ACCEPTED AUTHOR VERSION OF THE MANUSCRIPT: Is contamination of bovine-sourced material with bovine viral diarrhea virus still a problem in countries with ongoing eradication campaigns?

contamination of bovine-sourced material with bovine still a problem in countries with ongoing eradication Abstract In this report, we describe the detection of bovine viral diarrhea virus (BVDV) contamination in commercial animal-derived sera and vaccines against animal viral pathogens on the market in Poland. Antibodies against BVDV were detected in 4/45 sera samples (8.9%) using an ELISA test. The presence of BVDV antigen using ELISA was found using ELISA in 3/45 serum samples (6.6%) and 18/172 vaccine samples (10.5%). An RT-PCR was conducted using primers targeting two genome regions, the five prime untranslated region (5’UTR) and N-terminal protease (N pro ). BVDV RNA was detected in 33/45 (73.3%) of sera, and 11/172 samples (6.4%) of collected vaccines, of which one vaccine did not declare BVDV strain in its composition. A single serum showed the presence of an infectious virus and only one was contaminated with all 3 species of BVDV. The most frequent species in sera was BVDV-3 (75.5%), whereas in vaccines only BVDV-1 was identified. Sequence analysis showed that the tested commercial sera and one vaccine were contaminated by six genotypes of BVDV: -1a, 1b, -1c, -1d, -2a, and -3. Identification of BVDV and its genetic material in animal-derived products is important due to the possibility of pestivirus transmission as well as the chance of falsifying the results of a diagnostic test. It also demonstrates the necessity of rigorous monitoring of the bioproducts used in the laboratory and industry level.

of bovine-sourced materials. BVDV is considered one of the most important infectious diseases of cattle with respect to animal health and economic impact worldwide. Epidemiological data from Poland indicated that infection with BVDV is very common, with over 70% of dairy herds seropositive in bulk-tank milk testing (Kuta et al., 2013a). A similar study carried out by Rypuła et al. (2010) showed a high percentage of BVDV-positive animals, especially in large dairy herds. The presence of the BVDV in beef cattle aged less than 12 months in Poland was also reported by Wernicki et al. (2015). Furthermore, the constantly growing number of genetic variants recently identified in Poland shows that this problem is still relevant (Mirosław et al., 2019). Some European countries, including Denmark, Norway, Sweden, and Finland have recently eradicated or are almost free from BVDV. Switzerland, Austria, and Germany are in the advanced or final stages of BVDV eradication, whereas the Netherlands and Ireland have only recently implemented control programs (Wernike et al., 2017). Following these countries, Poland introduced a voluntary BVDV eradication program in early 2018. For the eradication program to be successful, it is crucial to control the risk factors for possible virus transmission including contamination in animal-derived products such as vaccines, which can introduce an infectious virus to a herd.
BVDV species 1 (Pestivirus A) and 2 (Pestivirus B), each comprising several subgenotypes, together with Classical Swine Fever Virus (CSFV, Pestivirus C), and Border Disease Virus (BDV, Pestivirus D) belong to the genus Pestivirus of the family Flaviviridae (Smith et al., 2017). BVDV-1 is the most common species of this virus around the world, as well as the most prevalent in Poland (Kuta et al., 2013b;Mirosław et al., 2019), whereas BVDV-2 is common in North and South America. However, the transmission of species 2 to European herds was described (Vilcek et al., 2005;Polak et al., 2014). Additionally, a novel putative pestivirus species was discovered in the strain named D32/00_'HoBi' in contaminated commercial FBS of Brazilian origin (Schirrmeier et al., 2004). It was followed by the detection of natural infection with this pathogen in southeast Asia, Italy, and Brazil (Stahl et al., 2007;Decaro et al., 2011;Cortez et al., 2006). This new species is tentatively called "HoBi-like", "BVDV-3" or "atypical pestivirus (Pestivirus H)" (Smith et al., 2017). All pestiviruses are structurally and genetically related and serological cross-reactivity between members of the genus has been reported (Bauermann et al., 2012). Based on the induced effect on cell culture, two biotypes of BVDV were distinguished: the noncytopathic (ncp) and cytopathic (cp). The key issue of the BVDV infection (uniquely ncp BVDV infection) is its ability to generate persistent infection though fetal infection in early gestation, these fetuses then serving as virus reservoirs. Infected fetuses are the source of high loads of virus, and therefore contaminate any subsequently produced FBS (Deregt and Loewen, 1995). BVDV almost always spreads by direct contact with persistently infected (PI) animals through introduction or mixing at a show, sale, feedlot or through a fence. Other sources of infection besides the PI animals include transiently infected (TI) animals, wild ruminants and nonbovine hosts, and BVDVcontaminated semen and embryos. Additionally, the spread of the virus via needles, nose tongs, hoof trimmers, and vaccine vials was also proved.
FBS is the most commonly used supplement in cell culture media since it contains high levels of nutrients, adhesion proteins, and growth factors (GF), among other vital components.
Cell cultures have wide clinical application in medicine, such as in the production of vaccines, diagnostic tests, and cell therapy (Hawkes, 2015). Previously conducted research has shown that pestivirus contamination is common in FBS (Audet et al., 2000;Polak et al., 2008;Laassri et al., 2018;Pecora et al., 2019) and can also be present also in bovine-sourced products for an animal or human use (Barkema et al., 2001;Giangaspero et al., 2001;Studer et al., 2002). The presence of BVDV was confirmed for instance in the measles, mumps, rubella, and polio vaccines administered to children (Giangaspero et al., 2001;Sasaki et al., 1996). Although pestiviruses are not considered zoonotic, their antigens were found in the feces of children showing signs of gastroenteritis and respiratory disorders (Yolken et al., 1989). Furthermore, BVDV was isolated from human leukocytes in patients in Belgium (Giangaspero et al., 1997).
Recent findings also confirmed the presence of peptide(s) from the polyprotein of a bovine-like viral diarrhea virus in Zika-positive brains of microcephalic fetuses during the 2015 outbreak in Brazil (Nogueira et al., 2016). In addition to exposing of animals and humans to BVDV, the contamination can affect in vitro biological and diagnostic tests, including those to diagnose infection with related viruses such as classical swine fever virus (CSFV) and human hepatitis C virus (HCV).
At present, with the common use of techniques such as heat inactivation, ultrafiltration, ethylene-imine inactivation, or gamma irradiation, the risk of transmission of pathogens has nearly been eliminated, but does still exist. The aim of the conducted research was to determine the potential contamination of commercial FBS and vaccines against animal viral and bacterial pathogens which are manufactured containing animal-derived products. The conducted research will allow estimation of how widely pestiviruses are unintentionally distributed in products presumed to be safe, and assessment of the risk of infection by using such products available on the Polish market. The safety of animal-derived products is a crucial issue in avoiding the risk of pathogen introduction into nonendemic regions or unintentional induction of an immune response in herds where an eradication campaign pursues a strategy excluding vaccination.
All commercial products were purchased from Polish market suppliers and were tested for the presence of antibodies against BVDV, BVDV antigen, and its genetic material.

