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1.
Elife ; 102021 04 09.
Article in English | MEDLINE | ID: covidwho-1389777

ABSTRACT

Virus propagation methods generally use transformed cell lines to grow viruses from clinical specimens, which may force viruses to rapidly adapt to cell culture conditions, a process facilitated by high viral mutation rates. Upon propagation in VeroE6 cells, SARS-CoV-2 may mutate or delete the multibasic cleavage site (MBCS) in the spike protein. Previously, we showed that the MBCS facilitates serine protease-mediated entry into human airway cells (Mykytyn et al., 2021). Here, we report that propagating SARS-CoV-2 on the human airway cell line Calu-3 - that expresses serine proteases - prevents cell culture adaptations in the MBCS and directly adjacent to the MBCS (S686G). Similar results were obtained using a human airway organoid-based culture system for SARS-CoV-2 propagation. Thus, in-depth knowledge on the biology of a virus can be used to establish methods to prevent cell culture adaptation.


Subject(s)
Epithelial Cells , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/genetics , Virus Cultivation/methods , Virus Internalization , Animals , Cell Line , Chlorocebus aethiops , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelial Cells/virology , Humans , Proteolysis , Respiratory System/cytology , Respiratory System/virology , Serine Proteases/metabolism
2.
Int J Mol Med ; 47(6)2021 06.
Article in English | MEDLINE | ID: covidwho-1181666

ABSTRACT

The Coronavirus Disease 2019 (COVID­19) pandemic has forced the scientific community to rapidly develop highly reliable diagnostic methods in order to effectively and accurately diagnose this pathology, thus limiting the spread of infection. Although the structural and molecular characteristics of the severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) were initially unknown, various diagnostic strategies useful for making a correct diagnosis of COVID­19 have been rapidly developed by private research laboratories and biomedical companies. At present, rapid antigen or antibody tests, immunoenzymatic serological tests and molecular tests based on RT­PCR are the most widely used and validated techniques worldwide. Apart from these conventional methods, other techniques, including isothermal nucleic acid amplification techniques, clusters of regularly interspaced short palindromic repeats/Cas (CRISPR/Cas)­based approaches or digital PCR methods are currently used in research contexts or are awaiting approval for diagnostic use by competent authorities. In order to provide guidance for the correct use of COVID­19 diagnostic tests, the present review describes the diagnostic strategies available which may be used for the diagnosis of COVID­19 infection in both clinical and research settings. In particular, the technical and instrumental characteristics of the diagnostic methods used are described herein. In addition, updated and detailed information about the type of sample, the modality and the timing of use of specific tests are also discussed.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Animals , Biosensing Techniques/methods , Clustered Regularly Interspaced Short Palindromic Repeats , Humans , Inventions , Microscopy, Electron/methods , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Virus Cultivation/methods
3.
Methods Mol Biol ; 2203: 223-229, 2020.
Article in English | MEDLINE | ID: covidwho-729909

ABSTRACT

All viruses have to overcome the innate immune response in order to establish infection. Methods have been developed to assay if, and how, viruses overcome these responses, and many can be directly applied to coronaviruses. Here, in vitro methods to determine how coronaviruses overcome this response are described.


Subject(s)
Coronavirus/growth & development , Coronavirus/metabolism , Virus Cultivation/methods , Animals , Cell Line , Coronavirus/pathogenicity , Host-Pathogen Interactions , Humans , Immunity, Innate/immunology , Interferons , Middle East Respiratory Syndrome Coronavirus/growth & development , Middle East Respiratory Syndrome Coronavirus/pathogenicity , RNA, Viral , Viral Nonstructural Proteins , Virus Replication
4.
Jpn J Infect Dis ; 74(1): 48-53, 2021 Jan 22.
Article in English | MEDLINE | ID: covidwho-1116920

ABSTRACT

JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system affecting immunocompromised patients. The study of PML-type JCPyV in vitro has been limited owing to the inefficient propagation of the virus in cultured cells. In this study, we carried out long-term culture of COS-7 cells (designated as COS-IMRb cells) transfected with PML-type M1-IMRb, an adapted viral DNA with a rearranged non-coding control region (NCCR). The JCPyV derived from COS-IMRb cells were characterized by analyzing the viral replication, amount of virus by hemagglutination (HA), production of viral protein 1 (VP1), and structure of the NCCR. HA assays indicated the presence of high amounts of PML-type JCPyV in COS-IMRb cells. Immunostaining showed only a small population of JCPyV carrying COS-IMRb cells to be VP1-positive. Sequencing analysis of the NCCR of JCPyV after long-term culture revealed that the NCCR of M1-IMRb was conserved in COS-IMRb cells without any point mutation. The JCPyV genomic DNA derived from a clone of COS-IMRb-3 cells was detected, via Southern blotting, as a single band of approximately 5.1 kbp without deletion. These findings suggest the potential of using COS-IMRb-3 cells as a useful tool for screening anti-JCPyV drugs.


