Kennedy J, Hoeltig D, Becher P, Baechlein C.
This chapter is a manuscript in preparation.
Contribution as first author:
Experimental work: Sample preparation of serum and excretion samples, RNA isolation, screening for PPgV RNA presence by TM qRT-PCR, RT-PCR and sequencing of PPgV in serum samples, cloning of plasmids and bacterial expression of PPgV proteins, establishment of purification protocol for PPgV NS3h protein, establishment of SDS-PAGE and Western blot protocols for testing serum sample reactivity with PPgV proteins, screening of serum samples for PPgV-specific antibodies by ELISA.
Evaluation and scientific presentation of the results: Analyses and graphical presentation of qRT-PCR results, presentation of Western blot and antibody-ELISA results. Scientific writing: preparation of the manuscript, tables and figures.
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Abstract
Porcine pegivirus (PPgV) is a positive-sense ssRNA virus belonging to the Pegivirus genus within the Flaviviridae family that can cause persistent infections in its host. The association of PPgV infection with disease remains unknown, but viral genome has been detected in up to 15.1% of pigs from the United States, China and Europe. Thus far, there are no reports on the virus transmission routes and assays for the detection of PPgV-specific antibodies (Abs) are not available. Therefore, this study investigated serum and excretion samples from PPgV viremic pigs to determine possible virus shedding. Additionally, we expressed PPgV non-structural protein 3 helicase domain (NS3h) and C-terminally truncated E2 (E2t) intracellularly in E. coli for use in Ab detection. Purified NS3h showed reactivity with porcine serum in Western blot (WB) and indirect enzyme-linked immunosorbent assay (ELISA), and crude E2t (from bacterial inclusion bodies) was reactive in WB. Thus both, NS3h and E2t, appear to be immunogenic and are viable candidates for the further validation of ELISA methods.
Detection of PPgV RNA in serum and excretion samples of PPgV viremic pigs and their piglets revealed one piglet born of a sow, which cleared viremia by day 69 of gestation, was positive in serum directly after birth (before colostrum intake), indicating intrauterine infection. Lack of PPgV genome detection in excretion samples suggested that PPgV may rather be transmitted horizontally via the blood-borne route.
This study reports the first methodological steps towards investigating the immune response induced by PPgV and provides insights into possible viral transmission routes.
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Introduction
Porcine pegivirus (PPgV) was first discovered in 2016 in apparently healthy domestic pigs from Germany (Baechlein et al., 2016). It belongs to the genus Pegivirus, which comprises a group of positive-sense, single-stranded RNA viruses within the Flaviviridae family (Smith et al., 2016). Persistent pegivirus infections have been found in humans and horses and were also present in three pigs from Germany, in which viral RNA was detected for up to 22 months (Berg et al., 1999, Tanaka et al., 1998, Kapoor et al., 2013a, Baechlein et al., 2016). As in other pegiviruses, the pathogenicity of PPgV is so far unknown. Although PPgV RNA has been found in porcine serum samples from a farm with pigs exhibiting vesicular disease and lameness, there is no clear association between PPgV infection and disease, and most frequently the virus has been detected in apparently healthy pigs with no clinical signs of viral infection (Yang et al., 2018, Baechlein et al., 2016, Kennedy et al., 2019).
PPgV is widely distributed and has been detected in domestic pigs from North America, China, and different countries in Europe (Yang et al., 2018, Lei et al., 2019, Chen et al., 2019, Baechlein et al., 2016, Kennedy et al., 2019). The highest RNA detection rate was reported in 11 of 67 (16.4%) clinical serum or tissue samples from China (Chen et al., 2019). However, reports on PPgV-specific antibody (Ab) detection are lacking and viral genome detection alone may result in the underestimation of the abundance of PPgV occurrence in domestic pig herds and further potential hosts.
