viruses are the same as those of the coronavirus isolated from the patients' organs. The research results obtained so far indicate to a large extent that coronavirus may be the pathogen causing SARS.

On April 11, Xinhua News Agency announced the findings of two researchers, Zhu Qingyu and Qin Ede, from the Institute of Microbiology and Epidemiology of the Academy of Military Medical Sciences. At the end of February, the institute isolated and identified the coronavirus from an autopsy specimen of a SARS patient. As of March 21, further evidence about the coronavirus has been obtained through research in serology, immunology, molecular biology and other aspects. On April 9, the sequences of the four isolated coronavirus strains were determined. These results were announced on 16 April.

On April 12, Dr. Holt and his research team from the British Columbia Cancer Institute in Vancouver, Canada released the genome sequence of the suspected SARS pathogen.

On April 14, Dr. Anderson's research team at the CDC in Atlanta also completed genome sequencing and published it online

The sequencing results of the two research groups were basically consistent.

On April 16, David Hyman, WHO Executive Director for Infectious Diseases, announced that through the joint efforts of global scientific researchers, a mutated coronavirus was officially confirmed to cause SARS.

On April 17, the WHO held a press conference in Geneva and announced that it had completed the final step of identifying the pathogen,

which is the fourth step in the "Koch's presumption." A research team led by Dr. Albert Osterhaus of Erasmus University in the Netherlands successfully infected experimental monkeys with the new coronavirus. The research team then isolated the virus from the infected monkeys and cultured it in the laboratory. It shows that after a month of collaborative efforts, scientists around the world have initially identified the pathogen of SARS [2-9].

On April 22, China released a map of the coronavirus responsible for SARS [3][7][10].

1.2 Progress in decoding the SARS virus genome

1.2.1 Canadian Smith Genetic Science Center

On April 12, Canadian scientists decoded the genes of the suspected SARS pathogen and headed toward The first step has been taken to develop SARS diagnostic methods and develop SARS vaccines and drugs.

The Smith Center for Genetic Sciences in Vancouver joins the global fight against SARS. Center director Marra said that genetic coding is the basic information needed by scientists to develop diagnostic testing methods. After the Smith Genetic Science Center cracked the genetic code, it immediately published it on the international network (http://www.bcgsc.bc.ca) for use by other scientists around the world.

1.2.2 University of Hong Kong

Yuen Kwok-yong, director of the Department of Microbiology of the University of Hong Kong, said that the Faculty of Medicine of the University of Hong Kong has completed the genetic sequence determination of the coronavirus that caused SARS and determined that it is a brand-new virus, which is suspected to be caused by Animals spread it to humans. As for which animals it is, it remains to be studied. Yuan Guoyong believes that this discovery can help improve current rapid testing methods. Previously, researchers at the Chinese University of Hong Kong had handed over the partial genetic sequence of the new coronavirus they had cracked to the World Health Organization's "SARS" working group on the evening of the 13th.

1.2.3 U.S. Centers for Disease Control and Prevention

On April 14, the U.S. Centers for Disease Control and Prevention announced that it had completed its investigation of the SARS virus believed to be responsible for the global epidemic

< p>Sequencing of the new coronavirus genome. The genetic sequencing results are basically the same as those from a Canadian laboratory. After comparing the sequencing results from the two institutions, it was discovered that the difference was that their sequencing results had 15 extra nucleotides, which would be a significant start for continued sequencing work. The sequencing results were obtained after 12 days of work with the joint efforts of 10 scientists and many technical staff. The researchers cultured the throat secretions of one of the SARS patients in African green monkey kidney cells, purified the nucleic acid sequence of the coronavirus that caused the disease, and then amplified and sequenced it. The new gene sequence has 29,727 nucleosides, which is within the typical RNA boundary of the coronavirus family, which generally has 29,000 to 31,000 nucleosides. Dr. Julie Gerberding, director of the US Centers for Disease Control and Prevention, said that determining the genetic sequence of a new virus is very important for both the treatment and prevention of the disease. Using information about genetic sequences, laboratory research on antiviral drugs can begin, serve as the basis for developing vaccines, and diagnostic tests can be developed for early detection of cases. The nearly identical results from the U.S. and Canadian studies are important and suggest the viruses may have come from the same source, since the samples were collected from different individuals infected in different countries. But officials from the U.S. Centers for Disease Control and Prevention emphasized that the analysis of the virus is far from complete. Coronaviruses can mutate rapidly. Researchers need to combine viruses isolated from cell cultures with those from SARS patients. Viruses obtained from diseased tissues are compared, and the genetic sequencing work carried out will speed up the comparison work.