Subject(s)
JC Virus/growth & development , JC Virus/genetics , Leukoencephalopathy, Progressive Multifocal/virology , Virus Cultivation/methods , Animals , Blotting, Southern/methods , COS Cells , Chlorocebus aethiops , DNA Replication , DNA, Viral/isolation & purification , Hemagglutination , Humans , Transfection , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Replication
5.
J Clin Lab Anal ; 35(4): e23735, 2021 Apr.
Article in English | MEDLINE | ID: covidwho-1092109

ABSTRACT

BACKGROUND: The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has generated a pandemic with alarming rates of fatality worldwide. This situation has had a major impact on clinical laboratories that have attempted to answer the urgent need for diagnostic tools, since the identification of coronavirus disease 2019 (COVID-19). Development of a reliable serological diagnostic immunoassay, with high levels of sensitivity and specificity to detect SARS-CoV-2 antibodies with improved differential diagnosis from other circulating viruses, is mandatory. METHODS: An enzyme-linked immunosorbent assay (ELISA) using whole inactivated virus cultured in vitro, was developed to detect viral antigens. WB and ELISA investigations were carried out with sera of convalescent patients and negative sera samples. Both analyses were concurrently performed with recombinant MABs to verify the findings. RESULTS: Preliminary data from 10 sera (5 patients with COVID-19, and 5 healthy controls) using this immunoassay are very promising, successfully identifying all of the confirmed SARS-CoV-2-positive individuals. CONCLUSION: This ELISA appears to be a specific and reliable method for detecting COVID-19 antibodies (IgG, IgM, and IgA), and a useful tool for identifying individuals which have developed immunity to the virus.


Subject(s)
Antigens, Viral , COVID-19 Serological Testing/methods , COVID-19/diagnosis , SARS-CoV-2 , Virus Cultivation/methods , Animals , Antibodies, Viral/blood , Antigens, Viral/chemistry , Antigens, Viral/immunology , Antigens, Viral/isolation & purification , Blotting, Western , COVID-19/immunology , COVID-19/virology , Chlorocebus aethiops , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/immunology , Coronavirus Nucleocapsid Proteins/isolation & purification , Enzyme-Linked Immunosorbent Assay/methods , Humans , Phosphoproteins/chemistry , Phosphoproteins/immunology , Phosphoproteins/isolation & purification , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/isolation & purification , Vero Cells
6.
Appl Microbiol Biotechnol ; 105(4): 1421-1434, 2021 Feb.
Article in English | MEDLINE | ID: covidwho-1052958

ABSTRACT

Similar to the recent COVID-19 pandemic, influenza A virus poses a constant threat to the global community. For the treatment of flu disease, both antivirals and vaccines are available with vaccines the most effective and safest approach. In order to overcome limitations in egg-based vaccine manufacturing, cell culture-based processes have been established. While this production method avoids egg-associated risks in face of pandemics, process intensification using animal suspension cells in high cell density perfusion cultures should allow to further increase manufacturing capacities worldwide. In this work, we demonstrate the development of a perfusion process using Madin-Darby canine kidney (MDCK) suspension cells for influenza A (H1N1) virus production from scale-down shake flask cultivations to laboratory scale stirred tank bioreactors. Shake flask cultivations using semi-perfusion mode enabled high-yield virus harvests (4.25 log10(HAU/100 µL)) from MDCK cells grown up to 41 × 106 cells/mL. Scale-up to bioreactors with an alternating tangential flow (ATF) perfusion system required optimization of pH control and implementation of a temperature shift during the infection phase. Use of a capacitance probe for on-line perfusion control allowed to minimize medium consumption. This contributed to a better process control and a more economical performance while maintaining a maximum virus titer of 4.37 log10(HAU/100 µL) and an infectious virus titer of 1.83 × 1010 virions/mL. Overall, this study clearly demonstrates recent advances in cell culture-based perfusion processes for next-generation high-yield influenza vaccine manufacturing for pandemic preparedness. KEY POINTS: • First MDCK suspension cell-based perfusion process for IAV produciton was established. • "Cell density effect" was overcome and process was intensified by reduction of medium use and automated process control. • The process achieved cell density over 40 × 106 cells/mL and virus yield over 4.37 log10(HAU/100 µL).


