PCR and peptide based PCMV detection in pig – development and application of a combined testing procedure differentiating newly from latent infected pigs

Porcine cytomegalovirus (PCMV) is widely distributed in pigs and difficult to detect due to latency. PCMV infection of source pigs was associated with early graft failure after cardiac and renal xenotransplantation into nonhuman primates. Importantly, PCMV infection of the first genetically modified pig heart into a human may have contributed to the reduced survival of the patient. Sensitive and reliable assays for detection of latent PCMV infection are thus indispensable. Here, we report the development of five peptide‐induced rabbit antisera specific for PCMV glycoprotein B (gB) and their validation for detection of PCMV in infected pig fallopian tube (PFT) cells by immunofluorescence and electron microscopy (EM). The anti‐gB antibodies were also used for detection by Western blot analysis of PCMV purified from the supernatant of infected PFT cells. Sera of infected versus non‐infected pigs have been compared. In parallel, PCMV viral load in blood samples of the animals was quantified by a novel highly sensitive nested‐PCR and qPCR assay. A combination of four partly overlapping peptides from the gB C‐terminus was used to establish a diagnostic ELISA for PCMV gB specific pig antibodies which is able to differentiate infected from non‐infected animals and to quantify maternal antibodies in neonates. The combination of a highly sensitive nested PCR for direct virus detection with a sensitive peptide‐based ELISA detecting anti‐PCMV gB‐antibodies, supplemented by Western blot analysis and/or immunohistochemistry for virus detection will reliably differentiate pigs with active infection, latently infected pigs, and non‐infected pigs. It may significantly improve the virologic safety of xenotransplantation.


INTRODUCTION
Porcine cytomegalovirus (PCMV; porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV); Suid betaherpesvirus 2 (SuBHV2, SuHV-2)), is a human betaherpesvirus-6 and -7 (HHV6 and HHV7) related roseolovirus. It is a large, enveloped double stranded DNA virus composed of 128.367 bp (RefSeq NC_022233) with 80 protein coding genes. It is widely distributed in domesticated pigs that are the natural host as well as in wild boars. 1,2 The virus is routinely detected in nasal and ocular secretions, cervical fluid and urine being directly transmitted from one animal to another. 3,4 Events of congenital transmission have been described. [5][6][7] Except for neonates, PCMV causes mild clinical signs, described as "inclusion body rhinitis" because of its histological appearance. [8][9][10][11][12][13] As no human infections have been reported, PCMV is not seen as a zoonotic threat. Nonetheless, its potential as a zoonotic virus in human is uncertain as PCMV is associated with endothelial activation, consumptive coagulopathy and accelerated renal and cardiac xenograft rejection [14][15][16][17][18] ; under some conditions PCMV may infect human cells in vitro. 5,[19][20][21][22][23] For xenotransplantation (XTx) using pigs as donors, PCMV increasingly came into focus since organs, for example, hearts and kidneys, derived from PCMV positive donor animals failed early and exhibited significantly reduced survival after transplantation into baboons. 14,18,22,24,25 Furthermore, PCMV infection of the first genetically modified pig heart used for compassionate transplantation into a human may have contributed to the demise of the patient after 2 months. 26,27 Therefore, the generation of pigs as source animals for xenotransplantation free of PCMV is mandatory. 28 Since PCMV, like HHV6 and HHV7, resides intracellularly, 5  In this study, polyclonal rabbit antibodies against five synthetic peptides of 15 or 16 aa of the highly abundant and immunogenic PCMV envelope glycoprotein B (gB). [29][30][31] were generated. Four peptides are localized in the C-terminus 29 and one in the midsection. 32 The antibodies were validated for immunofluorescence (IF) and Western blot (WB) analyses. Furthermore, a peptide-based ELISA for the detection of anti-PCMV antibodies in serum was established. In addition and as a control, pig fallopian tube cells (PFT) infected with PCMV (PFT-PCMV) that also produce porcine endogenous retrovirus (PERV) 33 have been used for virus propagation and isolation of PCMV from cell-free supernatant. Non-infected PFT cells served for comparison. Using these novel tools, we demonstrated that maternal antibody levels in PCMV negative neonates decrease within the first weeks as long as they stay non-infected. Furthermore, we localized PCMV by electron microscopy (EM) showing that viruses are released from the nucleus and endogenized for cellular hitchhike being transmitted from cell to cell by close contact, as described for other herpesviruses.
The combination of serological as well as nucleic acid and cell-based assays given here represents a sensitive and robust testing regimen for monitoring the PCMV status of pig stocks and for identifying PCMVfree source pigs for xenotransplantation.