BVDV antibody detection
The presence of BVDV antibodies in sera was tested with a commercial enzyme-linked immunosorbent assay (ELISA) kit. An Ab ELISA based on the pestivirus envelope protein E rns (BVDV Total Ab Test, IDEXX, Liebefeld-Bern, Switzerland) was used according to the manufacturer's instructions. The test provides specificity and sensitivity of respectively 97.1% and 96.7% compared with the virus neutralization test (VNT) (Lanyon et al., 2013).

BVDV antigen detection
A commercial ELISA which detects all three species of BVDV, based on the BVDV E rns antigen (BVDV Ag/Serum Plus, IDEXX, Liebefeld-Bern, Switzerland) was used. Tests were carried out on sera and vaccine samples according to the manufacturer's protocol. This test demonstrates specificity of more than 99.7% and a sensitivity of nearly 100% (Wolf, 2007;Van Maanen, 2001;Idexx Laboratories, 2005).

Total RNA extraction and BVDV genome detection
Extraction of viral nucleic acid from sera, vaccines, and potentially infected cell culture was performed with a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's spin protocol. Extracted RNA samples were stored at -80C prior to PCR tests. The samples were tested individually.

RT-PCR
A standard one-step RT-PCR was performed using a Transcriptor One-Step RT-PCR Kit (Roche, Mannheim, Germany) using the provided primers and adhering to the manufacturer's instructions ( Table 1). The final volume of RT-PCR mixture was 25 μl, comprising: 15.5 μl of RNase free water, 5 μl of reaction buffer, 1 μl of each primer (10 μM), 0.5 μl of enzyme mix, and 2 μl of RNA template. The reaction conditions were set at 50˚C for 30 min and 94˚C for 7 min, followed by 10 cycles of 94˚C for 10 s, 53˚C for 30 s, 68˚C for 30 s, then followed by 25 cycles of 94˚C for 10 s, 53˚C for 30 s, and 68˚C for 33 s with a final extension step of 68˚C for 7 min. RT-PCR products were analyzed with a molecular weight standard on 1.5% agarose gel stained with ethidium bromide.

