Abstract
We investigated the porcine lymphotropic herpesvirus (PLHV) DNA presence in multiple organs of pigs. Biological samples (n = 136) included tissue fragments of the central nervous system, heart, kidney, liver, lungs, spleen, urinary bladder, and urine. Sixty-eight (50%) organs were PLHV DNA-positive. None of the urine samples were detected with the virus genome. Although the presence of the PLHV DNA in the urinary bladder and kidney has been detected, it was not possible to show whether urine can be considered an effective route of virus shedding. This study warns to the risk of PLHV zoonotic transmission by xenotransplantation of tissues of porcine origin.
Electronic supplementary material
The online version of this article (10.1007/s42770-020-00335-9) contains supplementary material, which is available to authorized users.
Keywords: Suid gammaherpesvirus, Pan-herpesvirus, PLHV, Swine
Introduction
Suid gammaherpesvirus 3, 4, and 5 are viral species of Macavirus genus, Gammaherpesvirinae subfamily within the Herpesviridae family. Suid gammaherpesvirus 3 and 4 were first reported in 1999 and the viral types were designated as porcine lymphotropic herpesvirus 1 and 2 (PLHV-1 and PLHV-2), respectively [1]. Later, another Suid gammaherpesvirus was found with considerable genomic differences relative to PLHV-1 and PLHV-2, and named Suid gammaherpesvirus 5 (porcine lymphotropic herpesvirus 3—PLHV-3) [2].
PLHVs affect domestic and feral pigs [2]. The pathogenicity of PLHV is not currently clear; however, studies have shown that PLHV-1 may be associated with post-transplantation lymphoproliferative disease (PTLD) in pigs [3, 4]. Studies have reported that it is possible that PLHV-2 and PLHV-3 are also associated with PTLD since these viral species share genomic and biological features with PLHV-1 [2]. The symptoms of PTLD in pigs are similar to those reported in humans, with lymphadenomegaly and leukocytosis. The major concern regarding PLHV infections is their potential xenozoonotic feature. To the knowledge of the authors, there are no pieces of evidence that PLHV can replicate in cells of human origin nor serological tests in human populations for screening PLHV-specific antibodies. Even though herpesviruses are generally species-specific, the risk of infection in the human recipient after transplantation is a concern due to different factors associated with both recipient and infectious agents, such as immunosuppression, inflammatory responses, and intracellular pathogens and latent viruses (reviewed in [5]). In vitro studies have shown that PLHV-1 and human herpesviruses may feature mutual interactions, potentially triggering either human herpesvirus reactivation or PLHV-1 gene expression [6]. Therefore, the risk of humans may be infected by these viruses after xenotransplantation procedures with organ or tissue of swine origin as well as the potential for pathogenicity and synergism with pre-existing herpesviral infections cannot be ruled out [6–8].
The first detections of PLHV-1, PLHV-2, and PLHV-3 were based on a pan-herpesvirus consensus PCR assay that targeted a highly conserved region of the herpesvirus DNA polymerase [9]. Since then, PLHV DNA has been detected mainly in peripheral blood mononuclear cells, lungs, and lymphoid organs of pigs in different countries [7]. The B cell lymphocyte is considered the main target for PLHV infection in pigs [2]. Studies showed that the prevalence of PLHVs is high in some European countries [1, 9, 10]. However, there are no reports on the circulation of this virus in the Americas. The aim of this study was to investigate the presence of PLHV DNA in different organs of pigs from Brazil.
Materials and methods
Organs included in this study were part of the collection of pig biological samples of the Laboratory of Animal Virology/DMVP/CCA/UEL and were stored at − 80 °C. Nineteen pigs that had a large number of organs/tissue fragments collected for routine diagnostic investigations were selected. Pigs were grouped according to their age into 4–18 (n = 6), 35–130 (n = 7), and 150–210 (n = 6) days old. Samples (n = 136) analyzed were tissue fragments of the cerebrum (n = 19), cerebellum (n = 19), brainstem (n = 17), spinal cord (n = 14), heart (n = 7), kidney (n = 15), liver (n = 8), lungs (n = 16), spleen (n = 8), and urinary bladder (n = 7). Additionally, urine samples from the six pigs aged 150–210 days were also included in the analysis.
