An Outline of Testing Procedures Used for the Detection of Porcine

Circoviruses in Cell Substrates and Starting Materials

David Onions, PhD MRCVS FMedSci FRSE

Chief Scientific Officer

Technical Brief

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An Outline of Testing Procedures Used for the Detection of Porcine Circoviruses 1

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IntroductionThe purpose of this document is to indicate how

BioReliance currently tests cell substrates and raw

materials for freedom from contamination by porcine

circovirus 1 & 2 (PCV1 & 2), including additional

custom approaches that may be of value in specific

circumstances.

Porcine Circovirus (PCV) is a member of the Circoviridae,

a family of small unenveloped, closed circular ssDNA,

viruses. PCV-1 is ubiquitous in pig populations world-

wide and appears to be non-pathogenic. In contrast,

pathogenic forms of PCV-2 have emerged over the

last 2 decades and are believed to be a major causal

agent of the economically important, post weaning

multisystemic wasting syndrome. While PCV 1 is regarded

as non pathogenic in pigs, there are grounds for caution

in assuming that it will always be non-pathogenic in

other species. There are many examples of viruses that

are non pathogenic in their hosts but which cause

serious diseases in heterologous hosts, for instance the

non-pathogenic ovine herpesvirus OHV-2 causes fatal,

malignant catarrhal fever in cattle. Secondly, single

stranded DNA viruses have shown a propensity to

change hosts and pathogenicity following relatively few

mutations, as evidenced by the evolution of the canine

parvovirus, the Kresse strain of porcine parvovirus and

the emergence of pathogenic PCV-2 strains.

PCV has been reported to infect Vero and HeLa cells as

well as porcine, bovine and ovine cells (Allan et al 1994,

Hattermann et al 2004, Kiupel et al 2005). The ability of

PCV to infect pluripotent cells should be considered

when evaluating the safety of products derived from

stem cells (Steiner et al 2008). PCV-1 often establishes

persistent, non-cytopathic infections and most banks

of the widely used PK-15 cells are infected. In contrast,

PCV-2 can be cytopathic, for instance in HeLa cells.

It should be noted that a bovine circovirus has been

described that has high nucleotide similarity to PCV-

2 strains. It is not known if this virus is truly endemic

in cattle populations or results from occasional cross

species transmission. The serological data are conflicting

but, as described below, we have occasionally detected

circovirus sequences in pooled bovine serum. Very

recently a fatal haemorrhagic diathesis in calves has

been described which may be aetiologically associated

with BCV infection (Kappe et al 2010). Tischer (1995)

reported the presence of antibody cross reactive

with PCV in sera of humans, cattle and mice but,

in a more recent survey, Ellis et al (2000) did not

detect antibody to PCV-2 in veterinarians and

others exposed to infected pigs. Porcine circovirus

sequences have been detected in porcine pepsin

intended for human use (Fenaux et al 2004) but

the significance of this observation has not been

established.

BioReliance recommends testing cells for a range

of porcine viruses if they have been exposed to

porcine trypsin at any point in their history; these

2 An Outline of Testing Procedures Used for the Detection of Porcine Circoviruses

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viruses are listed in the document Porcine Viruses of

Concern in Biotechnology. Trypsin is widely used in

tissue culture but is also employed to activate certain

viruses and, in the case of rotaviruses, infectivity is

probably dependent on trypsin treatment of the virus.

Proteolytic activation of the haemagglutinin (HA)

protein is indispensable for influenza virus infectivity

and trypsin has been used to activate influenza virus.

Where it is known that irradiation has been used, it

may not be necessary to test for all of the viruses listed

in Porcine Viruses of Concern in Biotechnology but a

particular feature of porcine circoviruses is their high

radioresistance, as shown in Figure 1, based on a study

sponsored by Life Technologies, BioReliance’s former

parent company (Plavsic & Bolin 2001). Consequently

irradiated trypsin may still be a PCV hazard.