Real-time RT-PCR
An in-house real-time RT-PCR which allows species differentiation was performed using AgPath-ID Kit reagents (Life Technologies, Waltham, MA, USA) according to the manufacturer's protocol in the StepOnePlus real-time PCR system (Life Technologies, Republic of Singapore). Details of the primers and probes are shown in Table 1. The assay was run using the following thermal program: 48˚C for 10 min (RT-step), 95˚C for 10 min, and 40 cycles of 95˚C for 15 s and 60˚C for 45 s. The reaction was performed for 42 cycles with a Ct (threshold cycle) value of 40 as a cut-off.

Assessment of BVDV infectious status in commercial sera
To determine if the virus could be infectious for living organisms, all BVDV antigens and genome-positive sera were used to prepare a growing medium for the propagation of Madin-Derby bovine kidney (MDBK) cell lines. Before the experiment, the medium and cell culture were checked for BVDV presence. Heat inactivation of sera was not carried out before testing in order to get reliable information about the effectiveness of virus reduction/removal by the manufacturer. MDBK cells were split twice with the addition of tested BVDV-positive sera. Before the third splitting, the cells were washed with pure EMEM medium and one growing medium with contaminated serum was excluded from further incubation. After 3 days, cells were harvested and frozen until analysis. RNA was extracted from cell culture samples, and an RT-PCR with the panpestivirus primer set and a real-time RT-PCR were conducted (details described in section 2.4.1).

Sequencing and phylogenetic analysis
The products of the standard RT-PCR with all primers listed in Table 1 were purified and sequenced as described previously (Kuta et al, 2013b). Phylogenetic analysis was done by the neighbor-joining (NJ) statistical method with the Kimura two-parameter model using MEGA software (version 5.03). The reliability of the constructed phylogenetic trees was evaluated by running 1000 bootstrap replications in the phylogeny test and bootstrap values greater than or equal to 70% were considered good support.

BVDV antibodies detection
One sample of FBS and two of NBS/NCS was detected as antibody-positive using the Ab ELISA test, which accounted for 6.6% of all serum samples. Two samples had very similar high values, and the third sample was weakly positive (B2). A doubtful result was obtained for one FBS (B1) sample, which was 2% of the total number of samples. Of the 45 serum samples, 91.1% (n=41) were negative, or the level of antibodies was below the limit of detection.
Additional designation of tested samples, given in Table 2.

BVDV antigen detection
Overall, 3 FBS samples (6.6%) were positive for BVDV antigen. One of those samples was doubtful (B1), one was weakly positive (B2), and one was negative (F2) for the presence of antibodies in the Ab ELISA.
BVDV antigen was also detected in 18 vaccines. Only one sample among the positive results was not a BVDV vaccine. According to the manufacturer's declaration, it was an inactivated monovalent IBR vaccine.

RT-PCR
A standard RT-PCR was conducted using four pairs of primers targeting two regions of the BVDV, namely 5'UTR and N pro . These genome  Table 2.
Among vaccine samples, the genetic material of the BVD virus was detected in 4 BVDV vaccines. The fifth vaccine in which the BVDV genome was amplifying was an IBR marker live vaccine, which gave in total 0.58% of the screened products. The IBR vaccine was not the same vaccine that was positive in the Ag ELISA, and not the same manufacturer.
To check the repeatability of all positive results, we re-extracted serum and vaccine samples and conducted an RT-PCR. The same results were obtained.

Real-time RT-PCR
In the real-time RT-PCR test, an additional 8 sera were positive for the presence of the BVDV genome over the 26 detected by the standard RT-PCR test. The results revealed that only one sample, M1 FBS of Australian origin was contaminated with all 3 species of BVD virus. The most frequent virus species in the analyzed sera was BVDV-3 (75.5%), followed by BVDV-1 (40%) and BVDV-2 (11.1%). Of all sera, 44.4% (n=20) were positive only to a single BVDV species, which was BVDV-1 or BVDV-3. BVDV-2 never occurred alone in the tested sera. In the cases where 2 species of BVDV were detected, most were 1 and 3 (n=8), fewer were 1 and 2 (n=3), and the last paired species were 2 and 3 (n=1).
Positive results were obtained only for 10 out of 17 BVDV vaccines. In these vaccines, the presence of BVDV-1 was confirmed and this was in accordance with the manufacturer's declaration. The contamination with BVDV of the IBR marker live vaccine which was detected by standard was confirmed by real-time RT-PCR, and BVDV species 1 was identified. As in the case of serum and vaccine samples, more positive results were obtained by using real-time RT-PCR than standard RT-PCR, which indicates greater usefulness of this method in routine diagnosis.