Individual tissue samples were mechanically disrupted with MagNa Lyser Instrument (Roche Diagnostics®, Mannheim, Germany), homogenized in 0.01 M phosphate-buffered saline (PBS), pH 7.2, and clarified by centrifugation at 2000×g for 10 min. Nucleic acid extraction was performed from 500 μL of proteinase K pre-treated tissue suspensions using the phenol/chloroform/isoamyl alcohol followed by the silica/guanidine isothiocyanate method [11, 12]. For the urine samples, 250-μL aliquots were submitted to the silica/guanidinium isothiocyanate extraction method [12]. The extracted nucleic acid was eluted in 50 μL of diethylpyrocarbonate-treated water (DEPC) and stored at − 20 °C until analyses.
The first screening assay for the presence of PLHV DNA was performed by PCR assay with consensus primers (pan-herpesvirus) for the partial amplification of the herpesviral DNA polymerase gene, as previously described [13]. Following, to determine PLHV type, all the samples that presented positive results for PLHV DNA with the pan-herpesvirus primers were submitted to three single-round PCR assays with specific primers for PLHV-1 (213-S/215-AS) [1, 10], PLHV-2 (208-S/212-AS) [1, 10], and PLHV-3 (886-S/886-AS) [2], with expected product sizes of 393 bp, 334 bp, and 148 bp, respectively.
Five positive samples for each of the PLHVs were randomly selected for sequencing analysis in order to confirm the identity of the amplicons. The amplicons were purified using the PureLink® Quick Gel Extraction and PCR Purification Combo Kit (Invitrogen™ Life Technologies, Carlsbad, CA, USA), according to the manufacture’s instructions, quantified by a Qubit® Fluorometer (Invitrogen™ Life Technologies, Eugene, OR, USA), and sequenced in both directions using an ABI 3500 Genetic Analyzer Sequencer with the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems™, Foster City, CA, USA). Reactions were performed in final solutions of 10 μL containing 1x BigDye Terminator v3.1 ready premix, 1x BigDye Terminator v3.1 Sequencing Buffer, and 5 μM of each primer (forward and reverse). The quantities of PCR templates were determined according to the product size in 2 ng/μL (< 300 bp) or 4 ng/μL (> 300 bp). Sequence quality analyses and consensus sequences were assembled using Phred/Phrap/CAP3 software (http://asparagin.cenargen.embrapa.br/phph/). Similarity searches were performed with sequences deposited in GenBank using the Nucleotide Basic Local Alignment Search Tool—BLASTn software (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Nucleotide search was performed using the standard database and the megablast algorithm (word size 28). The identity of the nt sequences was confirmed by comparison with reference sequences available in the public database (GenBank). Sequence alignment and identity matrix creation were performed using BioEdit software version 7.2.5. A phylogenetic tree based on nucleotide (nt) sequences representative of diverse gammaherpesviruses was obtained using the maximum-likelihood method and the Kimura 2-parameter model [14], which provided statistical support with 1000 bootstrap replicates using the MEGA package (version 7.0) [15].
Results and discussion
PLHV DNA was not detected in any of the samples collected from pigs at 4–18 days of age. Conversely, 12 of 13 pigs aged 35–130 (6/7) or 150–210 (6/6) days presented the PLHV DNA in at least two different organ samples. According to the samples, 68/136 (50%) of the tissue fragments presented the virus DNA. The organs most frequently detected with the PLHV DNA were the lymphoid organs (liver and spleen), followed by the urinary bladder, lungs, and heart. However, none of the urine samples was detected with the virus genome. Table 1 presents the PLHV DNA detection in the organ samples of pigs in each age group.
Table 1.