Circoviruses, like parvoviruses also display high

resistance to other physicochemical treatments and are

not inactivated by treatment at pH 3.0.

Recently, a new genus of closed circular ssDNA viruses

has been described, the anelloviruses. These include

TTV virus of humans and the porcine torque teno viruses.

Until recently little attention has been paid to these

viruses but tests for these viruses are now requested

by European authorities, when evaluating the safety of

cell substrates used for veterinary vaccines. Recently,

porcine torque teno viruses have been detected in a

human drug product as well as in veterinary vaccines

(Kekarainen et al 2009). In 2010 Li et al. described a

new putative genus the Cyclovirus genus with multiple

species widely distributed in animals and humans.

The role of these viruses in disease remains to be

determined.

Testing RegimesThe testing regime for cells is essentially straightforward.

BioReliance utilises a validated, real time, QPCR which

detects PCV 1 & 2 and BCV. Independent confirmation

is provided by immunofluorescence assays for viral

capsid antigens. Where necessary, further evidence of

replication can be obtained by electron microscopy,

although this is not a sensitive method for persistent

infections, and by analysis of spliced transcripts of the

replicase gene.

PCR DetectionBioReliance currently employs a validated QPCR assay

(protocol 107031GMP.BUK) for the detection of porcine

circovirus 1 & 2 and bovine circovirus. The assay is

validated to detect 100 genome copies per reaction

but trend analysis indicates it will regularly detect 10

genome copies.

Key points to be kept in mind when using PCR to detect

PCV in cell substrates are:

• Ifpossiblethecellsshouldbeattheendoflogphase

of growth when harvesting for PCR analysis.

• Porcine trypsin should not be used in the culture

period as residual PCV sequences from the high level

present in porcine trypsin can give false positives.

There is also a theoretical possibility of detection of

0

0.5

1

1.5

2

2.5

3

0 kGy 30 kGy 45 kGy

Lot 1075791

Lot 1073592

Lot 1073596

Figure 1 Inactivation of Porcine Circovirus by Gamma Irradiation

An Outline of Testing Procedures Used for the Detection of Porcine Circoviruses 3

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residual BCV sequences although, in our experience

the levels of this virus are low or absent in most

bovine sera. The cells should be washed several times

in non-serum medium and removed by scraping or

with a recombinant trypsin like Life Technologies’

Tryple™.

While there has been a recent example of a PCV positive

cell substrate used in a veterinary vaccine, we have not

detected a positive Vero cell bank. We are currently tracing

through our data but of the four most recent commercial

Vero cell banks none were positive. One of the banks

was identified as CCL81 but the origin of the other 3 was

not specified by the client. During the validation of our

assay we included CCL81 cells and they were negative

on that occasion. While sequences in porcine trypsin

are abundant, the relatively infrequent infection of non-

porcine cell lines is likely attributable to the reduced,

albeit not complete, inactivation by irradiation and

the observation that high virion numbers are required

to infect cells (Tischer et al 1987). This is supported by

findings that in vivo infection of gnotobiotic pigs can be

orders of magnitude more sensitive than in vitro assays

(Allan, Personal Communication).

Immunofluorescent detectionImmunofluorescence for capsid antigens provides

the simplest method to confirm infection of cell

substrates. We utilise both monoclonal antibodies to

PCV-2 and polyclonal antibodies reactive with PCV-1.

This assay was regularly employed in the past but was

discontinued based on commercial demand. However,

it is currently being re-established. Infected PK-15 cells

are used as a positive control with appropriate negative

cells. Key factors in using IF confirmation are:

• Thecellsmustbemitoticallyactive.

• Porcine trypsin should not be used in preparing

the cells and irradiated bovine serum or serum free

medium should be used during pre-test culture.

• Treatmentwith300mMglucosaminefor1hour,3to

6 hours after replating the cells (or after infecting the

cells) can enhance antigen expression by up to 7 fold

~20 hours later (Tischer et al 1987).