Subject(s)
Influenza A Virus, H1N1 Subtype/physiology , Virus Cultivation/methods , Virus Replication/physiology , Animals , Bioreactors , Dogs , Madin Darby Canine Kidney Cells
7.
J Clin Microbiol ; 58(8)2020 07 23.
Article in English | MEDLINE | ID: covidwho-999208

ABSTRACT

Real-time reverse transcription-PCR (RT-PCR) is currently the most sensitive method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19). However, the correlation between detectable viral RNA and culturable virus in clinical specimens remains unclear. Here, we performed virus culture for 60 specimens that were confirmed to be positive for SARS-CoV-2 RNA by real-time RT-PCR. The virus could be successfully isolated from 12 throat and nine nasopharyngeal swabs and two sputum specimens. The lowest copy number required for virus isolation was determined to be 5.4, 6.0, and 5.7 log10 genome copies/ml sample for detecting the nsp12, E, and N genes, respectively. We further examined the correlation of genome copy number and virus isolation in different regions of the viral genome, demonstrating that culturable specimens are characterized by high copy numbers with a linear correlation observed between copy numbers of amplicons targeting structural and nonstructural regions. Overall, these results indicate that in addition to the copy number, the integrity of the viral genome should be considered when evaluating the infectivity of clinical SARS-CoV-2 specimens.


Subject(s)
Betacoronavirus/growth & development , Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Virus Cultivation/methods , Betacoronavirus/genetics , COVID-19 , COVID-19 Testing , COVID-19 Vaccines , Correlation of Data , Humans , Nasopharynx/virology , Pandemics , Pharynx/virology , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2
8.
Influenza Other Respir Viruses ; 14(2): 204-209, 2020 03.
Article in English | MEDLINE | ID: covidwho-825975

ABSTRACT

BACKGROUND: Cell-based influenza vaccines can solve the problem of the frequent occurrence of egg adaptation-associated antigenic changes observed in egg-based vaccines. Seed viruses for cell-based vaccines can be prepared from clinical specimens by cell culture; however, clinical samples risk harboring respiratory viruses other than influenza virus. Therefore, it is necessary to investigate the patterns of co-infection in clinical samples and explore whether cell culture technology can selectively propagate influenza viruses from samples containing other respiratory viruses. METHODS: A total of 341 clinical specimens were collected from patients with influenza or influenza-like illness and analyzed by ResPlex II assay to detect 18 respiratory viruses. The patterns of co-infection were statistically analyzed with Fisher's exact test. The samples with double or triple infections were passaged in suspension MDCK cells (MDCK-S), adherent MDCK cells (MDCK-A), and LLC-MK2D cells. Cell-passaged samples were analyzed by ResPlex II assay again to investigate whether each cell line could amplify influenza viruses and eliminate other respiratory viruses. RESULTS: Double infections were detected in 8.5% and triple infections in 0.9% of the collected clinical specimens. We identified four pairs of viruses with significant correlation. For all samples with double and triple infection, MDCK-S and MDCK-A could selectively propagate influenza viruses, while eliminating all contaminating viruses. In contrast, LLC-MK2D showed lower isolation efficiency for influenza virus and higher isolation efficiency for coxsackievirus/echovirus than MDCK-S and MDCK-A. CONCLUSIONS: Both MDCK-S and MDCK-A are considered suitable for the preparation of influenza vaccine seed viruses without adventitious agents or egg-adaptation mutations.


Subject(s)
Madin Darby Canine Kidney Cells/virology , Orthomyxoviridae/isolation & purification , Virus Cultivation/methods , Animals , Cell Line , Dogs , Humans , Orthomyxoviridae/growth & development , Viral Vaccines
9.
J Transl Med ; 18(1): 362, 2020 09 23.
Article in English | MEDLINE | ID: covidwho-788689

