Enterovirus and Acute Flaccid Myelitis Research Materials

Figure 1: Human rhabdomyosarcoma (RD) cells (ATCC® CCL-136™) infected with EV-D68, US/MO/14-18947 (BEI Resources NR-49129) showing signs of cytopathic effect in the right panel. The left panel shows mock infected cells. Magnification 10x brightfield.

In support of Enterovirus D68 (EV-D68) vaccine development research, BEI Resources has performed initial screening of cell lines amenable for human vaccine development. The following study assesses the suitability of cell lines for virus propagation in the presence of serum and after adaptation to serum free (SF) conditions. A guidance on how to adapt cell lines to serum free media is provided.

Enterovirus and Acute Flaccid Myelitis
Enterovirus D68 (EV-D68) has reemerged as a cause of severe respiratory infections in young children around the world and as a newly emerged neuropathogenic threat. First isolated in the United States (US) from children with serious respiratory illness in 19621, only sporadic clusters of EV-D68 infection were reported globally in the last decade2 until 2014 when an unusually large outbreak occurred in the US. During this outbreak (and in subsequent 2016 and 2018 outbreaks), a subset of children with laboratory confirmed EV-D68 related severe respiratory illness also developed an acute flaccid myelitis (AFM). Detection of virus in the spinal fluid of AFM affected children3 is rare but recently, researchers have found new evidence linking AFM to enteroviruses due to the presence of higher levels of EV specific antibodies in cerebrospinal fluid of children with AFM compared to non-AFM controls4,5.

BEI Resources Screening of Cell Lines for EV-D68 Propagation in Media Supplemented with Serum
Typically, EV-D68 is propagated in human rhabdomyosarcoma (RD) cells (ATCC® CCL-136™) at 33°C. However, this cell line is not amenable for vaccine production and additionally is maintained in serum containing media. Consequently, BEI Resources has performed an initial screen of suitable cell substrates for EV-D68 vaccine development. Five cell lines were tested for EV-D68 susceptibility and production rates to assess the successful propagation using two EV-D68 isolates originally grown in RD cells that are available in BEI Resources (see Table 1 for details).

Prior to infection, the cell lines were maintained at 37°C. Viral growth media contained 2% fetal bovine serum (FBS) (ATCC® 30-2020™) and the ATCC® recommended base media for each cell line (Table 1). The cells were infected with each viral strain using a multiplicity of infection (MOI) of 0.01 and incubated at 33°C. When approximately 90% of the cells showed cytopathic effect (CPE), the virus was harvested by scraping the monolayer into the supernatant. The infected cell lysate was clarified by centrifugation at 2400g and the pellet underwent three freeze-thaw cycles to release the virus which was then added back to the supernatant. The viruses were titrated in RD cells by the 50% Tissue Culture Infectious Dose (TCID50) method and calculated by the Reed-Muench method. If a virus infected cell line displayed no signs of CPE after an extended incubation period of 12-14 days, the absence of virus was confirmed by performing an immunofluorescence assay (IFA) with a virus specific antibody. Furthermore, they underwent two blind passages with extended incubation to confirm that the cell line did not support growth of the virus. All infected cell lines were subjected to three total serial passages with the second and third passage utilizing a 1:100 dilution of virus inoculum in media rather than MOI. For a direct comparison of virus susceptible cell lines using MOI for passage 1, virus titration was performed RD cell line (Table 1).

The cells lines A549, MRC-5 and HEK-293 T/17 support growth of both EV-D68 viral isolates. These results are comparable to the same viral isolates grown in RD cells. HEK-293 cells supported growth of US/MO/14 18947 but showed reduced virus fitness for the three passages tested and did not support growth of the US/KY/14-18953 virus. CHO cells expressing the ICAM-1 receptor was not susceptible to the two isolates used in the cell screening assessments.
 