Porcine lymphotropic herpesvirus DNA detection in organ samples of 19 pigs according to the age groups
| Organ samples | Days of age (total number of pigs) | Total (n = 12/19) | ||
|---|---|---|---|---|
| Positive/number of samples tested | ||||
| 4–18 (n = 6) | 35–130 (n = 7) | 150–210 (n = 6) | ||
| Cerebrum | 0/6 | 5/7 | 3/6 | 8/19 |
| Cerebellum | 0/6 | 4/7 | 3/6 | 7/19 |
| Brainstem | 0/6 | 4/5 | 2/6 | 6/17 |
| Spinal cord | 0/6 | 2/4 | 3/4 | 5/14 |
| Heart | NA | 2/2 | 2/5 | 4/7 |
| Kidney | 0/6 | 3/3 | 4/6 | 7/15 |
| Liver | NA | 2/2 | 6/6 | 8/8 |
| Lung | 0/6 | 4/4 | 6/6 | 10/16 |
| Spleen | NA | 2/2 | 6/6 | 8/8 |
| Urinary bladder | NA | 1/1 | 4/6 | 5/7 |
| Urine | NA | NA | 0/6 | 0/6 |
| Total | 68/136 | |||
NA, not available
According to the different PLHV types, single infections by PLHV-1 or PLHV-2 were detected in two (#4 and 6) and one (#1) pigs, respectively. Double infection with PLHV-1 and PLHV-2 was detected in pig #11. All the other animals were detected with triple infections by PLHV-1, PLHV-2, and PLHV-3 (Table 2).
Table 2.
Porcine lymphotropic herpesvirus (PLHV)-1, PLHV-2, and PLHV-3 DNA detection in different organ samples of the 13 pigs aged 35 to 210 days
| Organ sample | PLHV type | Pig number | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 30–135 days old | 150–210 days old | |||||||||||||
| #1 | #2 | #3 | #4 | #5 | #6 | #7 | #8 | #9 | #10 | #11 | #12 | #13 | ||
| Cerebrum | PLHV-1 | − | − | + | + | − | + | + | + | − | + | − | + | − |
| PLHV-2 | − | − | − | − | − | − | + | − | − | + | − | − | − | |
| PLHV-3 | − | − | − | − | − | − | − | + | − | − | − | + | − | |
| Cerebellum | PLHV-1 | − | + | + | + | − | + | + | + | − | + | − | + | − |
| PLHV-2 | − | − | − | − | − | − | − | − | − | + | − | + | − | |
| PLHV-3 | − | + | + | − | − | − | − | − | − | − | − | + | − | |
| Brainstem | PLHV-1 | NA | NA | + | + | − | + | + | + | − | + | − | − | − |
| PLHV-2 | + | − | − | − | + | − | − | + | − | − | − | |||
| PLHV-3 | + | − | − | − | + | + | − | − | − | − | − | |||
| Spinal cord | PLHV-1 | NA | − | NA | + | − | + | NA | + | NA | + | − | + | NA |
| PLHV-2 | − | − | − | − | − | + | − | − | ||||||
| PLHV-3 | − | − | − | − | + | − | − | + | ||||||
| Heart | PLHV-1 | NA | NA | + | NA | NA | NA | + | + | − | + | − | − | NA |
| PLHV-2 | − | + | − | − | + | − | − | |||||||
| PLHV-3 | + | − | + | − | − | − | − | |||||||
| Kidney | PLHV-1 | + | NA | + | NA | NA | NA | + | + | − | + | − | + | + |
| PLHV-2 | − | − | + | − | − | + | − | + | + | |||||
| PLHV-3 | − | + | − | + | − | − | − | + | − | |||||
| Liver | PLHV-1 | NA | NA | + | NA | NA | NA | + | + | + | + | + | + | + |
| PLHV-2 | − | + | − | − | + | + | + | + | ||||||
| PLHV-3 | + | − | + | + | − | − | + | + | ||||||
| Lungs | PLHV-1 | + | + | + | NA | NA | NA | + | + | + | + | + | + | + |
| PLHV-2 | + | + | + | + | − | + | + | + | + | + | ||||
| PLHV-3 | − | + | + | − | + | + | − | − | + | + | ||||
| Spleen | PLHV-1 | NA | NA | + | NA | NA | NA | + | + | + | + | + | + | + |
| PLHV-2 | + | + | + | − | + | + | + | + | ||||||
| PLHV-3 | + | − | + | + | + | − | + | + | ||||||
| Urinary bladder | PLHV-1 | NA | NA | + | NA | NA | NA | NA | + | − | + | − | + | − |
| PLHV-2 | − | − | − | + | − | + | + | |||||||
| PLHV-3 | + | + | − | − | − | + | − | |||||||
NA, not available
+, positive
−, negative
Five samples positive for each of the PLHVs were randomly selected for sequencing analysis (Fig. 1). The megablast analysis provided 8 hits for PLHV-1, 6 hits for PLHV-2, and 4 hits for PLHV-3. These results are summarized in the Online Resource 1. All the PLHV-1 nt sequences obtained in this study were 100% identical to each other. Similar results were obtained for PLHV-2. Therefore, one sequence of PLHV-1 and one sequence of PLHV-2 were selected to represent the Brazilian strains herein. When compared with each other, PLHV-1 and PLHV-2 strains in this study were 94.7% nt similar. The comparison of the Brazilian PLHV-1 (Table 3) and PLHV-2 (Table 4) strains with other nt sequences from reference strains revealed similarities from 99 to 100% (Online Resource 2). As regards to PLHV-3, the nt sequences were 100% identical to each other and with the reference strains (Table 5). The phylogenetic tree constructed with gammaherpesvirus sequences from different host species confirmed the specificity of the amplicons in this study, showing that the Brazilian PLHV strains grouped together with representative sequences of PLHV-1, PLHV-2, and PLHV-3 (Fig. 2).