ABSTRACT

BACKGROUND: Since the first outbreak of SARS-CoV-2, the clinical characteristics of the Coronavirus Disease 2019 (COVID-19) have been progressively changed. Data reporting a viral intra-host and inter-host evolution favouring the appearance of mild SARS-CoV-2 strains are since being accumulating. To better understand the evolution of SARS-CoV-2 pathogenicity and its adaptation to the host, it is therefore crucial to investigate the genetic and phenotypic characteristics of SARS-CoV-2 strains circulating lately in the epidemic. METHODS: Nasopharyngeal swabs have been analyzed for viral load in the early (March 2020) and late (May 2020) phases of epidemic in Brescia, Italy. Isolation of SARS-CoV-2 from 2 high viral load specimens identified on March 9 (AP66) and on May 8 (GZ69) was performed on Vero E6 cells. Amount of virus released was assessed by quantitative PCR. Genotypic characterization of AP66 and GZ69 was performed by next generation sequencing followed by an in-depth in silico analysis of nucleotide mutations. RESULTS: The SARS-CoV-2 GZ69 strain, isolated in May from an asymptomatic healthcare worker, showed an unprecedented capability of replication in Vero E6 cells in the absence of any evident cytopathic effect. Vero E6 subculturing, up to passage 4, showed that SARS-CoV-2 GZ69 infection was as productive as the one sustained by the cytopathic strain AP66. Whole genome sequencing of the persistently replicating SARS-CoV-2 GZ69 has shown that this strain differs from the early AP66 variant in 9 nucleotide positions (C2939T; C3828T; G21784T; T21846C; T24631C; G28881A; G28882A; G28883C; G29810T) which lead to 6 non-synonymous substitutions spanning on ORF1ab (P892S; S1188L), S (K74N; I95T) and N (R203K, G204R) proteins. CONCLUSIONS: Identification of the peculiar SARS-CoV-2 GZ69 strain in the late Italian epidemic highlights the need to better characterize viral variants circulating among asymptomatic or paucisymptomatic individuals. The current approach could unravel the ways for future studies aimed at analyzing the selection process which favours viral mutations in the human host.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/virology , Genetic Variation , Pneumonia, Viral/virology , Amino Acid Substitution , Animals , Betacoronavirus/isolation & purification , Betacoronavirus/physiology , COVID-19 , Chlorocebus aethiops , Coronavirus Infections/epidemiology , Cytopathogenic Effect, Viral/genetics , Cytopathogenic Effect, Viral/physiology , Genome, Viral , Humans , Italy/epidemiology , Mutation , Pandemics , Phylogeny , Pneumonia, Viral/epidemiology , Polymorphism, Single Nucleotide , SARS-CoV-2 , Vero Cells , Viral Proteins/genetics , Viral Proteins/physiology , Virus Cultivation/methods , Virus Replication/genetics , Virus Replication/physiology , Whole Genome Sequencing
10.
PLoS One ; 15(9): e0238614, 2020.
Article in English | MEDLINE | ID: covidwho-771793

ABSTRACT

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and associated with severe respiratory illness emerged in Wuhan, China, in late 2019. The virus has been able to spread promptly across all continents in the world. The current pandemic has posed a great threat to public health concern and safety. Currently, there are no specific treatments or licensed vaccines available for COVID-19. We isolated SARS-CoV-2 from the nasopharyngeal sample of a patient in Turkey with confirmed COVID-19. We determined that the Vero E6 and MA-104 cell lines are suitable for supporting SARS-CoV-2 that supports viral replication, development of cytopathic effect (CPE) and subsequent cell death. Phylogenetic analyses of the whole genome sequences showed that the hCoV-19/Turkey/ERAGEM-001/2020 strain clustered with the strains primarily from Australia, Canada, England, Iran and Kuwait and that the cases in the nearby clusters were reported to have travel history to Iran and to share the common unique nucleotide substitutions.


Subject(s)
Betacoronavirus/isolation & purification , Pandemics , Virus Cultivation/methods , Animals , Australia , Betacoronavirus/genetics , Betacoronavirus/physiology , COVID-19 , Canada , Cell Line , Chlorocebus aethiops , Contact Tracing , Coronavirus Infections , Cytopathogenic Effect, Viral , DNA, Complementary/genetics , DNA, Viral/genetics , England , Genome, Viral , HeLa Cells , Humans , Iran , Kuwait , Macaca mulatta , Nasopharynx/virology , Phylogeny , Pneumonia, Viral , SARS-CoV-2 , Sequence Analysis, DNA , Travel , Turkey/epidemiology , Vero Cells , Virus Replication
11.
Euro Surveill ; 25(25)2020 06.
Article in English | MEDLINE | ID: covidwho-649992

ABSTRACT

The advent of COVID-19, has posed a risk that human respiratory samples containing human influenza viruses may also contain SARS-CoV-2. This potential risk may lead to SARS-CoV-2 contaminating conventional influenza vaccine production platforms as respiratory samples are used to directly inoculate embryonated hen's eggs and continuous cell lines that are used to isolate and produce influenza vaccines. We investigated the ability of these substrates to propagate SARS-CoV-2 and found that neither could support SARS-CoV-2 replication.