DNA and PERV viral RNA (vRNA) purification
PCMV DNA and genomic DNA (gDNA) was purified either from cells or blood samples using the QIAamp DNA Blood Mini Kit (Qiagen, Germany) in accordance to the manufacturer's instructions. For whole blood or Li-heparin blood samples have been immediately frozen after collection at <20 • C and freshly thawed right before purification.

PCR and qPCR
PCMV was detected by nested PCR and if positive quantified by qPCR using the primers and conditions described ( Table 2). Porcine GAPDH (pGAPDH) or PERV respectively were used as a control for sample quality. 34

Nested-PCR and qPCR detection limits
The detection limit for qPCR in comparison to nested-PCR of PCMV gB was determined by serial dilutions of PCMV co-purified with pig gDNA and serial dilutions of plasmid vector that was also used as standard for qPCR. Primer and position of the inserts are listed (Table 1).

Purification of virus isolates (VI) from cell supernatant
For WB analysis and detection studies virus containing supernatants were harvested while reaching an average activity of approximately 10 5 copies/µL PCMV. Quantification occurred by qPCR using the PCMV gB and pol specific primers ( Table 1). Supernatants of PFT respectively PFT-PCMV cells were subsequently filtered through 0.45 µm Minisart, non-pyrogenic, single use filters (Sartorius, Germany) and stored frozen at −80 • C until further processing. Purification occurred by ultracentrifugation through a 20% sucrose cushion. 38 Viral pellets were resuspended in PBS o/n at 4 • C. Ultracentrifugation was repeated once for 1.5 h at 30.000 rpm (SW32 Ti-Rotor, Beckmann, Germany) in PBS. The final volume was adjusted to a viral copy number of approximately 5 × 10 6 copies/µL PCMV and 5 × 10 6 copies/µL PERV and stored at −80 • C until further analysis.   (Table 1).

Generation of peptides and peptide-based antibodies
The selection of five 15 aa long PCMV gB peptide sequences ( following rabbit immunization protocols for the generation of the corresponding serum derived IgG antibodies. Uncoupled peptides were applied for ELISA studies to test pig sera (Table 3). IgG serum antibodies were designated as: a552, a554, a789, a180, and a181. Those have been purified by affinity chromatography, quantified by Bioanalyzer TA B L E 2 Custom designed synthetic peptides generated by Eurogentec, used for the immunization of rabbits and peptide ELISA studies for the detection of PCMV gB directed antibodies.

Peptide-KLH conjugate/rabbit (r) Peptide (p)
Non-conjugated peptide synthesized for the program b Immunization Program TA B L E 3 Serum binding to PCMV gB specific peptides. ELISA was performed on individual peptides and a combination (p554, p789, p180, and p180) testing rabbit anti-peptide directed and purified IgG as well as pig serum antibody binding of PCMV positive and negative pigs (s#). according to the manufacturer's standard operational procedures.

2.9
Immunohistochemistry using gB specific antibodies and pig sera

Electronmicroscopy of PCMV in PFT cells2
EM of PCMV positive PFT cells was performed according to standard procedures as described previously. 40