1.2.4 The Beijing Institute of Genomics, Chinese Academy of Sciences and the Institute of Microbial Epidemiology, Academy of Military Medical Sciences

The Beijing Institute of Genomics, Chinese Academy of Sciences and the Institute of Microbial Epidemiology, Academy of Military Medical Sciences work together to On April 16, 2003, the genome decoding of four coronavirus strains isolated from different SARS cases was completed. The results showed that the length of this virus is about 30,000 base pairs, which is basically consistent with the sequences reported by Canada and the United States. It belongs to a new type of coronavirus. This result is only two days later than the time when Canadian scientists announced that they had deciphered the coronavirus gene. sky.

The successful determination of the entire genome sequence of the coronavirus has laid a solid foundation for tracking the source of the coronavirus and developing diagnostic preparations, vaccines and therapeutic drugs for SARS, and has taken an important step forward in the prevention and treatment of SARS in my country [ 2][3][4][5][8][10].

2 Researchers isolated and identified pathogens

In this joint search for pathogens by relevant laboratories around the world, Hong Kong researchers took the lead in making a breakthrough. They used traditional virus culture, Serological detection technology and modern molecular genetic methods identified the pathogen - a new coronavirus - in 50 SARS patients. In addition, analysis of samples from the control group further supported their argument about the pathogen: among 40 respiratory samples from patients with other respiratory diseases, not a single sample was detected to contain the RNA of the new coronavirus; for blood donors, None of the 200 serum samples contained antibodies to the virus.

These findings also support the views put forward by two other research institutions. The U.S. Centers for Disease Control and Prevention (CDC) and relevant research institutions in Toronto, Canada, also isolated this new coronavirus from SARS patients. , and believe that the virus is linked to the SARS outbreak. German researchers discovered the new coronavirus in the first three patients and further collected samples from patients in Hanoi, Vietnam for testing and analysis. The results also support this conclusion.

2.1 Research results of an international laboratory network

2.1.1 Hong Kong, China

A research team led by Professor Peiris of the Department of Microbiology of the University of Hong Kong conducted a study on patients in Hong Kong Research was conducted to find the pathogen

.

The team selected 50 SARS patients who met the revised WHO definition admitted to three emergency hospitals in Hong Kong, collected

throat swab samples and serum samples from all patients, and selected some patients. Serum and excretion samples were collected in the severe and convalescent stages respectively. In addition, they obtained a patient's lung tissue sample and conducted virus culture isolation, reverse transcription PCR (RT-PCR), conventional tissue autoradiography and electron microscopy. The microbiological test results of other patients' throat swab samples, feces and serum samples

were used as controls.

The researchers initially conducted routine blood tests, biochemical tests and microbiological tests, and conducted bacterial culture and serological tests on blood samples and throat swab samples

respectively. Swab samples for rapid fluorescent antigen testing to determine whether the pathogen is a common respiratory infection virus, and use a variety of cells to culture to isolate the pathogen; use clinical samples to directly perform RT -PCR to detect influenza A virus and human paramyxovirus infection. In addition, an ELISA

method was used on cultured cells to detect the presence of chlamydia.

The breakthrough in the research was the observation of coronavirus-like particles in samples from two patients. One of the samples came from a 53-year-old male patient. Coronavirus RNA was detected in his throat swab samples, lung biopsies and other samples. The patient himself

had coronavirus The viral antibody titer increased significantly (1/200~1/1600). Another sample was taken from a 42-year-old female patient. The PCR test was positive for coronavirus, and her own serum antibodies changed (1/150~1/1600).