Table 1. Susceptibility of five cell lines growth in media containing 2% FBS using two EV-D68 isolates

Cell line

Virus growth media
(Base Media) + 2% FBS

Virus isolates *

Passage 1 Titer
(in RD cells)

 Control: RD cells

 (ATCC® CCL-136™)

 DMEM

 (ATCC® 30-2002)

 US/MO/14-18947

 1.58 x107

 US/KY/14-18953

 1.58x107

 MRC-5

 (ATCC® CCL-171™)

 EMEM

 (ATCC® 30-2003)

 US/MO/14-18947

 1.58x107

 US/KY/14-18953

 8.89x106

 A549

 (ATCC® CCL-185™)

 F-12K

 (ATCC® 30-2004)

 US/MO/14-18947

 1.58x107

 US/KY/14-18953

 8.89x105

 HEK-293 T/17

 (ATCC® CRL-11268™)

 DMEM

 (ATCC® 30-2002)

 US/MO/14-18947

 2.81x107

 US/KY/14-18953

 1.58x106

 HEK-293

 (ATCC® CRL-1573™)

 EMEM

 (ATCC® 30-2003)

 US/MO/14-18947

 4.5x104

 US/KY/14-18953

 No growth

 CHO-ICAM-1

 (ATCC® CRL-2093™)

 RPMI-1640

 (ATCC® 30-2001)

 US/MO/14-18947

 No growth

 US/KY/14-18953

 No growth

*US/MO/14-18947 is available in the BEI Resources catalog as NR-49129 and US/KY/14-18953 as NR-49132


BEI Resources Screening of Cell Lines for EV-D68 Propagation in Serum Free Media
BEI Resources performed a second screen of cell lines under serum free conditions to assess the growth of EV-D68 . Four of the cell lines (MRC-5, A549 and HEK-293 T/17 and 293) were adapted to grow in serum free conditions by weaning serum and switching into chemically defined media with supplements . All the cell lines were grown in viral growth media (base media recommended for each cell line) supplemented with 2% FBS to serve as controls. An additional control included use of RD cells grown in base media with 2% serum. (see Table 2 for details)

Serum Free (SF) adapted cell lines were grown in appropriate growth media at 37°C and infected with the EV-D68 isolate US/MO/14-18947 at a MOI of 0.001. All cell lines were incubated at 33°C for three passages with each successive passage infected at MOI of 0.001. All control cell lines were similarly infected. At the end of each passage virus from all cell lines were titrated (see Table 2). Note that viral titers were determined by a standard assay using RD cells.
 

Table 2. Comparison of EV-D68 growth in cell lines adapted to serum free conditions
Cell Line Media Titration in RD Cells
Passage 1 Passage 2 Passage 3

 Control: RD cells

 DMEM (+2% FBS)  1.58x107  8.89x106  5.0x106

 MFC-5

 

 EMEM (+2% FBS)  8.89x106  5.0x106  1.58x106
 SF media: PC-1 media  2.81x106  1.58x106  1.58x106

 A549

 

 F-12K (+2% FBS)  2.81x107  1.58x107  2.81x107
 SF media: PC-1 media  1.58x107  2.81x106  2.81x106
 HEK-293 T/17

 DMEM (+2% FBS)

 8.89x107  2.81x107  1.58x107

 SF media: EX-CELL®
 293 Serum-Free Media

 2.81x103  8.89x105  2.81x106

 

Infection and viral production of the EV-D68 US/MO/14-18947 virus isolate was successful over the course of three passages in serum free conditions for three cell lines, A549, MRC-5, and HEK-293 T/17, however HEK-293 did not support the growth of the virus. The CPE for HEK-293 T/17 in SF adapted cells is difficult to discern as the cells lose anchorage dependence. As a result, confirmation of viral infection in HEK-293 T/17 SF adapted cells requires IFA.

Summary of BEI Resources Cell Screening for EV-D68 Propagation
Using two EV-D68 isolates, it was observed that two cell lines A549 and MRC-5 successfully supported virus growth after adaptation to serum-free conditions for use in downstream vaccine developmental needs. Both the cell lines were found to be robust and tolerated the serum weaning process while maintaining their morphology and viability.

For guidance and protocols on serum free adaptation of cell lines A549, MRC-5, HEK-293 and HEK-293 T/17, please visit our Knowledge Base.

For information on the availability of virus isolates grown in serum free A549 adapted cells, please email Contact@BEIResources.org.
 

