Viral metagenomic analysis of bushpigs (Potamochoerus larvatus) in Uganda identifies novel variants of Porcine parvovirus 4 and Torque teno sus virus 1 and 2
© Blomström et al.; licensee BioMed Central Ltd. 2012
Received: 19 March 2012
Accepted: 5 September 2012
Published: 11 September 2012
As a result of rapidly growing human populations, intensification of livestock production and increasing exploitation of wildlife habitats for animal agriculture, the interface between wildlife, livestock and humans is expanding, with potential impacts on both domestic animal and human health. Wild animals serve as reservoirs for many viruses, which may occasionally result in novel infections of domestic animals and/or the human population. Given this background, we used metagenomics to investigate the presence of viral pathogens in sera collected from bushpigs (Potamochoerus larvatus), a nocturnal species of wild Suid known to move between national parks and farmland, in Uganda.
Application of 454 pyrosequencing demonstrated the presence of Torque teno sus virus (TTSuV), porcine parvovirus 4 (PPV4), porcine endogenous retrovirus (PERV), a GB Hepatitis C–like virus, and a Sclerotinia hypovirulence-associated-like virus in sera from the bushpigs. PCR assays for each specific virus combined with Sanger sequencing revealed two TTSuV-1 variants, one TTSuV-2 variant as well as PPV4 in the serum samples and thereby confirming the findings from the 454 sequencing.
Using a viral metagenomic approach we have made an initial analysis of viruses present in bushpig sera and demonstrated for the first time the presence of PPV4 in a wild African Suid. In addition we identified novel variants of TTSuV-1 and 2 in bushpigs.
Wild animals are carriers of a number of pathogens that have the potential to infect the human population and/or domestic animals. The intensity of contact between wildlife, livestock and human population is increasing due to a number of factors, primarily human and livestock population growth leading to encroachment onto wildlife habitat [1, 2]. During recent decades a number of pathogen crossovers from wildlife to humans and livestock have occurred resulting in emerging diseases, such as SARS, Hantavirus Pulmonary syndrome 1, Nipah virus disease 1, and Hendra virus-induced diseases among others. However, it is also evident that transmission can occur both ways i.e. pathogens may spill-over to wildlife from humans and/or from livestock. One example of this is the spill-over of Canine distemper virus from domestic dogs (Canis familiaris) to African wild dogs (Lycaon pictus) in Serengeti in 1991 leading to local extinction of wild dogs in the area . Apart from being carriers of novel viruses with the potential to cause disease in naïve domestic hosts, wildlife may also act as reservoirs for known viral pathogens – for example there are a number of wildlife reservoirs for foot and mouth disease virus such as African buffalo (Syncerus caffer), reindeer (Rangifer tarandus) and wild boar (Sus scrofa) . However, information on the viral flora in wildlife is typically scarce or non-existent.
Traditional viral detection methods, such as virus isolation, are often hindered by the inability to grow virus in cell culture. The divergence of many viruses and absence of a common viral marker gene makes detection difficult using standard molecular techniques including PCR and microarray as they are frequently target-specific through the use of specific primers, probes and/or antibodies. Viral metagenomics is a sequence, and culture-independent approach that has proven to be a valuable tool for the investigation not only of diseases of unknown etiology but also of the complete viral flora of different reservoirs and vectors. By providing insights into a wide range of unknown potential pathogens and revealing novel aspects of biodiversity, metagenomics is able to detect and characterise novel pathogens [4–6].
In many rural parts of the developing world, domestic livestock are kept in free-range systems, potentially allowing contact with wild animals. In some parts of Uganda, free-range scavenging by pigs is frequent. At the same time wild species of suidae such as bushpigs (Potamochoerus larvatus), with a wide distribution in Eastern and Southern Africa, live and move at the interface between the national parks and the farmland where there is an opportunity for interaction and sharing of pathogens with domestic relatives. Bushpigs are considered to be possible natural reservoirs for African swine fever virus , but less is known about what other viruses bushpigs might carry. Therefore, to investigate whether bushpigs are carriers of known and or unknown porcine viruses we have in this study investigated the viral flora of bushpig sera.
Results and discussion
Viral hits of the 454 reads after assembly and blastn/x searches
75 - 83%
48 - 75%
68 - 85%
40 - 86%
No hit - 72%
40 - 76%
GB Hepatitis C virus
Sclerotinia hypovirulence associated virus
Porcine endogenous retrovirus
80 - 95%
86 - 89%
Parvoviruses are small single-stranded linear DNA viruses with a genome of approximately 5000 nucleotides, which have been found in a number of species such as human, swine, cattle and gorilla . In swine, porcine parvovirus (PPV) is a known agent that causes reproductive failure . However, in recent years a number of new parvoviruses – porcine hokovirus (PHoV) , porcine bocavirus (PBoV)  and porcine parvovirus 4 (PPV4)  - have been discovered in pigs, with their potential involvement in disease currently unknown.
Torque teno sus virus
Torque teno virus was discovered 1997 in a serum sample from a patient with posttransfusion hepatitis of unknown etiology using representational difference analysis . Since then the virus has been detected and characterised in a number of species such as primates, cats, dogs and pigs , but the role of these viruses in disease development is still controversial. These viruses have small (approximately 2.8 kb) circular DNA genomes. Torque teno virus in pigs is divided into two different species, Torque teno sus virus 1 and 2, and prevalence studies have shown that TTSuV is widely spread in pig populations across the world [16, 17]. In a previous study , we have found that 51.6% of a sample population of domestic pigs in Uganda were carrying one or both these TTSuV variants. Although, most studies have targeted domestic pigs, TTSuV have also been found in wild boar in Europe .
