Materials and methods - Biological characterization of porcine pegivirus

Samples

Serum samples available for Ab detection originated from apparently healthy domestic pigs from Europe (Germany, Italy, Poland, Great Britain, Switzerland and Serbia) and Taiwan, which were used in a previous study on PPgV RNA detection rate (Kennedy et al., 2019). They were collected between 2014 and 2018 within the

framework of national veterinary health management in concordance with national legal and ethical regulations.

Additionally, serum as well as nasal swabs, oral swabs, and fecal and urine samples were collected from six healthy, originally PPgV positive pigs (animals A-F) from a farm in Lower Saxony, Germany, and from initially PPgV negative control pigs (animals G, H, J, and K) from the Farm for Education and Research in Ruthe of the University of Veterinary Medicine Hannover. All animals were housed in the Clinic for Swine and Small Ruminants and Forensic Medicine and Ambulatory Service of the University of Veterinary Medicine Hannover after the initial PPgV RNA screening and were sampled every 2-3 weeks for a maximum of 46 weeks (see Figure 3-4). Excretion samples were taken from all animals at each sampling time point from week 7 to week 46.

Animals A and B, both sows, were inseminated in week 20 while both animals were PPgV RNA positive. All 13 piglets from sow A (P1-P13) and 13 piglets from sow B (P14-P26) were sampled after birth (before colostrum intake), and every 2 weeks thereafter for a maximum of 12 weeks. Samples taken from piglets included serum, nasal, oral and fecal swabs. For the detection of PPgV RNA in piglet excretion samples, swabs of three piglets of each sow were selected for each sampling week. Two vaginal swab samples were taken from each sow during the birth of their piglets. Sampling was approved by Lower Saxony’s official authorities (LAVES AZ 15A602) and was carried out in accordance with German legislation (TierSchVersV).

All swab samples were soaked in 1 ml cell culture media containing antibiotics for a minimum of 2 hours (h), after which media was used for RNA isolation, or stored.

Feces were diluted 1:10 in phosphate buffered saline (PBS), vortexed vigorously and centrifuged for 20 min at 4000 × g, and supernatant was stored. All samples were stored at -80 °C until use.

41 RNA isolation and PCR

Viral RNA was isolated from 200 µl of serum (100 µl of serum from piglets in week 36 due to limited volume available), urine, fecal swab supernatant, or media using the IndiMag Pathogen Kit (Indical Bioscience, Germany) and the KingFisher Duo Prime extractor (Thermo Scientific, Germany) and was eluted in 100 µl elution buffer.

Quantitative reverse-transcription PCR (qRT-PCR) was used to detect PPgV RNA as previously described (Kennedy et al., 2019). Control RNA was added to excretion samples and detected by qRT-PCR (primers EGFP-1-F

(5’-GACCACTACCAGCAGAACAC-3’), and EGFP-2-R

(5’-GAACTCCAGCAGGACCATG-3’) and probe EGFP-HEX

(5’-[HEX]-AGCACCCAGTCCGCCCTGAGCA-[BHQ1]-3’)) to confirm successful RNA isolation, as previously described (data not shown) (Hoffmann et al., 2006).

Complementary DNA of the viral genomic RNA of PPgV isolate PPgV_903/Ger/2013 (GenBank accession number KU351669.1) was synthesized using SuperScript II Reverse Transcriptase (Invitrogen, Germany). PCR for the amplification of the genomic regions encoding the predicted NS3 helicase domain and the truncated E2

was performed using primers PPgV/NS3H/fwd/19

(5’-ATTACTCGAGGTGGTCCCCTGGGCCAACATGCCTCAGGA-3’) and

PPgV/NS3H/rev/20

(5’-CCGCAGATCTGTCATACCACAATCAGTCACAGTGTCA-3’), or

PPgV/E2T/fwd/17 (5’-ATTACTCGAGCTTCTGCTCCTTGCTGCTGCTG-3’) and PPgV/E2T/rev/18 (5’-CCGCAGATCTGTAGGAAACTGGTCTGTGTACTCAT-3’), respectively, containing appropriate restriction sites for subcloning (XhoI and BglII, underlined).