The researchers inoculated the two samples with cultured cells. After 2-4 days, circular refractive diseased cells appeared, indicating that

there was pathogen isolation, and the isolated pathogens were not recognized. Reagent plate reactions for common viruses. The cell culture extract was subjected to

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high-speed centrifugation, and then negative staining was observed under an electron microscope. It was found that the enveloped virus had an irregular shape and a diameter of about 80-90nm. Table< /p>

The facial features are similar to those of coronavirus. Electron microscopy of ultrathin sections of infected cells revealed the presence of similar virus particles in the cytoplasm and cell membrane surface, and the viruses isolated from the two patients were very similar in size and morphological characteristics.

In order to obtain the genetic sequence information of the newly isolated virus, the researchers conducted random RT-PCR, cloned and sequenced the characteristic chromosomal bands of the virus-infected cells

, and conducted a database in GenBank. Comparison of homologous sequences revealed that an unknown sequence was found

in the 30 clones tested. Analysis of this DNA sequence revealed that it has low homology with coronaviruses, but the amino acid sequence deduced from it has high homology with bovine coronavirus and murine pneumonia virus RNA polymerases of the Coronaviridae family. (57%),

Phylogenetic study of the protein sequence shows that the new virus is highly related to coronavirus Group2. Indirect immunofluorescence detection was used to measure the viral serological response

. The results showed that seroconversion occurred in all 32 patients in the severe and convalescent stages, indicating the presence of coronavirus. The phenomenon of increased virus antibody titers.

The research team also conducted RT-PCR and serum antibody titer testing for human paramyxovirus, and the results were negative, and no other pathogens were

detected. Therefore, the research team believes that the isolated coronavirus is the pathogen or an essential factor of SARS. However, it remains to be seen whether other microbial or non-microbial cofactors play a role[11][12 ].

2.1.2 Germany

The initial SARS outbreak occurred in Asia, from which the disease spread to other continents due to intercontinental travel. Given that SARS disease

is a new disease and that humans know nothing about its causative agent, initial research efforts focused on the identification of the causative agent.

WHO organized an international laboratory network to focus the research efforts of relevant countries to find the pathogen of SARS. German research institutions, which are part of the network, are also conducting research on the identification of pathogens.

The research team initially selected samples from three people in the same family: a 32-year-old male patient, his

wife and his mother-in-law. The man was a Singaporean physician who had treated a SARS patient and became infected. He subsequently infected his wife and mother-in-law. The three people came to the United States from Singapore for some reason. The doctor developed symptoms while in the United States. He informed his Singaporean colleague, who reported it to the WHO, who transferred the three people on their flight back to Singapore. Station—

Frankfurt quarantined them. Relevant German researchers obtained their respiratory tract samples and blood samples, and then the team also obtained samples from 18 other suspected or possible SARS cases from Asia, as well as 21 people who had been exposed to SARS

Samples from patients but not infected persons.

The researchers first conducted PCR testing on the samples from the above three patients to identify the presence of Diplococcus pneumoniae, Chlamydia pneumoniae, human cytomegalovirus, parainfluenza virus, and influenza Viruses, human paramyxoviruses, rhinoviruses, and human coronaviruses

Known respiratory pathogens such as OC43 and 229E types; perform antigen ELISA on respiratory samples to detect the presence of pneumonia

cocci, influenza viruses and respiratory syncytial virus, and serological testing was performed on blood samples; in addition, researchers

negatively stained respiratory samples and blood samples for electron microscopy observation, and cultured cells on the samples Vaccination.

The researchers extracted RNA from sputum samples and analyzed it with random RT-PCR technology. Some of the designed PCR primers

contained degenerate sites, and most of the primers The 3' end is a T base so that DNA polymerase can take effect even if the bases at the end of the primer do not completely match. The BLAST tool was used to compare the homology of the clonal amplification products.

The research team conducted multiple tests on samples from three patients, and the test results for known respiratory pathogens

were mostly negative. When observing the respiratory samples under an electron microscope, they found rare Paramyxovirus-like particles, but several subsequent PCR tests for the Paramyxovirus family were negative. Six days after the sputum samples were inoculated with cultured cells, the researchers found that there were diseased cells in the culture medium. They then performed RNA extraction and performed RT-PCR on the extracted RNA. About 20 clones were amplified and cloned.

