Why should I test my CHO cell culture for virus infections?


The answer is:

CHO cells can be infected in bioreactors with viruses such as Vesivirus 2117!

The presence of a virus in a bioreactor can inflict major losses for biotech and pharmaceutical companies involved in the manufacturing of protein based biologicals and pharmaceuticals.

Contaminations of bioreactors with Vesivirus 2117

are detrimental to the production of biologicals.

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To avoid cross-contamination it is therefore a smart idea to test all cell cultures for the presence of viruses routinely during key production steps. The design and synthesis of specific molecular probes can help with this task.

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Cell Line Authentication at Biosynthesis Inc.

Call us at 1-800-227-0627 or you can visit our website at “www.biosyn.com

Cell Line Authentication

The Genzyme case illustrates why regular testing is a smart idea !

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In 2009 Genzyme Corp., the Cambridge biotechnology giant has detected a virus at its Allston plant that has forced it to halt the production at its manufacturing plant.

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The contamination was caused by a virus strain called “Vesivirus 2117”.

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Let me cite the news from BioPharm in June 17, 2009 here:

“Genzyme Corporation has halted production at its manufacturing plant in Allston Landing, MA, because it has detected a virus that impairs cell growth in one of the six bioreactors there, the company announced yesterday. The company will sanitize and fumigate the facility, and expects to resume production by the end of July.

The virus strain, Vesivirus 2117, is known to interfere with the growth of Chinese hamster ovary (CHO) cells and is believed to have been introduced through a cell culture nutrient. Genzyme confirmed that this virus was the cause of declines in cell productivity last year at its facilities in Allston and Geel, Belgium, which were subsequently addressed.

The company was able to detect the virus in this case using a PCR assay it developed after standard tests were unable to identify the cause of the previous productivity declines. Genzyme has added this assay to its standard screening.

The virus, a member of the calicivirus family, has not been shown to cause human infection, and the company was unable to infect any of three human cell lines that it tested. Nonetheless, the US Food and Drug Administration has asked the company to run the assay on finished lots. The company is also in contact with the EMEA and other regulatory authorities around the world.”

A swift identification of the exact root or cause for a contamination of a bioreactor is of paramount importance to avoid losses during the production of biological products such as monoclonal antibodies and/or other recombinant proteins that use Chinese hamster ovary (CHO) cell cultures for their production. This is critical to determining needed appropriate corrective and preventive actions in real time.

The use of specific cell culture infectivity assays allows identification of the presence of a viral contamination. Ultimately PCR-based methods allow to determine the nature of the virus type and to distinguish the contamination from other cytoxic agents.However, when the traditional strategies fail a mass spectrometry-based proteomic technique may be needed to identify the cause of the infection.

Qiu and coworkers were able to identify and quantify Vesivirus 2117 particles in complex bioreactor fluids using a mass spectrometry-based proteomic technique. The Genzyme research group reported the identification and quantitation of Vesivirus 2117 particles in bioreactor fluid from infected Chinese hamster ovary cell cultures using global protein sequencing by mass spectrometry in combination with multi-dimensional liquid-chromatography.  The researchers were able to identify six virus specific peptides following mass spectrometric data acquisition and rigorous data analysis. The peptides were found to be fragments of two structural proteins, the capsid protein pre-cursor and a small structural protein, from the same species, the Vesivirus 2117. The use of stable heavy isotope-labeled peptides as internal standards allowed the researchers to determine the absolute concentration of the Vesivirus particles in the bioreactor fluid as well as the ratio of two capsid proteins (VP1:VP2) in the particles as approximately 9:1. Finally, the scientists confirmed the identification of the Vesivirus 2117 by RT-PCR. 

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The combination of newer, more sensitive testing methods, such as the use of mass spectrometry- combined with more traditional and PCR-based assays, allow for the detection and identification of new types of viruses that are potentially infecting cell cultures.

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Furthermore if the genomic sequences of viruses are available global protein sequencing using ultra-sensitive mass spectrometry can survey for multiple infectious agents simultaneously. In addition unique information regarding genomic variation of the isolate contaminations, e.g a virus, may also be obtained. Therefore MS-based proteomics should be considered as an additional or alternative tool together with other existing test methods when a bioreactor contamination occurs.

The vesivirus belongs to the family of calciviruses and is a positive-sense, single-stranded RNA virus. The calcivirus family includes the Vesivirus, Sapovirus, Lagovirus, and Norovirus. They infect a wide variety of mammals and can cause an array of different diseases. The feline vesivirus, calicivirus (FCV), is associated with a range of conditions such as upper respiratory tract disease in cats, while noroviruses and sapoviruses are reported to cause gastroenteritis. The human noroviruses represent a major health problem as well. It is estimated that this virus is responsible for up to 21 million cases of gastroenteritis in the United States every year and 200,000 deaths among children in developing countries.