Expression of PPgV NS3 helicase and truncated E2 recombinant proteins

The NS3h (811 bp) and E2t (994 bp) amplicons were cloned into a pET-19B vector (Novagen, Germany) downstream of a polyhistidin tag. Plasmid and inserts were digested using appropriate restriction enzymes (Thermo Scientific) and were purified using the GeneJet Gel Extraction kit (Thermo Scientific) followed by ligation with T4 ligase (New England Biolabs, Germany). Recombinant E. coli Top10 clones were grown in LB-medium and were selected with ampicillin (50 µg/ml). Plasmid integrity was confirmed by PCR and sequencing.

For protein expression, One Shot BL21(DE3)pLysS chemically competent E. coli (Invitrogen) were used according to the manufacturer’s instructions. Fresh cultures were added to 200 ml LB-medium containing 50 µg/ml ampicillin and cultured at 37

°C and 250 rpm. At an optical density at 600 nm of 0.6, bacterial expression was induced with 1 mM Isopropyl-beta-D-thiogalactopyranosid (IPTG) for 2 h. Bacteria were centrifuged at 4000 × g for 20 min at 4 °C. Cell pellets were weighed and frozen at 20 °C. B-PER complete protein extraction reagent (Thermo Scientific) was used for lysis of bacteria and soluble and insoluble cytoplasmic fractions were analyzed for the presence of NS3h and E2t proteins in Coomassie blue stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and Western blot. For E2t protein, the insoluble cytoplasmic fraction was washed four times with B-PER diluted 1:10 and the remaining pellet was resuspended in 1% SDS for use in Western blot.

Purification of PPgV NS3 helicase recombinant protein

Soluble proteins containing NS3h were diluted 1:10 in fast protein liquid chromatography (FPLC) buffer (20 mM sodiumhydrogenphosphate, 500 mM sodiumchloride, pH 7.4) and imidazole was added to a final concentration of 40 mM.

The diluted sample was filtered through a 0.22 µm filter membrane. Protein was purified using the Äkta Pure FPLC and a HisTrap excel 1 ml column (GE Healthcare,

Sweden). After sample application (flow rate 1 ml / min), the column was washed with 20 ml FPLC buffer containing 40 mM imidazole and protein was eluted in 10 fractions of 0.5 ml each of FPLC buffer containing 500 mM imidazole. Elution fractions were analyzed by SDS-PAGE and Coomassie blue staining, as well as WB. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific). Fractions containing high concentrations of target protein were pooled and stored at 4 °C containing 0.1% sodium acid as preservative.

SDS-PAGE, Coomassie blue staining and Western blot

To visualize bacterial protein expression and to confirm FPLC-purification of NS3h protein, diluted bacterial lysate containing crude NS3h or E2t protein, as well as eluted fractions of NS3h, were analyzed on 12.5% SDS-PAGE gels stained with Coomassie blue or blotted onto PVDF membranes in a semidry electroblotting procedure.

Membrane blocking, Ab incubation and washing steps were carried out with TBS-0.1%

Tween20-buffer. PVDF membranes were blocked overnight with 2% Amersham ECL Prime Blocking Reagent (GE Healthcare). Membranes were then incubated with Anti-His6 mouse monoclonal Ab (Roche, Germany) diluted 1:100 for 1 h at RT and then washed four times for 15 min. Polyclonal Rabbit Anti-Mouse Immunoglobulins / HRP (Dako, Denmark) diluted 1:2,000 was then added and incubated again for 1 h at RT, followed by four more steps of washing. Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare) was used for the visualization of bound Ab.

Serum samples were tested on WB membranes obtained after SDS-PAGE and transfer of purified NS3h or crude E2t was performed as described above. Membranes were blocked as described above and incubated with serum diluted 1:500 in blocking reagent for 1 h on a shaker at RT. Membranes were washed and anti-pig IgG (whole molecule)-Peroxidase antibody produced in rabbit (Sigma Aldrich, Germany) diluted 1:30,000 was added for 1 h at RT, followed by another round of washes. Bound Ab was

detected using ECL Prime or ECL Select Western Blotting Detection Reagents (GE Healthcare).

