Logistics for Rapid Isolation of Viruses From Humans
Abstract
An important aspect of microbiological surveillance is the ability to access live viruses for microneutralization assays, which enables the study of viral characteristics and mechanisms in vitro and production of positive controls for diagnostic methods. During the COVID-19 pandemic, the Public Health Agency of Sweden established a protocol for the rapid collection of clinical samples and subsequent isolation of novel virus variants.
Introduction
The COVID-19 pandemic has shown the profound impact of infectious disease outcomes on our society, not only for individuals at risk but also for the healthcare system.1 Intrinsically, all lessons learned related to pandemic preparedness—defined by the World Health Organization as “a continuous process of planning, exercising, revising and translating into action national and sub-national pandemic preparedness and response plans”2—should be prioritized by healthcare practitioners.
In January 2020, the Public Health Agency of Sweden (PHAS) and the national CBRN (chemical, biological, radioactive, and nuclear) Defence Centre, which is part of the Swedish Armed Forces, conducted a multiagency high-readiness exercise comprising the rapid deployment and diagnostic adaptation of a mobile biological field analysis laboratory.3 Apart from establishing diagnostic capability, the exercise resulted in the creation of mobile sample support teams. These teams were specifically trained in biosafety and could therefore safely collect and transport samples acquired through self-sampling.
The Swedish Armed Forces supported the PHAS from March 13, 2020, to January 31, 2023, with the logistic capability of the mobile sample support teams. Additionally, the National CBRN Defence Centre provided a command and control function in support of the PHAS. The main scope was for the Swedish Armed Forces to provide support for sampling as well as national cross-sectional surveys and similar surveys and studies of importance to PHAS.4
The COMMUNITY (COVID-19 Immunity) Study is one such important study, which began in April 2020 at Danderyd Hospital in Stockholm. This longitudinal study enrolled 2,149 healthcare workers who were followed for frequent measurement of SARS-CoV-2 antibodies and recurrent polymerase chain reaction (PCR) screening. Because symptomatic healthcare workers were unable to leave their PCR sample at the hospital due to the risk of contagion, the Armed Forces mobile sample support teams collected participants’ samples in their homes; this improved the participation rate in the COMMUNITY Study.5
Lessons Learned on Pandemic Preparedness
One of our main lessons learned during the COVID-19 pandemic comes from establishing a protocol for rapid isolation of novel SARS-CoV-2 subvariants. We initially used this protocol in December 2021 to isolate a SARS-CoV-2 Omicron BA.1 subvariant. We further developed the protocol by establishing a standard operating procedure and refined it in conjunction with 2 studies conducted in 2022.6,7 The current version of the protocol is presented in the Table.
Step | Description |
---|---|
Contact | Rapidly identify individuals possibly infected with a new SARS-CoV-2 variant. Contact each individual and ask if they would be willing to participate in a study by donating a sample. If they reply yes, obtain informed consent. |
Sample | Quickly enable the participant to self-sample by swabbing in oropharynx, lower nasal cavity, and saliva. Use mobile sample support teams to transport sampling materials and samples. |
Register | Register and store samples at the Public Health Agency of Sweden. |
Isolate | In a BSL-3 laboratory, inoculate samples on cells for 10 days or until strong CPE. Visually inspect cells daily for CPE using microscope. When CPE is noted, sample supernatants for qPCR for at least 2 consecutive days (with change of medium). If no CPE is observed, collect supernatants at day 10 for qPCR. |
Culture | Store supernatants from samples showing CPE at -80°C (passage 1). Inoculate part of the supernatant onto fresh cells in T175 flasks for growth of virus stock (passage 2). |
Sequence | Perform whole genome sequencing of the virus stock (passage 2) as well as the original sample to identify virus variant and possible mutations developed during the passage of the virus in cells. |
Abbreviations: BSL-3, biosafety level 3; CPE, cytopathic effect; qPCR, quantitative polymerase chain reaction.
We have also learned lessons from previous pandemics, particularly within 2 research fields: host immune responses and viral pathogenesis. These fields enable the detection of emerging novel virus strains in the populace and basic research on virus evolution.8
In addition, surveillance is a core component of disease prevention and control that benefits health security. Active surveillance can contribute to identifying risk profiles, which allows for adequate resource mobilization and health system planning.1 Furthermore, rapid sharing of biological materials at a global level is key; the World Health Organization BioHub System addresses this need.9
Challenges to Applying Lessons Learned
Although current dominant Omicron subvariants are believed to be less pathogenic than earlier variants, parts of the population are still vulnerable (eg, due to an inability to produce neutralizing antibodies). Adding to the challenge is the risk that novel and more pathogenic SARS-CoV-2 variants may emerge, as coronaviruses have very high mutation rates.10 To better understand the pathogenicity of a new virus (or a viral variant) and host immunity against it, rapid access to live viruses is crucial during an outbreak. Microneutralization tests (MNTs)—the gold standard for detecting neutralizing antibodies and evaluating the potential of a novel virus (or variant) to evade immune responses—requires a live virus.