Subject(s)
Chickens/immunology , Coronavirus/physiology , Influenza Vaccines/immunology , Influenza, Human/prevention & control , Madin Darby Canine Kidney Cells , Receptors, Virus/metabolism , Virus Cultivation/methods , Virus Replication , Animals , Betacoronavirus , COVID-19 , Cell Line , Chickens/virology , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Dogs , Eggs , Humans , Pandemics , Pneumonia, Viral , SARS-CoV-2 , Severe Acute Respiratory Syndrome
12.
J Mol Diagn ; 22(7): 871-875, 2020 07.
Article in English | MEDLINE | ID: covidwho-630204

ABSTRACT

As the coronavirus disease 2019 (COVID-19) pandemic sweeps across the world, the availability of viral transport medium (VTM) has become severely limited, contributing to delays in diagnosis and rationing of diagnostic testing. Given that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA has demonstrated stability, we posited that phosphate-buffered saline (PBS) may be a viable transport medium, as an alternative to VTM, for clinical real-time quantitative PCR (qPCR) testing. The intra-individual reliability and interindividual reliability of SARS-CoV-2 qPCR were assessed in clinical endotracheal secretion samples transported in VTM or PBS to evaluate the stability of the qPCR signal for three viral targets (N gene, ORF1ab, and S gene) when samples were stored in these media at room temperature for up to 18 hours. We report that the use of PBS as a transport medium allows high intra-individual and interindividual reliability, maintains viral stability, and compares with VTM in the detection of the three SARS-CoV-2 genes through 18 hours of storage. This study establishes PBS as a clinically useful medium that can be readily deployed for transporting and short-term preservation of specimens containing SARS-CoV-2. Use of PBS as a transport medium has the potential to increase testing capacity for SARS-CoV-2, aiding more widespread screening and early diagnosis of COVID-19.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , RNA, Viral/analysis , Saline Solution/chemistry , Specimen Handling/methods , Virus Cultivation/methods , COVID-19 , COVID-19 Testing , Coronavirus Infections/prevention & control , Coronavirus Infections/virology , Humans , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Pneumonia, Viral/virology , Predictive Value of Tests , Preservation, Biological , RNA, Viral/genetics , SARS-CoV-2
13.
Virology ; 548: 39-48, 2020 09.
Article in English | MEDLINE | ID: covidwho-597506

ABSTRACT

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is the agent responsible for the coronavirus disease 2019 (COVID-19) global pandemic. SARS-CoV-2 is closely related to SARS-CoV, which caused the 2003 SARS outbreak. Although numerous reagents were developed to study SARS-CoV infections, few have been applicable to evaluating SARS-CoV-2 infection and immunity. Current limitations in studying SARS-CoV-2 include few validated assays with fully replication-competent wild-type virus. We have developed protocols to propagate, quantify, and work with infectious SARS-CoV-2. Here, we describe: (1) virus stock generation, (2) RT-qPCR quantification of SARS-CoV-2 RNA; (3) detection of SARS-CoV-2 antigen by flow cytometry, (4) quantification of infectious SARS-CoV-2 by focus-forming and plaque assays; and (5) validated protocols for virus inactivation. Collectively, these methods can be adapted to a variety of experimental designs, which should accelerate our understanding of SARS-CoV-2 biology and the development of effective countermeasures against COVID-19.


Subject(s)
Betacoronavirus/physiology , Virus Cultivation/methods , Virus Inactivation , Animals , Antigens, Viral/analysis , Betacoronavirus/genetics , Betacoronavirus/growth & development , Betacoronavirus/immunology , Cell Line , Chlorocebus aethiops , Containment of Biohazards , Culture Media , Flow Cytometry , RNA, Viral/analysis , Rats , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2 , Vero Cells , Viral Plaque Assay , Virus Replication
14.
J Infect Public Health ; 13(7): 901-905, 2020 Jul.
Article in English | MEDLINE | ID: covidwho-548405

ABSTRACT

At present the whole world is facing pandemic of the Coronavirus disease (COVID-19); caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This disease has rapidly spreads across the world from its origin of Wuhan, China and affected millions people worldwide and make them to remain in their homes. The knowledge of available laboratory methods is essential for early and correct diagnosis of COVID-19 to identify new cases as well as monitoring treatment of confirmed cases. In this review we aim to provide the updated information about selection of specimens and availability of various diagnostic methods and their utility with current findings for the laboratory diagnosis of SARS-CoV-2 infection. This will guide the healthcare professionals and government organizations to make strategy for establishing diagnostic facilities for SARS-CoV-2 infections.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Immunoassay/methods , Pneumonia, Viral/diagnosis , Real-Time Polymerase Chain Reaction/methods , Virus Cultivation/methods , COVID-19 , Coronavirus Infections/prevention & control , Coronavirus Infections/virology , Humans , Nasopharynx/virology , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Pneumonia, Viral/virology , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2 , Specimen Handling
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