Selection of gB specific peptides and generation of peptide-induced antibodies
The sequences selected for the generation of synthetic peptides, namely p552, p554, p789, p180, and p181 (Table 2) are based on diagnostic sequences published before 29,32 (Figure 1). The first peptide (p552) was derived from the DNA sequence used for PCR detection of PCMV in the first clinical pig islet xenotransplantation trial in New Zealand 32 and is located in the midpart of gB (aa 321 to 376; AF268041.1). In addition, four partly overlapping peptides (p554, p789, p180, p181) were selected from the C-terminus of gB (aa 770 to aa 859; FJ595497.1) that was used before, in an indirect blocking ELISA for the detection of anti-PCMV gB antibodies in pigs. 29 The sequences of these five selected epitopes have been analyzed for aa variations within the group of PCMV and for homologies to HHV6 by NCBI Database analysis (Supplement S1 and S2). They revealed little variation ( Figure 1A) within PCMV and high mismatches to HHV6. The five peptides have been synthesized on basis of the epitopes selected ( Figure 1B). They represent conserved aa sequences within gB and are, including the antibodies derived (Table 2), intended for a broad diagnostic use.

Establishment of a testing system on the basis of PFT and PFT PCMV positive cell lines
For analysis of the presence and infectivity of PCMV and suitability of peptide-induced anti-PCMV gB antibodies ( Table 2, a552, a554, a789, a180, and a181), a cell line-based testing system was established ( Figure 2). Wild-type (wt) PFT cells were compared with PCMV infected PFT (PFT-PCMV) cells. 16 The cells have been tested for the presence or absence of PCMV gb and pol by PCR (Figure 2, embedded images; for primers used see Table 1) and their morphology was compared by microscopy ( Figure 2A structure, surrounded by a lipid layer, that is, characteristic for roseoloviruses ( Figure 3A-D). The viruses are located in the nucleus as well as in the cytosol ( Figure 3A,B); it was shown that cytoplasmic PCMV ( Figure 3C) invades the cellular endosomes for transport and cellular exocytosis just like HHV6 ( Figure 3D). This mechanism for PCMV has been described for roseoloviruses in general. 41 Evaluation of peptide-induced polyclonal rabbit IgG preparations and pig sera for immunofluorescence studies of PFT-PCMV cells. The suitability of PCMV gB epitope specific antibodies a552, a554, a789, a180 and a181 for PCMV detection was tested in vitro on PFT and PFT-PCMV cell lines (Figure 4). Noninfected PFT cells showed no cross-reactivity with the antibodies tested ( Figure 4A)

PCMV gB peptide-based ELISA for detecting anti-PCMV antibodies
The same peptides (except for p552) used to develop rabbit anti-PCMV gB antibodies were used to establish an ELISA for quantifying anti-PCMV antibodies in pig serum (Table 3). ELISA plates were coated with individual peptides or with a combination of the peptides p554, p789, p180, and p181. All peptides bound anti-PCMV antibodies with little nonspecific binding of secondary antibody. Sera from pigs that have been PCR positive for PCMV (Table 4) exhibited weak binding to all individual peptides except p180 that strongly bound (Table 3). In general, the quality of binding was notably enhanced when using a combination of p554, p789, p180, and p181. Negative controls revealed only a weak background. As such, usage of a peptide combination was maintained and the cut-off for positive responders was set to 0.3 OD 405 . Data on the detection limits of qPCR compared to nested-PCR (1-10 copies/µL) used for the detection of positive pigs are provided as a supplement (Supplement S3 Tab. 1, Supplement S3 Fig. 1).

Screening of pig sera for PCMV gB specific antibodies and PCR results for blood samples
With the ELISA based on the combination of the four peptides p554, p789, p180, and p181 (Table 3)

3.6
Results of a blocking ELISA preincubating pig sera with soluble gB derived peptides The specificity of serum antibody binding to synthetic peptides was tested while pre-incubating pig sera that have also been tested by WBanalysis and ELISA with 5 µg or 50 µg peptide-mix per sample before testing them on an ELISA plate coated with the same combination.
Pre-incubation of sera with increasing peptide concentrations lead to a reduced antibody binding to p554, p789, p180, and p181, comparable to non-infected pigs ( Figure 6). In case of the positive control serum (#6732), pre-incubation only led to a limited inhibition of serum antibody binding.