However, several obstacles need to be overcome to allow for rapid isolation of novel viruses and the use of MNTs. First, PCR diagnostics are often performed after inactivation of samples, which precludes the use of these samples for isolation attempts. Second, to increase the possibility of successful isolation of a novel viral strain from a patient, the turnaround time from sampling to inoculation of samples on a cell culture in a laboratory should be as short as possible; if possible, freeze-thawing of the samples prior to isolation should be avoided. Third, work with Risk Group 3 viruses (or a new pathogenic and yet unclassified virus) requires a biosafety level 3 laboratory and trained personnel. Fourth, well-established protocols for isolation of new virus isolates and for MNTs are needed.11 Finally, potential hosts/carriers of a novel virus or variant need to be available; otherwise, it might take a long time before the immune response can be analyzed.
Application of the PHASProtocol
The COVID-19 pandemic has been characterized by the emergence of novel SARS-CoV-2 variants and their subvariants, as exemplified by the rapidly evolving Omicron.7,12 This accentuated the need to promptly understand the virus’ potential evasion of immune responses. From the PHAS’ perspective, the deployed mobile sample support teams and access to an available cohort through the COMMUNITY Study provided an opportunity to rapidly isolate new virus variants for MNTs. The PHAS promptly initiated its protocol in June 2022, which encompassed a solution of all above mentioned challenges related to MNTs, when Omicron emerged; however, with the variant’s continued spread, the need for MNTs was less relevant.
The PHAS invited 1,412 of the COMMUNITY Study participants to complete a screening survey to analyze point prevalence and identify circulating variants. These healthcare workers were asked if they had tested positive for SARS-CoV-2 in an antigen and/or PCR test within the previous 5 days.13 Nine participants who reported being SARS-CoV-2 positive were included for sampling after providing informed consent.
Upon activation of the protocol, samples were collected at participants’ homes using appropriate biosafety measures. The samples were transported to a biosafety level 3 laboratory and stored overnight in a refrigerator. Sample collection was conducted within 24 hours after initial participant contact. All samples were then inoculated onto A549-ACE2 cells (Figure) and incubated at 5% CO2, 37°C, and 100% humidity up to 10 days in a Sanyo MCO-20AIC CO2 Incubator (Sanyo Electric Biomedical Co., Ltd., Tokyo, Japan).

Five of the 9 samples caused cytopathic effect, observed 3 to 5 days post-inoculation. Supernatants (Dulbecco’s Modified Eagle’s Medium [Thermo Fisher Scientific, Bothell, Washington] supplemented with 7.5% fetal bovine serum, 10X antibiotic-antimycotic, 0.6 μg/mL penicillin, 60 μg/mL streptomycin, 2mM L-glutamine, 20mM HEPES) (passage 1) were collected from these cells and used for propagation of viral stocks (passage 2). Aliquots of passage 1 were also stored at −80°C as a backup for later propagation of stocks. The PHAS’ subsequent sequencing of passage 1 and 2 identified 5 SARS-CoV-2 variants based on Phylogenetic Assignment of Named Global Outbreak Lineages (pangolin) version 4.1.3, pangolin-data version 1.15.1 (SARS-CoV-2 lineages, Edinburgh, United Kingdom): BA.4.1, BA.5.1, BA.5.2, BA.5.2.1, and BE.1.
Conclusion
The implementation of this protocol for rapid isolation of a virus, alongside the availability of logistic capacity with strong knowledge of biosafety and a readily available cohort of participants who had possibly been exposed to the new variant, facilitated the rapid and successful isolation of the Omicron subvariant BA.5 in Sweden. Our success is based on a multisectoral approach to address the challenge of possible immune evasion from a new variant. All required assets were already in place when BA.5 emerged in Sweden, and sampling commenced just hours after the decision to activate the protocol was made. This cooperation illustrates how a multidisciplinary and multisectoral approach can be used to address a complex public health challenge, which cannot fully be managed by one actor alone.
We propose further study of multisectoral cooperation at national levels to address specific pandemic-related challenges. Identified theoretical synergetic effects should be tested in multisectoral readiness exercises, applying our protocol along with adequate sampling support and relevant cohorts to test. Where successful, our protocol for isolation of new virus variants can be implemented in national response plans aimed at pandemic preparedness and resilience for emerging threats.
Acknowledgments
We would like to thank all the participants in the survey for volunteering to perform self-sampling.
References
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© The Author(s) 2024. Published by Mary Ann Liebert, Inc.
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History
Published online: 21 October 2024
Published in print: September/October 2024
Published ahead of print: 18 September 2024
Accepted: 4 December 2023
Revision received: 1 November 2023
Received: 22 July 2023
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