As shown in Table1, our data indicated the presence of both TTSuV-1 and 2 in the investigated serum samples. Both the TTSuV-1 and 2 sequence reads were located in the major open reading frame (ORF1) and all reads showed significant similarity to the respective virus in both blastn and blastx analyses.
TTSuV-1 and 2 have previously, as mentioned, been detected both in domestic pigs across the world [16–18] and wild suidae in Europe  and now for the first time in bushpigs on the African continent. Studies on the genetic variability of TTSuV-1 and 2 have shown a higher genetic diversity in the coding regions compared to the untranslated region . The sequence analysis of both the bushpig-derived TTSuV and those from GenBank shows a high genetic variability among the different TTSuV-1 and TTSuV-2 and also shows the co-infection of two different TTSuV-1 variants and one TTSuV-2.
Endogenous retroviruses are integrated in the host genome and all vertebrates investigated have been shown to carry retroviral sequences. It is estimated that up to 10% of animal genomes are retroviral elements . Also the bushpig genome has been investigated and confirmed to contain PERV [22–24].
By running the specific PERV PCR on both DNA and on DNase treated RNA we could as expected see that all the bushpigs had both integrated proviral DNA and expressed PERV RNA, indicating active viral transcription and replication. The sequenced PERV products (GenBank accession number JX566717-719) showed a high similarity (85 - 89%) to those available in GenBank.
Through investigating sera collected from bushpigs in Uganda by viral metagenomics, we have for the first time showed the presence of PPV4 in a wild Suid on the African continent. The region of PPV4 investigated indicates a sequence divergence relative to previously described PPV4. In addition, novel TTSuV-1 and 2 variants were found. Further sequence analysis and prevalence studies can be used to define the genetic relationships of these viruses and their distribution in both domestic pigs and in wildlife.
The sera from three bushpigs collected from Lake Mburo National Park, Uganda in March 2010, as part of a research project on African swine fever epidemiology were used in this study. The animals were captured using game capture nets (50x3m, 150 mm square mesh, 3.5 mm nylon braid khaki, ALNET Ltd, South Africa) with assistance from Uganda Wildlife Authority (UWA) staff, and sedated with zolazepam and tiletamine (Zoletil forte vet 50 mg/ml + 50 mg/ml, Virbac Laboratories, France) before blood sampling from the saphenous vein. The Ministry of Agriculture Animal Industry and Fisheries and Uganda Wildlife Authority, together with Makerere University are mandated to carry out animal disease investigations in livestock and wildlife in the country. This is done by veterinarians who handle the animals under internationally recognized guidelines.
Sample preparation, nucleic acid extraction and random PCR
Fifty microliters of serum was aliquoted for the RNA and DNA extraction respectively. One hundred and fifty microliter of 1x DNase buffer (Roche, Mannheim, Germany) was added to each aliquot of serum after which the sample was treated with nucleases - 100 U DNase (Roche, Mannheim, Germany) and 2 μg RNase (Invitrogen, Carlsbad, CA, USA) for two hours at 37°C in order to degrade the host nucleic acid. Trizol was added to one of the two aliquots and RNA was extracted using a combination of Trizol and Qiagen RNAeasy kit. DNA was extracted using the Qiagen DNAeasy mini extraction kit according to the manufacturer’s instructions and eluted in 50 μl elution buffer (EB). The DNA and RNA were amplified by random PCR as described earlier . Before sequencing, the primers were cleaved using EcoRV (NEB, Ipswich, MA, USA) and the cleaved product was purified using the Qiagen PCR purification kit (Qiagen, Hilden, Germany) and eluted in 30 μl EB.
High-throughput sequencing and data analysis
The purified product was sequenced on 1/8th of a pico titre plate using the 454 technology by Roche at Inqaba Biotech (South Africa). The sequences were analysed through quality check and removal of very short sequences before being assembled using CLC genomic workbench v4.6 (http://www.clcbio.com/index.php). Blastn and blastx searches were performed through the Camera 2.0 portal [26, 27] and searches through NCBI (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). The viral blast hits with an e-value of 10-4 or lower were further analysed.
Confirmation PCRs, sequencing and phylogenetic studies
Primers for verification
Sequence 5’- 3’
TCC CAG CAG AAG ATG TAG TC
GGA TGG TGG CCT CTA CTA C
GCA GCA TAA CGC TAG GCT G
AGA GGA AAT GGG CTA CCT G
CTC TGA TAA TGT ATT ACT GGT C
AAG AAA GAT CCT TCT GTT ACA
GB Hepatitis C virus_F
CTG CCT CAA CGT TGA GGC AG
GB Hepatitis C virus_R
ACG ACG TAG CAG TGG TAG AT
Sclerotinia hypovirulence associated virus_F
CGC ATC ACG GTC AAG TTT GA
Sclerotinia hypovirulence associated virus_R
TTA CAT TGC TTC GTC GAC TTC
ATG GGA GCT GGG TCC AAT C
CCT TAC GTT TGA CTC TCG AC
We would like to thank our colleagues Denis Muhangi and Susan Ndyanabo for technical assistance in the laboratory and Uganda Wildlife Authority staff for assistance in the field. Financial support for this study was provided by the Swedish International Development Cooperation Agency (Sida; SWE-2009-081), the Swedish research Council Formas (221-2009-1984), the Swedish Ministry of Foreign Affairs as part of its special allocation on global food security (through the Swedish University of Agricultural Sciences, SLU) and by the Award of Excellence awarded to SB by SLU.
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