To support your Enterovirus research, BEI Resources has several isolates available
NR-51430 Enterovirus D68, Fermon          
NR-51844 Enterovirus D68, USA/MO-18949 mouse-adapted
NR-51845 Enterovirus 71, Tainan/4643/98 mouse-adapted          
NR-51996 Enterovirus D68, USA/2018-23087          
NR-51997 Enterovirus D68, USA/2018-23088          
NR-51998 Enterovirus D68, USA/2018-23089 
NR-51999 Enterovirus A71, USA/WA/2016-19522
NR-52000 Enterovirus A71, USA/2018-23092
NR-52009 Plasmid pBR322 Containing cDNA from Enterovirus D68, US/MO/14-18947, Infectious Clone pBR-49129
NR-52010 Plasmid pUC19 Containing cDNA from Enterovirus D68, US/MO/14-18949, Infectious Clone pUC-49130
NR-52011 Plasmid pUC19 Containing cDNA from Enterovirus D68, US/IL/14-18952, Infectious Clone pUC-49131
NR-52013 Enterovirus D68 US/MO/14-18947 (produced in serum-free A549 cells)
NR-52268 Homo sapiens Lung Carcinoma Cells (A549), Serum-Free
NR-471 Human Enterovirus 71, Tainan/4643/1998
NR-472 Human Enterovirus 71, MP4
NR-49129 Enterovirus D68, US/MO/14-18947
NR-49130 Enterovirus D68, US/MO/14-18949
NR-49131 Enterovirus D68, US/IL/14-18952
NR-49132 Enterovirus D68, US/KY/14-18953
NR-49133 Enterovirus D68, US/IL/14-18956
NR-4960 Genomic RNA from Human Enterovirus 71, Tainan/4643/1998
NR-4961 Genomic RNA from Human Enterovirus 71, MP4
NR-49134 Genomic RNA from Enterovirus D68, US/MO/14-18947
NR-49135 Genomic RNA from Enterovirus D68, US/MO/14-18949
NR-49136 Genomic RNA from Enterovirus D68, US/IL/14-18952
NR-49137 Genomic RNA from Enterovirus D68, US/KY/14-18953
NR-49138 Genomic RNA from Enterovirus D68, US/IL/14-18956

 

Coming Soon - Available in Spring of 2020
NR-52014        Enterovirus D68, US/KY/2014-18953 (serum free A549 cells)
NR-52015        Enterovirus D68, USA/2018-23087 (serum free A549 cells)
NR-52016        Enterovirus D68, USA/2018-23088 (serum free A549 cells)
NR-52017        Enterovirus D68, USA/2018-23089 (serum free A549 cells)
NR-52375        Plasmid, Enterovirus D68, USA/Fermon (molecular clone)
NR-52376        Plasmid, Enterovirus D68, USA/1989-23220 (molecular clone)
NR-52377        Plasmid, Enterovirus D68, USA/2010-23230 (molecular clone)
NR-52378        Plasmid, Enterovirus D68, USA/2016-19504 (molecular clone)
NR-52379        Plasmid, Enterovirus D68, USA/2018-23088 (molecular clone
NR-52380        Plasmid, Enterovirus D68, USA/2018-23252 (molecular clone)
 

The following isolates obtained from AFM cases will be available in Summer of 2020
Enterovirus D68, USA/2018-23201
Enterovirus D68, USA/2018-23263
Enterovirus D68, USA/2018-23206
Enterovirus D68, USA/2018-23209
Enterovirus D68, USA/2018-23216


References

1.            Schieble JH et al. 1967. A probable new human picornavirus associated with respiratory diseases. Am J Epidemiol 85:297–310.

2.            Eshaghi A et al. 2017. Global Distribution and Evolutionary History of Enterovirus D68, with Emphasis on the 2014 Outbreak in Ontario, Canada. Front Microbiol. 8:257.

3.            https://www.cdc.gov/non-polio-enterovirus/about/ev-d68.html#seasonal-circulation

4.            Mishra N et al. 2019. Antibodies to enteroviruses in cerebrospinal fluid of patients with acute flaccid myelitis. mBio 10:e01903-19.

5.            Schubert RD et al. 2019. Pan-viral serology implicates enteroviruses in acute flaccid myelitis. Nat Med 25: 1748–1752

 

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