DISCUSSION
In this study we generated synthetic peptides of PCMV gB mainly based on diagnostic sequences published. 32,29 The peptides are localized in the median and C-terminal part of gB described as highly specific for antibody binding. These peptides have been used for rabbit immunization to generate polyclonal antibodies for detection of PCMV The analysis shows a clear band at approximately 70 kDa for a554, a789, a180, and a181 but not for a552. Since the Mw of 859 aa gB is predicted with 97.8 kDa, according to its sequence, and the peptide antibody binding sites are located to the C-terminus of gB, a precurser protein and N-terminal cleavage of the core protein is supposed. (C) Cell lysate (CL) and VI generated from PFT (CL (−); VI (−)) and PFT-PCMV (CL (+); VI (+)) cell lines incubated with pig serum from a PCMV positive tested female sow #6732. Blot shows five distinct bands that respond to the serum. On basis of peptide directed antibodies gB is predicted at 70 kDA and indicated by arrow. (D) In accordance to #6732 blood sera of three different PCR positive and three PCR negative pigs have been tested and compared for PCMV-VI specific antibodies. The results show that all PCMV negative animals inherit varying amounts of anti PCMV directed antibodies indicating the presence of remaining maternal antibodies. In comparison piglet #6249 and piglet #6253 have been positively confirmed for PCMV by PCR and qPCR and show a strong antibody response. Piglet #7649 was PCR negative but also inherits a high antibody level. Since the blood sample analyzed was shortly taken after birth maternal antibodies are assumed.
of corresponding cell types that carry PCMV during latency remains important for successful identification of infected pigs. 23 The Results of the diagnostic, peptide ELISA testing sera of young and adult pigs for PCMV peptide-directed antibodies. For analysis a combination of four PCMV gB based peptides (p554, p789, p180, and p181) was used. Peptide ELISA of pre-incubated pig sera pre-incubation 50µg pre-incubation 5µg not pre-incubated F I G U R E 6 Peptide blocking ELISA. The specificity of serum antibody binding to synthetic peptides p554, p789, p180, and p181 used for ELISA was analyzed by pre-incubating pig sera to be tested with two concentrations of the same peptide-mix. Pre-incubation with 50 µg peptide lead to a complete reduction of the binding that is comparable to the background received with serum samples from PCMV free, uninfected pigs. The inhibition for pig #6732 revealed to be incomplete due to a more complex antibody pattern and higher diversity. required. The testing regimen should be applied to mother sows at (e.g.)

PCMV
half year intervals and, at day of delivery. Early weaning of piglets from positive mothers with combined testing (serology and nested PCR) at predefined intervals (e.g., d15-20 [<d20] for maternal antibodies and >d40 for clearance). Additional testing will be required just prior to xenotransplantation (e.g., 7 days prior XTx) when any increase of antibody should be detected and/or PCMV should be detectable in blood based on the timing of primary infection/transmission from the mother within 4-8 weeks (Figure 7). To exclude false negative PCR results it is suggested to retain a second sample that is in place for a retest. In addition, a resampling may be required in case of a second unclear result.
These results show that a combination of multiple methods is suitable for PCMV detection and monitoring in pig and piglets intended for xenotransplantation. A serum-ELISA and nested-PCR of PCMV in serum-and blood-samples can be supplemented by WB, immunofluorescence and qPCR. In the future, improved diagnostic tools can be

IV.
F I G U R E 7 Serum-ELISA and nested-PCR for PCMV gB detection in piglets intended for Xenotransplantation (XTx). Decreasing maternal antibodies are indicated by blue and newly formed, infection-related antibodies by red arrow. Piglets should be tested within d20 (first sampling) for the presence of maternal antibodies that diminish after d40 (second sampling) and shortly (e.g., 7 days) before transplantation (third sampling). A final testing and analysis of blood and serum taken at transplant day is recommended (final sampling). The results of the serum-ELISA need to be confirmed by nested-PCR monitoring virus copy number. generated based on gB epitope mapping, with investigation of the host immune response, and by differentiation of early and late antigens in addition of gB as a marker antigen. Such approaches will provide insights into PCMV infection and the development of strategies for safer XTx including animal facilities free of PCMV.

AUTHOR CONTRIBUTIONS
All authors contributed substantially to research design, the acquisition, analysis, and interpretation of data. This does include the drafting of the paper as well as critical revision and approval of the submitted and final version.