Infectious status of BVDV in FBS
After incubation of cell cultures with 33 of BVDV-positive sera in Ag ELISA, standard RT-PCR, and real-time RT-PCR, it could be confirmed that none of the samples contained the cpe biotype of the BVD virus, which was in accordance with the manufacturer's declaration.
The frozen cell culture samples were screened for the presence of genetic material of BVDV and only one was positive for it. A positive result in the RT-PCR and real-time RT-PCR (Ct=30 for BVDV-2) indicated the presence of the infectious BVD virus in that particular FBS sample (H7), which had been able to infect the cell culture and replicate. With regard to the information contained in the product specification, the serum batch was standard quality and not gamma irradiated. The obtained RT-PCR product (BVDV isolated in cell culture) was sequenced, analyzed, and compared to the sequence obtained straight from the FBS without propagation in cell culture. The sequence identity for these samples was 90.7% and both were classified as BVDV-2a. The highest similarity to the sequence from FBS possible to find using the BLAST NCBI tool was demonstrated by the isolate UEL12-Br/17 (GenBank: MG004720) isolated first in southern Brazil in 2017. This also corresponded to the origin of the tested serum, which was claimed to be South America.

Phylogenetic analysis
The sequences of virus strains used in phylogenetic analyses and their GenBank accession numbers are shown in Figures 1, 2, and 3. Phylogenetic analysis was made to determine the species and subtypes of the isolated BVDV strains. The viral RNA load from only 15 sera and 5 vaccine samples within the 5'UTR region and 12 sera samples within N pro was sufficient to obtain nucleotide sequences for genetic analysis. Phylogenetic analysis of the 5'UTR and N pro partial sequences from sera combined with additional sequences from GenBank identified 6 genotypes of BVDV, which were BVDV-1a, BVDV-1b, BVDV-1c, BVDV-1d, BVDV-2a, and BVDV-3. Interestingly, there were some mismatches between species and subtypes, of BVDV strains regarding the analyzed genome region, namely 5'UTR and N pro . Only in the cases of 2 isolates from sera were the same species and subtypes obtained in both analyzed region. The explanation for these differences is a mixture of many subtypes of BVDV in one serum batch. This is indicated by the presence of many signals present in the background of the main sequence chromatogram. Each genotype showed significant genetic diversity as reflected by the number of nodes and length of branches in trees among the same subtype. The set of panpestivirus primers has a weaker ability to detect atypical pestiviruses (Vilcek et al., 1994), so that is why none could be seen on the tree built on the base of the 5'UTR region. Thanks to the possibility of using dedicated primer pairs for amplification of the N pro region of atypical pestiviruses, a clade was created with five BVDV-3 isolates with clearly visible differences between them. Surprisingly only subtype BVDV-1d was detected, but the

