Limited environmental stability of infectious porcine endogenous retrovirus type C; Usage of reverse transcriptase in combination with viral RNA as markers for infectious virus

Porcine endogenous retroviruses (PERVs) are an integral part of the pig genome with infectious potential, as shown in vitro.


INTRODUCTION
Endogenous retroviruses (ERV) and related sequences are integral parts of the eukaryotic genome that evolved by retroviral infection of germlines millions of years ago. [1][2][3][4][5][6] A great proportion of beta-type ERV, including some classes of human endogenous retroviruses, are no longer replication competent. [7][8][9] Some retroviruses belonging to the gamma-retrovirus group have preserved their infectious capacities, such as murine leukemia virus (MuLV), koala retrovirus, and feline leukemia virus (FeLV). [10][11][12][13][14][15] Here, we focus on porcine endogenous retrovirus (PERV), which is also a gamma-retrovirus and an integral part of the pig genome. 16,17 It comprises three major classes, PERV-A, -B, and -C, present in the genome as proviruses. Most of them are truncated and impaired and not all pigs harbor PERV-C. 16 Nonetheless, a number of replication-competent full length PERV-A, -B, and -C viruses from different pig breeds and cell lines have been isolated and were tested in vitro. [18][19][20][21][22][23] Even if there is no documented in vivo infection or zoonosis, the relevance of PERV as an infectious agent is taken into account. [24][25][26][27] The absence of evidence remains a hurdle for xenotransplantation (XTx) since the development of pig-derived cell-based therapeutics inevitably requires maintenance of virus safety. 28  given by the absence of RT activity besides a basal background of RNA copies independent from infection. This RNA background is commonly known and has been described before. 35 The culture of cells was performed as described earlier. 36

Quantification of PERV
Detection and quantification of PERV-C env vRNA was performed as described 37

RT activity assay
RT activity in cell-free SN of infected cells and producer cells was detected as described 37 using the C-Type RT Activity Kit (Cavidi, Sweden) according to the manufacturer's instructions, protocol B. Samples were measured in triplicates and the mean value and standard deviation were determined.

Quantification of PERV-C RT activity
The storage of cell-free SN containing PERV-C(1312) and PERV-C(5683) from ST-IOWA producer cells at controlled room temperature ) and without (b[−]) sterilized wood litter, as used for large animal husbandry, led to a continuous reduction of viral enzymatic RT activity ( Figure 1, Table 1). The initial RT activity was determined with 217 mU/ml for PERV-C(1312) and 34 mU/ml for PERV-C(5683) in SN that was freshly harvested and 193 mU/ml for PERV-C(1312) and 56 mU/ml for PERV-C(5683) in SN that was collected before and stored at −80 • C until testing. Virus-containing SN showed approximately 33% mean reduction within 1 day when stored in b(−) compared to 66% when stored in b(+) flasks (Table 1). At Freshly harvested virus was more stable than virus from −80 • C stocks when comparing the d1 reduction data (Table 1). Virus-free SN from PERV-C negative ST-IOWA cells, used as control, remained negative.  (Table 1). Compared to RT activity, vRNA levels decreased much more gradually under b(−) conditions, and complete elimination was not attained after 57 days.

Quantification of PERV-C vRNA
Levels of 2 × 10 4 to 8 × 10 5 copies/µl PERV-C env vRNA remained, respectively. In general, vRNA sampled from freshly harvested PERV-C(5683) and PERV-C(1312) that were not stored at −80 • C before testing was more stable and was less affected by storage at ambient temperature than the viral RT (Table 1).

in vitro infection assay
Since mean RT activity was reduced by 96% in b(+) and by 83% in

DISCUSSION
Despite phylogenetic data on PERV, aspects of host pathogen interac- Since PERV-C is not present in all pigs but may recombine with PERV-A to give the recombinant human-tropic and high-titer PERV-A/C, 42 In summary, the decrease in RT activity was accompanied by a loss of infectivity (Figures 1 and 3A), indicating that the viral RT is not only necessary but also a suitable surrogate marker for viral infectivity being used for further evaluation. The infectivity of PERV-C did not depend directly on the presence of vRNA since PERV-C env vRNA was still available at high amounts during the whole experiment. The results clearly show that RNA, utilized as gold standard for PERV detection, is not the only defining functional parameter for analyzing the infectious risk of PERV. Only intact viruses have the ability to infect host cells and to integrate their genomes. The probability of unwanted genomic integration of foreign vRNA, while possible, is unlikely, based on the infection results presented here. These data provides initial insights toward PERV environmental stability and infectivity outside its host.

CONCLUSIONS
In this study, we analyzed the stability of two different PERV-C molecular clones, PERV-C(1312) and PERV-C(5683), in culture SN with and without sterile animal bedding material at controlled room temperature. Due to the fact that handling of genetically modified organisms or viruses requires an evaluation of potential environmental impact, we initiated this investigation to estimate how long PERV-C remains infectious at ambient temperature outside its host. This investigation is the first approach to determine the environmental stability of PERV-C and requires further analysis under conditions found in the actual barn husbandry setting. We showed in vitro by using a ST-IOWA cell based assay that even in cell culture medium at 100% humidity, the RT activity decreases continuously, resulting in a loss of infectivity within 7 days despite the presence of vRNA. The presence of vRNA without intact viral RT is not sufficient in itself to initiate a productive infection. For this reason, RT testing should be considered as an additional or surrogate marker to differentiate infectious PERV.