Fig. 1.
Electrophoresis (2% agarose gel) of porcine lymphotropic herpesvirus (PLHV)-1, PLHV-2, and PLHV-3 amplicons submitted to sequencing analysis. Lanes M = molecular marker (100 bp). Lanes 1 to 5 = PLHV-1 amplicons with 393 bp. Lanes 7 to 11 = PLHV-2 amplicons with 334 bp. Lanes 13 to 17 = PLHV-3 amplicons with 148 bp. Lanes 6, 12, and 18 = negative control
Table 3.
Percentages of similarities of the porcine lymphotropic herpesvirus 1 (PLHV-1) nucleotide sequence in this study and representative strains based on the partial (393 nt) polymerase gene
Table 4.
Percentages of similarities of the porcine lymphotropic herpesvirus 2 (PLHV-2) nucleotide sequence in this study and representative strains based on the partial (323 nt) polymerase gene
Table 5.
Percentages of similarities of the porcine lymphotropic herpesvirus 3 (PLHV-3) nucleotide sequence in this study and representative strains based on the partial (146 nt) polymerase gene
Fig. 2.
Molecular phylogenetic analysis by maximum likelihood method. The evolutionary history was inferred by using the maximum likelihood method based on the Kimura 2-parameter model [14]. Brazilian PLHV strains in this study are highlighted with a black-filled circle (PLHV-1/BRA-UEL349/2016, GenBank accession number MN873041), triangle (PLHV-2/BRA-UEL349/2016, GenBank accession number MN873042), and square (PLHV-3/BRA-UEL268/2016). The GenBank accession numbers of the reference sequences are presented in the tree. The tree with the highest log likelihood (− 1235, 0386) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying neighbor-join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 29,299)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 38 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 145 positions in the final dataset. Bootstrap values higher than 60% are shown. Evolutionary analyses were conducted in MEGA7 [15]
This study describes for the first time the detection of PLHV DNA in pigs of Brazil. The results are in agreement with previous studies that reported PLHV in different organs of pigs such as spleen, lung, liver, and kidney [16]. Currently, the availability of organs for allograft transplantation (among individuals of the same species) is lower than the demand. Therefore, several studies have sought alternative approaches, including xenotransplantation [17]. Swine is an option to solve the lack of organs for transplants since this animal species may represent a source of multiple organs for humans [18]. However, human xenotransplant recipients may be at risk of infection with animal viruses, including porcine endogenous retroviruses (PERV), porcine cytomegalovirus (PCMV), and PLHV. In this regard, there is a concern about the potential risk of human infection with these viruses in xenotransplantation using virus-contaminated organs or tissues of swine origin [8].