Different DNA fragments. After sequencing these fragments and searching with BLAST, three new fragments were found. The new fragments did not match the sequences in the database, but were determined by the new fragments. The amino acid sequence deduced from the fragment showed homology with the coronavirus family, indicating that a new coronavirus was isolated. The researchers compared the new fragment with the new viral nucleic acid sequence measured by the U.S. Centers for Disease Control and Prevention (CDC) and found homology. Immunofluorescence testing was performed on serum samples from these three patients as well as infected cultured cells to determine the presence of antibodies. Elevated IgG antibodies were detected in the serum of two patients, indicating the presence of a serological response to the new coronavirus.

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To test whether the new virus is related to SARS, researchers further collected data from possible or suspected SARS patients

and those who had been in contact with SARS patients However, samples from uninfected people (further collected samples were all from Hanoi, Vietnam), nested PCR analysis was performed on this batch of samples, and the result was that the proportion of virus found in possible SARS cases was 100% , 23% of suspected cases, but no virus was found in healthy contacts. These data may further prove the connection between the new coronavirus and SARS.

The research team also detected paramyxoviruses and pneumococci, but subsequent targeted PCR experiments were negative.

Chlamydial infection has been found in several patients but not in other SARS patients. Therefore, researchers are still unclear whether these pathogens act as causative factors or co-causing factors in SARS disease [13].

2.1.3 United States

The United States Centers for Disease Control and Prevention (CDC) is one of the research institutions of the international laboratory network organized by WHO. They

Identification of the SARS pathogen has also been launched. CDC samples came from SARS patients in Vietnam, Singapore, Thailand, Canada, Hong Kong, Taiwan and the United States, trying to identify the cause of the epidemic from a series of known pathogens.

< p>The source of the SARS outbreak.

Since the clinical symptoms of SARS patients are non-specific, initial research focused on the investigation of known respiratory pathogens

using a combination of detection methods. The researchers collected a variety of samples including blood, serum, nasopharyngeal swabs, cough fluid and organ tissues, cultured them in a variety of cells, and injected them into suckling mice. , to isolate pathogens;

observed cultured cells and suckling mice, and prepared sections of diseased cells or individuals for electron microscope observation;

conducted serological experiments on serum samples to detect Antibodies; general and special bacterial cultures were carried out, and molecular biology techniques such as PCR

, RT-PCR were also used; a variety of respiratory pathogens were screened, such as Yersinia, Mycoplasma, Chlamydia, rickettsia

, Legionella, influenza virus types A and B, parainfluenza virus family, etc.

The breakthrough in the research was the observation of coronavirus-like particles through electron microscopy. The cultured cells inoculated with the patient's respiratory tract samples showed cytopathic effects. Ultra-thin sections were made of the diseased cells and observed under an electron microscope. The findings were found in the diseased cells and cell membranes. Coronavirus-like particles: about 80-140nm in diameter, with complex structural protrusions of 20-40nm on the surface of the virus.

Using an electron microscope to observe the patient's bronchial washing fluid samples, it was also found that the coronavirus was present in many infected cells.

The researchers performed RNA extraction and RT-PCR on the diseased cells to amplify the new virus sequence. The primers were designed based on the sequence information of known coronaviruses in GenBank.

The amplified purified product was sequenced and analyzed, compared with the published coronavirus sequence, and analyzed using bioinformatics technology to obtain the evolutionary tree of the virus. Comparing the sequences of the new virus with other

coronaviruses and the amino acid sequences deduced from the sequences, it was found that the new virus has high homology with the coronavirus family group2

. However, analysis of the evolutionary tree shows that this virus is genetically different from other coronaviruses, indicating that the isolate is a new type of coronavirus. Serological testing of the samples found that infected cell sections reacted with the serum of suspected SARS patients in the recovery period

The serum of suspected SARS patients from Hong Kong, Bangkok and the United States showed specific reactions

>

Response, showing a transition from negative to positive or increased reactivity in the indirect fluorescent antibody experiment; ELISA antigen test was performed on the same batch of serum samples, and the reaction of the recovery phase samples was highly specific , the antibody titer also gradually increased.

