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Testing for Senecavirus A in Biological Products

Background

Senecavirus A (SVA), also known as Seneca valley virus, is a positive single stranded RNA 

virus belonging to Picornaviridae family. The virus causes disease in young pigs. The 

pathological lesions in infected animals are similar to reportable diseases such as foot and mouth 

disease, vesicular stomatitis, swine vesicular disease, and vesicular exanthema. Disease 

outbreaks have been reported in Australia, Brazil, New Zealand, China, and Canada. Outbreaks 

have increased in the United States rapidly over the last few years. 

At the 2017 annual meeting of the American Association of Swine Veterinarians (AASV), a 

biologics company reported SVA contamination in two lots of porcine-derived trypsin. The

identification was made by whole genome sequencing and virus isolation. The finding raised 

concerns with pork industry representatives regarding the possibility of SVA contamination in 

veterinary biological products, including those already licensed and in commerce. In response, 

the Center for Veterinary Biologics (CVB) immediately initiated testing vaccine samples 

available in the CVB repository that were known to be manufactured using porcine-derived 

ingredients. The repository contains samples of all vaccines on the market that have current 

expiration dates. 

Selection of Biological Product Samples

CVB prioritized the highest risk biologics for testing. Those included biologics used in swine 

and manufactured using swine serum and swine-derived trypsin. For their initial testing, CVB 

selected forty-two (42) samples of high risk biologic serials from the repository. 

Testing Procedure 

CVB included trypsin lots routinely used, including the two trypsin lots identified as 

contaminated during the AASV meeting. For positive controls, CVB used trypsin and vaccine 

samples spiked with control plasmids or with SVA. For subsequent testing, CVB incorporated

thirty-five (35) CVB repository available serials of inactivated autogenous and mycoplasma 

bacterins. 

CVB screened multiple cell lines. For the biologics sample testing, they chose the fast growing

and easily available chicken embryo fibroblast (CEF) cell culture substrate for SVA isolation.

They adopted the sensitive Fowler real-time rt-qPCR testing method for amplification and 

screening of viral RNA. RNA was extracted directly from the biologics samples, serum and 

trypsin samples, and the infected CEF cell cultures. Independent confirmatory testing was 

completed at Iowa State University (ISU) laboratory in Ames, Iowa and VS’ Foreign Animal 

Disease Diagnostic Laboratory (FADDL) in Plum Island, New York. 

Results and Discussion

The testing results are summarized in the Tables below. CVB testing found all biologics samples 

screened negative except for two vaccine serials manufactured by one firm that was positive for 
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SVA nucleic acid. Both of the vaccine serials tested positive in confirmatory testing at the ISU 

laboratory as well as at FADDL. Of the several trypsin and swine serum samples screened, CVB 

only found one serum lot positive by rt-qPCR. ISU and FADDL confirmed this result. This 

serum lot was used during the manufacturing of the two serials that were identified as SVA

contaminated in the initial CVB screening. All control samples performed as expected. To date, 

CVB has not found vaccine serials, serum, or trypsin samples positive for SVA contamination

during screening.

CVB further characterized the SVA present in two PCR positive vaccine serials and one PCR 

positive serum sample found during their initial screening. The quality of both the cDNA 

necessary for nucleic acid library preparation, and the viral RNA for direct sequencing was 

compromised because the contaminated serum used in the preparation of the two serials was 

inactivated by gamma irradiation at 47kGy. Thus, the whole genome sequencing (WGS) method 

failed to detect SVA in any of these three positive samples. However, PCR amplification and 

Sanger Sequencing attempts were useful in successfully generating an SVA-specific 1.8kb 

nucleic acid fragment. This SVA nucleic acid fragment was similar to the SVA-OH2 strain 

isolated in 2015 and available at GenBank with identification #KU058183.1. Despite the 

identification of SVA nucleic acid in these samples, CVB did not detect viable virus in any 

of the samples tested.

Table 1. Susceptibility of Various Cell Lines for SVA Infection

Cell Line Type SVA Titer in

Logs TCID50

Virus Amplification Identification by

CPE* IFA*

B-ST cells 8.2 + +

CEF 8.1 + +

CrFk 7 + +

DF-1 5.8 + +

GPC 9.5 + +

H1299 8.8 + +

MA-104 6.9 + +

MDBK-A 7.6 + +

MDCK 6.6 - +

McCoy 6.2 - +

PK-15N 7.5 + +

PK-13B 6 + +

VERO 7.3 + +

*CPE=Cytopathogenic effect; IFA=Immunofluorescent Antibody test
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Table 2. Biologics & Animal Origin Ingredients Test Results for SVA Contamination

Sample Type Serials/Lots/Sample

s

(Total #)

Serials/Lots/Samples Identified Positive by

Cell Culture Test 

(Total #)

PCR Method

(Total #)

Modified Live Virus 

Vaccines

42 0 2

Autogenous Vaccines 7 0 0

Mycoplasma Bacterins 28 0 0

Serum lots 4 0 1

Trypsin lots 6 0 0

Spiked CEF samples 2 2 NA

Spiked PCR Samples 7 NA 7

Conclusions

CVB could not reproduce the results presented at AASV meeting. CVB tested 77 biologics 

samples with the highest risk of contamination with SVA. CVB found two vaccine serials, which

were manufactured by a single firm using the same lot of swine serum, positive for SVA nucleic 

acid. CVB testing did not detect viable virus in any of the samples, and testing supports that 

the serum had been adequately irradiated to inactivate the virus. CVB found none of the 

screened porcine-derived trypsin samples positive for SVA. Based on CVB’s testing, it is

reasonable to conclude that there is no evidence to suggest viable SVA is present as an 

extraneous agent in any of the vaccines that were evaluated. Further, it is very unlikely that 

SVA was spread by contaminated vaccines.

The CVB has implemented SVA contamination screening for all incoming Master Seeds, 

incoming Master Cells, and materials with animal origin ingredients. The CVB published a draft 

notice recommending biologics manufacturers implement SVA contamination screening of all 

seed materials and animal origin ingredients. 

Also, the CEF cell culture system is non-permissive to FMD virus amplification. Plans are 

underway at the CVB to use lamb kidney and BHK21 cells for standardizing a test for viable 

SVA contamination detection, which will also detect FMD in animal origin ingredients. CVB is 

working with industry to review all testing of animal origin ingredients regulations and will 

propose updated guidance in the near future.