 Vesivirus structure

(Source: http://www.caliciviridae.com/vesivirus/vesivirus.htm)

The calicivirus genome of approximately 7.5 kilobases contains 2 to 4 open reading frames (ORFs). The exact nature of the ORFs depend on the type of genus. The major (VP1; ORF2) and minor (VP2; ORF3) capsid proteins of caliciviruses are usually translated from a subgenomic RNA. ORF1 encodes a large polyprotein precursor that is cleaved into mature nonstructural proteins NS1 to NS7 by the virus-encoded protease NS6pro. The mature proteins include an RNA-dependent RNA polymerase (RdRp) (NS7pol), an ATPase (NS3), and proteins that disrupt cellular trafficking (NS1-2 and NS4). Furthermore, proteolytic cleavage of the ORF1 polyprotein releases the NS5 protein, which is also known as VPg (viral protein genome linked). This caliciviral VPg is a 13- to 15-kDa protein that is found covalently attached to the 5’ terminus of the genomic and subgenomic RNAs. Covalently attached terminal proteins are also found at the 5’ ends of the genomes of other positivesense RNA viruses, including the Picornaviridae and Astroviridae, which are mammalian viruses, and several plant virus families, such as the Potyviridae, Comoviridae, and Nepoviridae, and they also occur in DNA viruses in the Adenoviridae and in members of the bacteriophage Podoviridae. In addition the VPg proteins of different viruses vary in size and sequence. 

The known virus genomes are available from the NIH/NCBI database:

Steller sea lion vesivirus: Chromosome: 1 ID: 6255 

Rabbit vesivirus: Chromosome: 1ID: 5867

Mink calicivirus: Chromosome: 1 ID: 15756

Feline calicivirus: Chromosome: 1 ID: 5044

Vesicular exanthema of swine virus: Chromosome: 1ID: 4888

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Below is an example of a PCR-based method useful for the identification of virus contamination:

Many real-time RT-PCR based detection methods have been developed in recent years and the scientific literature provides a voluble source to find examples that can be used as a starting point for the design of molecular probes needed for the identification assays.

Steps involved in this process of the identification of a new virus strain are: 

1.     Sample preparation

2.    RNA extraction

3.     cDNA synthesis

4.     Design of primer pairs and probes such as TagMan major groove binder (MGB) probes

5.     Plasmid preparation as a standard to calibrate the assay

6.     Development of the RT-qPCR assay

7.     And finally execution of the assay

References

Eoin N. Leen, K. Y. Rex Kwok, James R. Birtley, Peter J. Simpson, Chennareddy V. Subba-Reddy, Yasmin Chaudhry, Stanislav V. Sosnovtsev, Kim Y. Green, Sean N. Prater, Michael Tong, Joanna C. Young, Liliane M. W. Chung, Jan Marchant, Lisa O. Roberts, C. Cheng Kao, Stephen Matthews, Ian G. Goodfellow, Stephen Curry; Structures of the Compact Helical Core Domains of Feline Calicivirus and Murine Norovirus VPg Proteins. Journal of Virology p. 5318–5330.

Mingxiao M, Jinhua L, Yingjin S, Li L, Yongfei L (2013) TaqMan MGB Probe Fluorescence Real-Time Quantitative PCR for Rapid Detection of Chinese Sacbrood Virus. PLoS ONE 8(2): e52670. doi:10.1371/journal.pone.0052670.

Yongchang Qiu, Nathan Jones, Michelle Busch, Peng Pan, Jesse Keegan, Weichang Zhou, Mark Plavsic, Michael Hayes, John M. McPherson, Tim Edmunds, Kate Zhang, Robert J. Mattaliano; Identification and Quantitation of Vesivirus 2117 Particles in Bioreactor Fluids From Infected Chinese Hamster Ovary Cell Cultures. Biotechnol. Bioeng. 2013;110: 1342–1353.

Biosynthesis Incorporated

has 28 years experience in the analysis and synthesis of natural and

synthetic peptides, proteins, DNA and RNA oligonucleotides, bioconjugates, DNA and RNA probes

and other biomolecules of interest.

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Please call us at 1-800-227-0627

or visit our website at

www.biosyn.com

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We are eager to help you with all your scientific research needs!

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Categories: Bioanalysis, Bioinformatics, cell culture, Cell Development, Cell line authentication, cell line cross contamination, Cell Line Identification, Cell lines, DNA Analysis, FISH Probes, Mass spectrometry, Molecular Probes, Next-generation sequencing, Nucleoprotein, Oligonucleotide Synthesis, peptides, RNA, RNA Editing, RNA Synthesis, Sequencing, Synthesis, Synthetic peptides, Virus

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