Ab detection is an important diagnostic tool for HPgV, as viremia and envelope protein 2 (E2) Ab response are mutually exclusive markers of infection in most individuals (Stapleton et al., 2011). Because of this, investigations on the exposure rate of HPgV require both E2-Ab and RNA detection (Gutierrez et al., 1997, Thomas et al., 1998). Different serological assays have been established by expression of full-length or C-terminally truncated E2 protein in E. coli or mammalian cells (Chinese hamster
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ovary (CHO) and Baby hamster kidney (BHK-21) cells) (Dawson et al., 1996, Dille et al., 1997, Pilot-Matias et al., 1996a, Tacke et al., 1997a). In most developed countries, HPgV RNA detection rates and HPgV E2-Ab-positive rates in volunteer blood donors amount to 1-4% and 5-13%, respectively, with higher rates in developing countries (Blair et al., 1998, Gutierrez et al., 1997, Pilot-Matias et al., 1996a, Tacke et al., 1997b, Mohr and Stapleton, 2009, Williams et al., 2004). HPgV RNA and Ab are more prevalent in high risk groups, such as intravenous drug users or people suffering from other blood-borne or sexually transmitted infections, reaching exposure rates over 80%
(Williams et al., 2004, Scallan et al., 1998, Tacke et al., 1997b, Stapleton et al., 2011).
Clearance of HPgV from blood occurs within two years in most immunocompetent individuals and coincides with the development of protein conformation-dependent, long-lasting anti-E2 Abs (Berg et al., 1999, Tanaka et al., 1998, McLinden et al., 2006, Pilot-Matias et al., 1996b, Tacke et al., 1997b). Due to the fact that the Ab response appears to be restricted to E2, this antigen is assumed to possess the immunodominant epitopes (McLinden et al., 2006).
Equine pegivirus (EPgV) RNA has been detected in 0.8% to 14.2% of horses from the United States, China and Brazil (Kapoor et al., 2013a, Lyons et al., 2014, Lu et al., 2016, Agnello et al., 1999, Figueiredo et al., 2019). An ELISA for the detection of Abs against this virus was established using the non-structural protein 3 helicase domain (NS3h) and bacterial expression in E. coli (Lyons et al., 2014). In the study, 218 of 328 (66.5%) horses were positive for NS3h Abs in ELISA, of which 88% (192 of 218) were confirmed by Western blot (WB). Contrary to findings in HPgV with E2 Ab assays, in the NS3 Ab assay for EPgV, 10 of the 12 RT-PCR positive horses were also Ab positive (Lyons et al., 2014).
Transmission of human pegivirus can occur both parenterally through blood and sexual contact, as well as vertically (Dawson et al., 1996, Linnen et al., 1996, Schmidt et al., 1996, Feucht et al., 1996). The transmission of EPgV has not been studied in detail.
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However, transmission of Theiler’s disease associated virus (TDAV, Pegivirus E) between horses has been shown to be possible by experimental inoculation (Chandriani et al., 2013). Similarly, pegiviruses can be transmitted to different species of New World monkeys by experimental infection via the blood-borne route (Stapleton et al., 2011). Transmission routes of pegiviruses infecting bats were not extensively studied; however, viral RNA was detected in saliva of viremic bats, suggesting that horizontal or even zoonotic transmission may be possible (Epstein et al., 2010).
To our knowledge, an assay for the detection of PPgV-specific Abs has not been described and studies on virus transmission between pigs have not been reported.
Therefore, we expressed PPgV proteins NS3h and C-terminally truncated E2 (E2t) intracellularly in E. coli and implemented WB and indirect ELISA methods for the detection of PPgV-specific serum Abs. A serological assay will permit the investigation of PPgV infection characteristics, including time point of seroconversion, relevance and relation of IgM, IgG, and IgA Abs, as well as maternally derived Abs. During the study, PPgV field infected domestic pigs were monitored alongside their piglets and serum and excretion samples were obtained every 2-3 weeks for evaluation of PPgV RNA presence and potential serological responses. Taken together, this study provides the first insights into possible PPgV transmission routes and PPgV-specific Ab detection.