To the knowledge of the authors, this is the first report of PLHV DNA in pig biological samples consisting of the urinary bladder and central nervous system (CNS). As PLHVs are detectable in blood, mainly B cells [2], the detection of PLHVs in different organs supplied with blood, including urinary bladder and CNS, is not surprising. Although not always at significant titers, studies have shown that the herpesviruses may also be present in the urine of humans [19, 20] and other animal species, including swine [21, 22]. Although there is a presence of PLHV DNA in paired kidney and urinary bladder samples of pigs in this study and the consistent results from these tissues, the urinary shedding of PLHV was not evidenced herein. This may be explained by the likely latent condition of PLHV infection at the moment of the sample collection since during latency the viral DNA remains silent. In this case, it is possible to detect the viral DNA even without viral replication and shedding. Another explanation can be undetectable amounts of PLHV DNA in urine samples. It is possible to assume that, in this case, the kidneys and urinary bladder did not represent important targets during virus infection, with low replication rates and viral loads in the analyzed animals, and consequent undetectable DNA levels in urine. Finally, it is possible that the negative results obtained from the urine samples of these animals were influenced by the time elapsed between the animal's death, urine collection, and diagnostic test, leading to the degradation of the viral DNA with consequent false-negative results. Because the PCR assay detects only the viral DNA and does not provide evidence on viral replication, additional analysis based on complementary techniques, such as immunohistochemistry, in situ hybridization, and quantitative PCR should be conducted to confirm PLHV active infection and this previous hypothesis. Additionally, although the negative results from pigs at 4-18 days of age are in agreement with a previous study [23], we cannot rule out the possibility of the negative results be due to low PLHV genomic copies in the analyzed organs. Additional serological analysis also should be performed as a comparative assay to certainly assert the negative results, especially from pigs at this age group.
In summary, this study reports for the first time in Brazil the circulation of PLHV in pigs and is the first description of the virus DNA in samples of the urinary bladder and CNS. However, PLHV was not being excreted through the urinary pathway of animals included in this study and, therefore, it was not possible to show whether urine can be considered an effective route of virus elimination. Further studies under natural and experimental conditions of PLHV infection may contribute to the knowledge of the virus elimination pathways in pigs of different age groups. On the basis of the knowledge that transplantation of cells, tissues, and organs among different species is a potential solution for the current transplant demands and considering the xenotransplantation practice may thrive, a perspective may be raising pigs towards this goal. Tucker et al. [24] have shown that cesarean-derived pigs raised under controlled biosecure barrier environment are less detected with PLHVs. In this sense, studies are necessary seeking for improvements of the rearing conditions towards a PLHV-free environment. In addition, glucocorticoid-based immunosuppressive experimental studies may be a useful model to verify PLHV reactivation and/or shedding under stressful condition. Even causing asymptomatic infections in pigs, the study warns of the risk of zoonotic transmission of the virus. The monitoring of the health status of pigs regarding potentially xenotic pig viruses should be constantly performed, especially considering that heart valves and other tissues of pigs are used for various orthopedic procedures and that companies that provide porcine bioprostheses obtain the valves from slaughterhouses [18].
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Funding information
The authors thank the following Brazilian Institutes for financial support: the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES), the National Council of Technological and Scientific Development (CNPq), Financing of Studies and Projects (FINEP), and the Araucaria Foundation (FAP/PR). Alfieri AF, Alfieri AA, Headley SA, and Oliveira TES, are recipients of CNPq fellowships. Dall Agnol AM is a recipient of the FAP/PR fellowship. This study was funded by FAP/PR (grant number 88887.354496/2019-00).
Data availability
The datasets generated and analyzed during the current study are available in the GenBank under the accession numbers MN873041 (PLHV-1/BRA-UEL349/2016) and MN873042 (PLHV-2/BRA-UEL349/2016). The representative nt sequence of PLHV-3 (BRA-UEL268/2016) in this study is shorter than 200 bp and therefore was not submitted to the GenBank, but is available from the corresponding author on reasonable request. Additional data analyzed during this study are included in this article and its supplementary information files.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
The study was submitted to the Ethics Committee on Animal Experiments of the Universidade Estadual de Londrina and approved under the identification number 11363.2015.16. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
Footnotes
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Associated Data
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Supplementary Materials
(PDF 434 kb)
(PDF 447 kb)
Data Availability Statement
The datasets generated and analyzed during the current study are available in the GenBank under the accession numbers MN873041 (PLHV-1/BRA-UEL349/2016) and MN873042 (PLHV-2/BRA-UEL349/2016). The representative nt sequence of PLHV-3 (BRA-UEL268/2016) in this study is shorter than 200 bp and therefore was not submitted to the GenBank, but is available from the corresponding author on reasonable request. Additional data analyzed during this study are included in this article and its supplementary information files.