Viral metagenomics demonstrates that domestic pigs are a potential reservoir for Ndumu virus
- Charles Masembe1Email author,
- George Michuki2,
- Maria Onyango2,
- Cecilia Rumberia2,
- Martin Norling2, 4,
- Richard P Bishop2,
- Appolinaire Djikeng3,
- Stephen J Kemp2,
- Alan Orth2,
- Robert A Skilton3,
- Karl Ståhl1, 4 and
- Anne Fischer2, 5
© Masembe et al.; licensee BioMed Central Ltd. 2012
Received: 21 November 2011
Accepted: 18 September 2012
Published: 24 September 2012
The rising demand for pork has resulted in a massive expansion of pig production in Uganda. This has resulted in increased contact between humans and pigs. Pigs can act as reservoirs for emerging infectious diseases. Therefore identification of potential zoonotic pathogens is important for public health surveillance. In this study, during a routine general surveillance for African swine fever, domestic pigs from Uganda were screened for the presence of RNA and DNA viruses using a high-throughput pyrosequencing method.
Serum samples from 16 domestic pigs were collected from five regions in Uganda and pooled accordingly. Genomic DNA and RNA were extracted and sequenced on the 454 GS-FLX platform. Among the sequences assigned to a taxon, 53% mapped to the domestic pig (Sus scrofa). African swine fever virus, Torque teno viruses (TTVs), and porcine endogenous retroviruses were identified. Interestingly, two pools (B and C) of RNA origin had sequences that showed 98% sequence identity to Ndumu virus (NDUV). None of the reads had identity to the class Insecta indicating that these sequences were unlikely to result from contamination with mosquito nucleic acids.
This is the first report of the domestic pig as a vertebrate host for Ndumu virus. NDUV had been previously isolated only from culicine mosquitoes. NDUV therefore represents a potential zoonotic pathogen, particularly given the increasing risk of human-livestock-mosquito contact.
KeywordsMetagenomics Ndumu virus Pigs Reservoir Zoonoses
Serum samples from 16 domestic pigs were collected from five localities in Uganda (Figure 1). The sampling was carried out as part of a study on African swine fever (endemic in the region) from localities with suspected outbreaks of the disease. Serum samples from pigs originating from the same geographical locality were combined to generate a total of six pools from across the country (A-Moyo; B-Lira; C-Gulu; D-Mityana; E-Mpigi). The samples were filtered through a 0.22 μm filter followed by DNA and RNA extraction using a Qiamp DNA Mini kit (Qiagen) and TRIzol reagent (Invitrogen), respectively. DNA and RNA were amplified using the modified random priming mediated sequence independent single primer amplification (RP-SISPA) methodology . Each amplified sample was further processed as described for shotgun library preparation in GS FLX 454 technology. The sequencing reads were trimmed to remove SISPA primers and barcodes, and only reads with a length greater than 50 bp were retained. Low complexity repeats were masked using Repeatmasker (RepeatMasker Open-3.0.1996-2010 http://www.repeatmasker.org) and sequences with more than 50% repeats were excluded. The sequences in each pool were assembled using the Newbler assembler version 2.5.3 with default settings (Roche. Genome Sequencer FLX Data Analysis Software Manual. Mannheim, Germany: Roche Applied Science, 2007). Contiguous sequences (contigs) and reads which did not assemble into contigs were categorized using BLASTN and BLASTX homology searches against the non-redundant nucleotide and amino acid databases from NCBI (version June 2011). Taxonomic classification of each contig/read was investigated using MEGAN 4.0 .
A total of 289,038 reads with an average length of 175 nucleotides was obtained. After filtering for length and repeat content, 190,706 reads remained. Seventy-seven percent of the reads were assembled into contigs. Both BLASTN and BLASTX analyses gave similar results. Subsequently, the results that follow are from the BLASTN analysis.
For 62% of all sequences, there was no significant match within GenBank as defined by the above criteria. Among the sequences assigned to a taxon, 52%, whether DNA or RNA, mapped to the domestic pig host (Sus scrofa). Thirty six percent of the sequences also exhibited similarity with other mammalian genomes (17% to the family Bovidae and 6.5% to human); these additional matches were most likely a consequence of the currently incomplete status of the porcine genome.