ELISA

Nunc Medisorp ELISA plates (Thermo Scientific) were coated with 250 ng / well (ELISA²⁵⁰) or 100 ng / well (ELISA¹⁰⁰) of NS3h in 100 µl coating buffer (0.1 M NaCO3, pH 9.6) per well over night at 4 °C. Plates were washed three times with 360 µl PBS-0.05% Tween20 (PBS-Tw) and blocked with 4% skimmed milk powder in PBS-Tw for 2 h at room temperature (RT) and subjected to another round of washes. Porcine serum samples were diluted 1:25 in 4% skimmed milk powder in PBS-Tw and 100 µl per well were incubated for 1 h at 37 °C. Plates were washed again and anti-pig IgG (whole molecule)-Peroxidase produced in rabbit (anti-pig IgG, Sigma Aldrich) diluted 1:35,000 in 4% skimmed milk powder in PBSM-Tw was added and incubated for 1 h at 37 °C. After washing, tetramethylbenzidine (Sigma Aldrich) was added and plates were incubated in the dark for 10 min at RT. The reaction was stopped with 1 M hydrochloric acid and optical densities (ODs) were determined automatically (TECAN Sunrise Remote, Tecan, Switzerland) at a wavelength of 450 nm and a reference wavelength of 620 nm.

Results

NS3h protein purification

Purification of NS3h was achieved by immobilized metal ion chromatography (IMAC) in FPLC, which permitted elution of 0.67 mg protein / ml. Western blots and Coomassie blue stained gels were used to assess the elution and the purity of the target protein. Though weak bands of unspecific protein were visible at larger protein sizes, purity of NS3h was high (Figure 3-1).

Figure 3-1. Coomassie gel of NS3h protein before and after purification by IMAC. Lane 1 shows crude soluble protein that was applied onto the HisTrap column before dilution with FPLC buffer. Lane 2 shows flow through of FPLC, indicating proteins that did not bind to the column. Lanes 3, 4 and 5 show different fractions of eluted proteins with NS3h at a size around 37 kDa (compare Figure 3-2, lane 1: detection of NS3h by the anti-His⁶ Ab. Additional unspecific bands can be seen in lanes 3 and 4 at a protein size of ~70 kDa.

NS3h-specific serum antibody reactivity in Western blot and ELISA

PPgV RNA positive and negative serum samples were prescreened using ELISA250 to identify samples suitable for validation of WB and ELISA (data not shown). Further evaluation of 18 samples with varying reactivity in the preliminary ELISA²⁵⁰ was performed by WB and ELISA100. Clearly visible NS3h-specific bands at a protein size of ~37 kDa were seen following incubation with 5 of 18 samples (Figure 3-2). One sample evidenced unspecific bands at a protein size ~70 kDa, and variations in unspecific background reactions were visible, making evaluation of possible bands difficult in 6 samples. Differences between ODs of ELISA²⁵⁰ and ELISA¹⁰⁰ ranged from a factor change of 0.6 (sample in lane 12, Figure 3-2) to 0.92 (sample in lane 3, Figure 3-2; Table 3-1).

Figure 3-2. Western blots of purified NS3h protein incubated with serum samples as first antibody (Ab). Lane 1 shows the position of the NS3h-His fusion protein at a molecular weight of ~37 kDa using the anti-His6 Ab. Lanes 8, 11, 14, 17, and 18 evidence an NS3h-specific band. Background varies from low (lanes 7, 9, 13, 14, 15, and 19) to high (lanes 4, 5, and 18). Unspecific bands at a protein size ~70 kDa can be seen in lane 6.

Table 3-1. Characterization of selected serum samples. ELISA optical densities (OD) of serum samples and their reactivity with NS3h and background Western blot, as well as PPgV RNA detection. Samples are arranged in descending order of their OD in ELISA²⁵⁰(arrow). Detection of NS3h in WB is indicated as positive [+], uncertain [(+)], or negative [-].

E2t-specific serum antibody reactivity in Western blot

The expression of C-terminally truncated E2 was successfully achieved in the E. coli strain BL21(DE3)pLysS used here, and the protein was detectable in the insoluble cytoplasmic fraction of the bacterial lysate using the anti-His6 Ab in WB at a protein size of ~40 kDa (Figure 3-3, lane 1). Due to detection of the target protein E2t in the insoluble fraction indicative of expression in bacterial inclusion bodies, crude protein containing E2t was prepared for WB analyses by repeated washing of the insoluble protein pellet and final resuspension in 1% SDS.

Two samples (one of which is seen in lane 17 of Figure 3-2; the other is not shown here) that evidenced NS3h-specific Ab reactivity in WB and ELISA were implemented in WB with E2t crude protein (equivalent to isolation from 100 µl of bacteria), one of which evidenced an E2t-specific band (Figure 3-3, compare with lane 17 in Figure 3-2).