• Intensenuclearstainingisobservedinpositivecultures.

Alternative assay to detect replicating PCVWhen low level PCV PCR signals are obtained it is

possible to investigate whether active replication is

occurring through detection of splice variants of the rep

gene (Bratanich et al 2002). Replicase gene products

of 1kb, 600bp and 250bp, resulting from alternative

spliced transcripts are observed in cells with actively

replicating virus.

Analysis of raw materials for PCV & BCV and infectivity assays In our experience PCV sequences are present at a high

level in most batches of porcine trypsin. Illustrative

examples are shown in Table 1, p. 4. The positive bovine

products giving signals below the limit of quantification,

were confirmed as positive on re-amplification but

note the much lower level of sequences compared to

porcine trypsin. It is unlikely the origin of the positive

bovine serum is of relevance.

Negative IF control Positive culture

4 An Outline of Testing Procedures Used for the Detection of Porcine Circoviruses

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A standard PCR positive assay does not indicate whether

or not the genomes are encapsidated or present within

infectious particles. It is possible to evaluate :

• If the genomes are protected within capsids by

centrifugation through gradients, followed by

nuclease treatment to destroy free nucleic acid.

The capsids are then disrupted and the genomes

detected by PCR.

• Usingrollingcircleamplificationofthegenomesitis

possible to determine if the genomes are full length

or have been fragmented by irradiation.

Neither of these procedures are routine and they are

only used in investigations. An infectivity assay provides

a method of directly detecting infectious virus but there

are several factors to be considered;

• As indicated above in vitro infectivity assays are

relatively insensitive.

• Permissive cells like PCV negative clones of PK-15

cells should be used.

• Mitotically active cells are required and if an IF

endpoint is employed, glucosamine treatment

should be considered.

• PCR endpoints are sensitive alternatives but great

care is required to ensure that residual input virus is

not being detected. An early post infection sample

should be taken and an increase in genome copy

number demonstrated during the culture period.

MaterialQuantity of Starting

Material In ReactionResult Genome Equivalents

Porcine trypsin 3.2 ml Positive 4,673 ± 2,098

Bovine trypsin 5.31 mg Positive 9.62 ± 0*

Bovine insulin 3.2 mg Positive 2.71 ± 0*

BSA 1.4 mg Negative 0.00

FBS 1 (US) 3.2 ml Negative 0.00

FBS 2 (US) 3.2 ml Negative 0.00

FBS (French) 3.2 ml Negative 0.00

FBS 4 (South American) 3.2 ml Positive 5.74 ± 0*

FBS 5 (New Zealand) 3.2 ml Negative 0.00

FBS 6 (Australian) 3.2 ml Negative

Sentinel extraction control Negative

PCR negative control Negative

Positive standard 102 target

copiesPositive

Positive standard 101 target

copiesPositive

*Below level of quantification. Samples confirmed positive on re-amplification

Table 1. QPCR detection of PCV and BCV in Materials

An Outline of Testing Procedures Used for the Detection of Porcine Circoviruses 5

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preliminary characterisation and applications of monoclonal

antibodies to porcine circovirus. Vet Immunol Immunopathol. 1994

Nov;43(4):357-71.

Bratanich AC, Blanchetot A. PCV2 replicase transcripts in infected

porcine kidney (PK15) cells. Virus Genes. 2002 Dec;25(3):323-8.

Ellis JA, Wiseman BM, Allan G, et al. Analysis of seroconversion to

porcinecircovirus 2 among veterinarians from the United States and

Canada. J Am Vet Med Assoc. Dec 1 2000;217(11):1645-1646.

Fenaux, M., Opriessnig, T., Halbur, P. G., Xu, Y., Potts, B. & Meng, X. J.

(2004). Detection and in vitro and in vivo characterization of porcine

circovirus DNA from a porcine-derived commercial pepsin product.

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