FBS-B2 unknown 2
53099 USA FJ431189 Nevertheless, some companies that want to avoid the risk that their products will not be approved for use in the production of vaccines or other products irradiate raw FBS prior to testing for BVDV, without informing the biopharmaceutical companies. Moreover, they choose tests that are not able to detect inactivated viruses or that are less sensitive (Hawkes, 2016).
After comparison of the results from a standard RT-PCR (5'UTR and N pro ) with those of a realtime RT-PCR test, we can observe that the panpestivirus primer set was not able to detect all BVDV-3 positive samples, which confirms this finding. Also, it is congruent with the results obtained by others quite recently (Stahl et al., 2010;Peletto et al., 2012) and earlier by Vilcek et al. (1994) who emphasized the problems of intralaboratory contamination and doubtful specificity of primers used in RT-PCR.
It is worthy of note that in this study 73% (33/45) of the commercial sera were positive for at least one BVDV species, based on results in standard RT-PCR and real-time RT-PCR tests. Similarly to what we found for BVDV contamination in sera was previously reported by other research groups, where from 53.4% to 100% of tested sera were found contaminated with RNA of at least one pestivirus species (Laassri et al., 2018;Monteiro et al., 2018;Giammarioli et al., 2015), as they also were in our results. In the present work, a very large percentage (75.5%) of positive sera were contaminated with BVDV-3, confirming the results obtained in the study by Giammarioli et al. (2015). After comparing the place of origin of those sera we can conclude that they came from the same regions of the world. In contrast, Bauermann et al. (2014), who only studied sera of North American origin, found no evidence for the presence of BVDV-3. We could not confirm this, because of the lack of sera of this origin in our research.
Despite the fact that the majority of positive sera were contaminated with BVDV-3, most of BVDV's in Poland are representatives of species 1. According to studies from 2013 (Kuta et al., 2013b), the majority of isolates were subtypes 1b and 1d, while subsequent studies (Mirosław and Polak, 2019) showed the majority of 1b, 1g, 1f, and 1d. While no BVDV-3 infected animals have been detected in Poland, the presence of BVDV-3 in the country cannot be ruled out. Clinical signs are indistinguishable from infections with typical field strains and not all diagnostic tests are able to discriminate between typical BVDV and BVDV-3 infections.
Moreover, not every sample from a BVDV positive animal is classified for further analysis, which is often associated with a deficiency of sufficient material. Introduction of HoBi-like viruses could occur via live vaccines (Falcone et al., 2003) or insufficiently inactivated killed vaccines (Stahl et al., 2010). Previously, BVDV-2 contaminating the BoHV-1 marker vaccine was shown to cause wide spread outbreak of BVD in the Netherlands and Italy (Barkema et al., 2001). In this study, BVDV antigens were found in inactivated monovalent IBR vaccine, and BVDV-1d partial genome was in an IBR marker live vaccine, which further confirms previous reports and highlights the need for monitoring of bioproducts at each stage of production to prevent a possible outbreak of disease. However, in this particular case, the infectious status of both vaccines could not be checked, because it transpired that the adjuvants used in them were toxic to cell culture. Nevertheless the risk of disease or seroconversion after using such vaccines is high.
Despite the fact that in our studies over 70% of tested sera proved to be contaminated by BVDV, only one serum showed the presence of an infectious virus. It is possible that the presence of the detected specific antibodies against BVDV in high titers in the cases of 3 sera may have affected the result of virus isolation by neutralization, and resulted in false negative results. The virus was ncp, like most field isolates (Deregt and Loewen, 1995). Two nucleotide sequences were obtained from this serum, one directly from FBS by sequencing of the RT-PCR product, and the second after replication of the virus in cell culture for 3 passages. The large difference of sequence identity of 9.3% between these two sequences may be due to the fact that the given FBS sample was a pool of sera taken from many animals that may have been exposed to different isolates of the same BVDV species. For this reason, it could contain several virus isolates, as evidenced by the presence of additional peaks in the background in the obtained sequencing chromatogram (direct from FBS). Nevertheless, we obtained a clear sequencing chromatogram with no interference in the background for virus isolated in cell culture. This proves that FBS contains only one isolate of infectious BVDV. The serum in which the BVD infectious virus was detected was claimed to be of EU-approved origin. No connection was observed between the place of origin or purification method used and the occurrence of contamination, which was also noticed by Hawkes (2015). Many commercial sera were cleaned by filtration, but due to the small particle size of BVDV (45-55 nm), many viral particles could remain in commercial FBS after standard sterile filtration. It means that we should not use animal-derived products for diagnostic, medical or production purposes without using additional safety measures such as, for example, additional heat inactivation or gamma irradiation.
It seems that the perfect solution for avoiding pathogen contamination is using serumfree media. They were developed to cut down the use of animals in research and the medical industry. These products have met with great success, especially in the production of some proteins for medical use. Nevertheless, pharmaceutical companies, diagnostic centers, and researchers still depend on animal-derived products like FBS for most of their cell culture needs, because we are not yet able to replicate all growth factors present in FBS or to produce serum-free media on a large scale (Hawkes, 2015).
The current study provides evidence that the contamination of bioproducts with all species of BVDV, and especially the contamination of commercial FBS available on the local market and used in immunoprophylaxis and diagnostics in Poland, remains a threat for pestivirus transmission and may falsify the results of a diagnostic test based on commercial serum usage. While the risk of pestivirus infection through a vaccine seems low, the use of the vaccines contaminated with the BVDV antigen may provoke seroconversion, which may result in failure of a BVDV eradication program.