Figure 3-3. Western blot of crude E2t protein incubated with serum samples that showed NS3h-specific antibody (Ab) reactivity in Western blot and ELISA as first Ab.

Lane 1 shows the position of the E2t-His fusion protein at a molecular weight of ~40 kDa using the anti-His6 Ab. An E2t-specific band is visible in lane 2, while unspecific bands at a protein size ~35 kDa are visible in lanes 2 and 3.

PPgV RNA in serum and excretion samples of pigs and piglets

Six fattening pigs from a farm in Lower Saxony, Germany, were found to be PPgV RNA positive in serum and were moved to the Clinic for Swine and Small Ruminants and Forensic Medicine and Ambulatory Service of the University of Veterinary Medicine Hannover. Animals A and B were continually tested PPgV genome positive (samples taken every 2-3 weeks) by qRT-PCR for 38 and 27 weeks, respectively (Figure 3-4). Animals C, D, E, and F were PPgV RNA negative at the second sampling in week 3 and only animal D was again found PPgV RNA positive from weeks 13 to 33.

Additionally, excretion samples, including nasal and oral swabs, urine and feces, were taken at each time point (from week 7 to week 46) to assess possible routes by which

PPgV might be spread, all of which were found to be negative for PPgV RNA. Initially PPgV genome negative control animal G was found to be PPgV RNA positive in serum in week 17. Other negative control animals H, I and J were tested negative in serum and excretion samples throughout the study.

Animals A and B were inseminated in week 20. While animal A remained PPgV RNA positive for the duration of the pregnancy and until 12 days post-partum, animal B was last found PPgV genome positive in week 27, 50 days after insemination. In week 36, both sows bore 13 piglets each. PPgV RNA was detected in serum of one piglet (P8) directly after birth (before colostrum intake) in week 36 and contained 1,412 RNA copies / ml serum (Cq value 37.61). All other piglet serum samples were tested PPgV RNA negative by qRT-PCR. For each sampling week, excretion samples were selected from three piglets of each sow for detection of PPgV genome, and samples from serum RNA positive piglet P8 in week 36 were additionally included. However, all excretion samples from piglets were found to be PPgV RNA negative.

Figure 3-4. PPgV viral genome quantity in serum (RNA positive results only) during the course of infection in domestic pigs. Sampling periods of each animal, gestation of animals A and B, and piglet sampling are indicated below. Animals A-F were PPgV genome positive at the first sampling time point (week 0). Animals A and B were not sampled during the first four weeks of gestation. Negative control animals G-J were genome negative at their respective first sampling time point. Animals E, F, H, I, and J remained PPgV RNA negative for the duration of sampling, and animal G and piglet P8 were PPgV genome positive only in sampling week 17 and week 36 (piglet sampling directly after birth, before colostrum intake), respectively.

Discussion

Since the discovery of PPgV in recent years, studies have shown that the virus is widely distributed and RNA detection rates ranged from 0% in Serbia, Taiwan and Switzerland in one study to 15.1% in another study from the United States (Kennedy et al., 2019, Yang et al., 2018). Though PPgV infections can persist, genome detection alone may lead to underestimation of virus distribution, as animals with transient and past infections can be overlooked (Baechlein et al., 2016, Dawson et al., 1996).

However, an assay for the detection of Abs induced during PPgV infection, which would aid not only in the estimation of PPgV exposure, but also give deeper insights into the immune response, has not been described so far. Therefore, in this study, PPgV proteins were expressed intracellularly in E. coli to allow for the detection of PPgV-specific Abs using Western blot and indirect ELISA.

The helicase domain of PPgV NS3 was expressed in the soluble cytoplasmic fraction of E. coli, which facilitated the purification with IMAC under native conditions. Purity of the protein was optimal at 40 mM Imidazole during washing and binding and yielded elution with high protein concentrations. NS3h has a predicted protein size of 33.5 kDa, which coincides with results obtained here showing NS3h in Western blot at a size of ~37 kDa.

PPgV NS3h-specific Abs were detectable in porcine sera using Western blot, showing that PPgV NS3 can induce an Ab response in the porcine host. Results obtained here indicate that viremia and NS3h Ab response can be detected simultaneously, similar to observations in EPgV (Lyons et al., 2014). WB with purified NS3h protein evidenced unspecific bands, as they can also be seen in Coomassie blue stained SDS-PAGE gel (Figure 3-1), in one of 18 samples shown here (lane 6 in Figure 3-2). All other serum samples reacted only with NS3h, suggesting that protein purity is sufficient for the methods implemented here.