A small proportion (6.2%) of the sequences mapped to DNA and RNA viruses. For the DNA searches, besides African swine fever virus, which was identified in all pools, Torque teno viruses (TTVs) were identified in two pools (A and D). TTVs are ubiquitous species-specific viruses that are currently considered non-pathogenic and have been reported to infect swine with a high prevalence [10, 13].
From the RNA pools analyses, three pig serum pools (A, C, & D) showed sequences with significant sequence similarity to diverse porcine endogenous retroviruses. Of particular interest are sequences that showed highly significant identity (98%) to NDUV and were present in pools B and C of pig RNA samples from Gulu and Lira districts, respectively. To validate this observation, the distribution of the sequence reads used earlier to build contigs from the two pools were determined by BLASTN analysis. RNA pool B had 6,228 sequence reads which comprised of 2% virus, 1% bacteria, 57% Mammalia and 40% unknown. RNA pool C had 64,583 reads which comprised of 5% virus, 1% bacteria, 51% Mammalia and 43% unknown (Additional file 1: Figure S1). The Mammalia genomes were from pig, human and Bovidae. Bacteria reads in both pools had identities of less than 30 bases at below 65% identity to Eubacterium hallii and Xylella fastidiosa. The bacteria sequences may therefore have occurred by chance and hence not significant explaining why they were lost on assembly of reads to contigs. No read showed a match to the class Insecta.
In this study, a metagenomics approach was used to determine the variety of viruses in domestic pig (Sus scrofa) serum. In addition to the detection of mammalian sequences, it revealed the presences of some viruses for example Torque teno viruses (TTVs) and bacteria Eubacterium hallii and Xylella fastidiosa that have previously been found to occur regularly in pigs and plants, respectively. However, in the same study, a virus that has not been found before in pigs, the NDUMU (NDUV) virus, was detected. NDUV is a single stranded RNA arbovirus transmitted by mosquitoes and belonging to the Togaviridae family in the alphavirus genus. Very little is known about NDUV and its vertebrate hosts. It was isolated for the first time in South Africa in 1959 from Mansonia uniformis and later in Kenya from Aedes mcintoshi and A. ochraceus. Mice experimentally infected with NDUV do not survive the infection . Although antibodies to the virus have been identified in humans from several African countries, no human morbidity or mortality has yet been attributed to NDUV infection . However the genus Alphavirus comprises at least 24 members , among which are many viruses, which cause diseases in humans and other animals. Chikungunya virus is one example of an Alphavirus that was responsible for recent severe outbreaks of human disease in Eastern Africa. In humans, the symptoms associated with Alphavirus infections range from fevers and rashes, to transient or debilitating arthritis, or encephalitis [20, 21]. In this study, the domestic pig has for the first time been identified as a potential vertebrate host of NDUV. NDUV therefore represents a potential zoonotic agent, given the increasing risk of human-livestock-mosquito contact as the pig industry continues to intensify, and the pig population increases in Uganda. Our discovery indicates that a focused search for the virus using reverse-transcription PCR should now be performed in human communities associated with the domestic pig populations in which we have detected NDUV.
We thank the District Veterinary Officers and field support staff in the study districts for assistance during sampling, Dr. Denis Muhangi and Ms Susan Ndyanabo for technical assistance in the lab and in the field. We thank Jandouwe Villinger for designing NDUV-specific primers and Jandouwe Villinger, Dan Masiga and Etienne de Villiers for comments on earlier versions of the manuscript. We thank the anonymous reviewers who gave constructive criticisms to earlier versions of this manuscript.
Financial support was provided from the Swedish International Development and Cooperation Agency (Sida; SWE-2009-081), the Swedish research Council Formas (221-2009-1984), google.org through the AVID project. We gratefully acknowledge the financial support provided to the Biosciences eastern and central Africa Hub at the International Livestock Research Institute (BecA-ILRI Hub) by the Australian Agency for International Development (AusAID) through a partnership between Australia's Commonwealth Scientific and Industrial Research Organisation (CSIRO) and the BecA-ILRI Hub; and by the Syngenta Foundation for Sustainable Agriculture (SFSA), which made this work possible. We thank the German Federal Ministry of Cooperation and Development which supported Anne Fischer in this study.
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