After sample inoculation and cultured cells amplified the pathogen, electron microscopic observation found that the pathogen was a coronavirus. Molecular biology research

further determined the essential characteristics of the new virus. Serological experiments determined that it was related to the genus coronavirus. Therefore, researchers believe that the isolated new coronavirus may be the pathogen of SARS. However, the research team also pointed out that the new coronavirus antigen or viral RNA in the patient's lesion tissue cells has not been directly detected during the pathological process. At the same time, it cannot be confirmed that the new virus exists in all SARS patients. No coronavirus has been detected in some SARS patients, and further relevant research is needed [14][15].

2.1.4 Canada

Canada’s National Microbiology Laboratory and related research institutions organized a SARS research team to conduct research on SARS

disease.

After a follow-up investigation, researchers confirmed that the country’s original SARS patient had traveled to Hong Kong and was infected there.

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The patient returned to Canada and Spread the SARS disease into the country. The samples obtained by the researchers came from 9 patients who were directly or indirectly infected by the patient (who died). The researchers performed histopathological examination of one autopsy tissue sample and microbiological testing of samples from all nine patients.

Immunohistochemistry was performed on autopsy tissue samples to detect the presence of influenza viruses A and B, respiratory syncytial virus, adenovirus, hantavirus, measles virus, enterovirus,

All known common viruses such as flavivirus, as well as rickettsiae, Mycoplasma pneumoniae, Chlamydia pneumoniae, Yersinia, etc.

all test results were negative. Routine bacterial and fungal testing was performed on blood samples, respiratory samples, and urine samples from 9 patients, and these samples were cultured and the results were negative. Special testing for Legionella was also conducted. Also showed negative.

The researchers conducted molecular biology tests on common bacteria in respiratory samples, extracted DNA, and conducted tests on common

pneumoniae, Yersinia, Chlamydia and other microorganisms. The RT-PCR result also showed negative.

The researchers also conducted detection of common viruses, and conducted routine direct virus detection on all respiratory samples and stool samples of 9 patients, including electron microscopy and direct fluorescence. Antibody testing showed that influenza viruses A and B, parainfluenza viruses 1, 2, and 3, adenovirus, respiratory syncytial virus, etc. were not found in the samples. Similarly, for common respiratory viruses, researchers

also conducted molecular biology tests of viruses. Most of the test results showed negative, but for bronchial washing fluid samples and

throat swabs Nested RT-PCR on the samples identified human paramyxovirus and ruled out the possibility of laboratory cross-infection.

In addition, a new coronavirus was isolated from cultured cells inoculated with respiratory samples, and was isolated from samples of 5 of 9 SARS patients

, paramyxovirus was isolated from samples of 4 patients. So far, the research institution, the Hong Kong research team and the US CDC researchers have all reported the isolation of the new coronavirus from SARS patients. The researchers then performed RT-PCR on the new virus, amplified and cloned the nucleotide sequence of the virus, sequenced it

, and analyzed the sequencing results using bioinformatics technology: New The sequence of the virus is different from that of known coronaviruses,

However, the amino acid sequence deduced from this sequence has high homology (78%) with the amino acid sequences of several known coronaviruses,

The biological evolutionary tree analysis of the new virus shows that the new virus is not very close to the known coronavirus families (group1, group2 and group3)

.

Based on existing findings, the researchers concluded that both the isolated human paramyxovirus and the new coronavirus may be linked to

SARS disease, but it is not yet clear Whether the two act in combination, or the two viruses act alone, or even other undetected viruses are at work, further research is needed to confirm [16].

2.2 China

The research team led by Academician Hong Tao of the Institute of Virology Prevention and Control of the Chinese Center for Disease Control and Prevention conducted research on the pathogen of SARS

and observed it through electron microscopy Chlamydia and coronavirus particles were found in autopsy specimens of SARS patients, and their research was published in the "Chinese Medical Journal".

The samples selected by the research team came from 7 patients who died of SARS. The researchers collected organ samples from the deceased's organs

and prepared electron microscopy specimens and pathological specimens. Among the 7 deceased, 4 were from Guangdong, 1 was from Shanxi, 1 was from Beijing, and 1 was a Hong Kong patient who died in Chengdu, which is quite representative.

In order to isolate the pathogen, the patient's lung tissue extract

was inoculated into 293 cells (human embryonic kidney transformed cell line), and the patient's tissue and the isolated pathogen were immunologically identified and collected

< p>Indirect immunofluorescence and immunohistochemistry were taken