Comparison of WB and ELISA results indicates that samples showing a clear band in WB also have high ODs in ELISA. Nonetheless, ODs over 0.93 in ELISA²⁵⁰ were found in serum samples that did not detect a clear NS3h-specific band in WB. So far, it is unclear whether such high ELISA reactivities may be caused by ELISA background (compare lane 2 in Figure 3-2) or whether WB background conceals NS3h-specific bands. To yield better differentiation between Ab positive and negative samples, ongoing experiments target further improvement of ELISA and WB conditions to reduce background caused by serum components binding to ELISA plates and WB membranes unspecifically, such as changing washing conditions (i.e. higher concentrations of Tween20). Furthermore, E2t appears to be a promising immunogenic PPgV protein for implementation in indirect Ab ELISA. The necessity of measuring anti-E2 Abs, specifically those that are protein conformation-dependent, is evident in HPgV serology (Berg et al., 1999, Tanaka et al., 1998, McLinden et al., 2006, Pilot-Matias et al., 1996b, Tacke et al., 1997b). Experiments pertaining to the expression of soluble E2t or purification of E2t under denaturing conditions followed by refolding are ongoing.

This study also investigated possible transmission routes of PPgV. Analysis of PPgV genome in nasal discharge, saliva, urine and feces showed that these are not probable routes of virus shedding from persistently or transiently infected pigs. Likewise, vaginal swab samples of animals A and B taken during the birth of their piglets did not contain PPgV RNA, though animal A was viremic at the time, indicating that transmission during birth may be unlikely. However, detection of PPgV RNA in the serum of piglet P8 directly after birth suggests intrauterine infection. Sows were housed separately for birth of piglets and P8 was born of sow B, which cleared viremia before day 69 of gestation, making cross contamination of the piglet sample highly unlikely. Intrauterine infection of P8 likely took place before day 69 of the gestation, when PPgV RNA was detectable in serum of the sow. Clearance of the virus from serum of P8 by week 38 (at 16 days of age) could have been facilitated by maternally

derived Abs. None of the other piglets were found PPgV RNA positive for up to 10 weeks of age, suggesting that transmission from sow A, which was viremic at the time, to piglets did not occur. The fact that this phenomenon was only found in one of 26 piglets, and that the Cq value was relatively high (37.61), indicates that intrauterine infection is possible, but that the vertical transmission route is not efficient in pegivirus infection of pigs. This coincides with higher PPgV genome detection rates found in older animals than in piglets, as was seen in previous studies (Lei et al., 2019, Kennedy et al., 2019). Blood-borne transmission between pigs could for instance take place iatrogenically during vaccinations, drug injections or other treatments, through direct blood contact of wounded pigs, or due to cannibalism.

Results obtained here show that PPgV is most probabaly not transmitted by virus shedding in excretion, but that horizontal transmission by the blood-borne route and occasional intrauterine transmission, as can be found in HPgV, are more likely (Dawson et al., 1996, Linnen et al., 1996, Schmidt et al., 1996, Feucht et al., 1996). This study also provides first insights into the PPgV-specific immune response by detection of NS3 and E2-specific Abs in PPgV genome positive and negative porcine serum samples. The establishment of reliable indirect ELISAs will allow for high throughput Ab detection in serum samples and will increase the understanding of the immune response induced during pegivirus infection. Furthermore, investigation of the Ab presence in other species with improved test systems will give deeper insights into the host range and elucidate possible reservoir hosts like wild boar, where PPgV genome has not been detected so far (Kennedy et al., 2019).

References

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BAECHLEIN, C., GRUNDHOFF, A., FISCHER, N., ALAWI, M., HOELTIG, D., WALDMANN, K. H. &

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BERG, T., MULLER, A. R., PLATZ, K. P., HOHNE, M., BECHSTEIN, W. O., HOPF, U., WIEDENMANN, B., NEUHAUS, P. & SCHREIER, E. 1999. Dynamics of GB virus C viremia early after orthotopic liver transplantation indicates extrahepatic tissues as the predominant site of GB virus C replication.

Im Dokument Biological characterization of porcine pegivirus (Seite 51-71)