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      Porcine Hokovirus in Domestic Pigs, Cameroon

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          Abstract

          To the Editor: Since 2005, new parvoviruses forming a novel genus of the proposed name Partetravirus, within the subfamily Parvovirinae, have been described ( 1 ). Human parvovirus 4 (PARV4) with 3 different genotypes globally infects humans ( 2 ). A related porcine virus, hokovirus (HoV or porcine partetravirus), was found in wild boar and domestic pig populations in Germany, Romania, China, and the United States, with prevalences of 12%–47%, forming 1 common genotype ( 3 – 6 ). Prevalence figures from sub-Saharan Africa are not available. Furthermore, no information about possibly region-associated genotypes is available for porcine HoV, although it is for human PARV4 from the same genus. We therefore used samples (collected during February–March 2012) from a study investigating hepatitis E virus (HEV) in pigs from Cameroon ( 7 ) to analyze the occurrence of porcine HoV in pigs in Africa and to determine the respective genotype. Viral DNA was extracted from liver samples by using the RTP DNA/RNA Virus Mini Kit II (STRATECMolecular, Berlin, Germany) according to the manufacturer’s instructions. DNA samples were pooled, with each pool containing 3 different samples. A total of 94 pooled samples from 282 animals originating from 3 districts in Cameroon (Doula, Yaoundé, and Bamenda) were investigated by using quantitative real-time PCR ( 3 , 7 ). Samples from pools that tested positive were analyzed individually. We detected HoV in 65 (69%) of the 94 pooled samples: 2 (15%) of 13 from Bamenda, 39 (70%) of 56 from Douala, and 24 (96%) of 25 from Yaoundé. We used an online tool to estimate the individual prevalence from pooled samples for fixed pool size and perfect test with exact 5% upper and lower CIs (http://epitools.ausvet.com.au/content.php?page = PooledPrevalence). A pool size of 3 with a total of 94 pooled samples and 65 positive samples resulted in an estimated general prevalence of 32.4% (95% CI 27%–39%). For Bamenda, the estimated prevalence was 5.4% (95% CI 1%–16%); for Douala, 32.8% (95% CI 25%–41%); and for Yaoundé, 65.8% (95% CI 44%–87%). From 94 positive pools, a total of 184 samples were available for individual testing: 6 from Bamenda, 110 from Douala, and 68 from Yaoundé; 12 were missing. Using the results from the negative tested pools and the individual testing, we found an estimated general prevalence of 47% (128/270). The regional prevalence was 10% (4/39) for Bamenda, 41% (65/160) for Douala, and 83% (59/71) for Yaoundé. These prevalences are higher than the estimates, but lie within the regional estimates within the range of the CI determined with the online tool. The discrepancy in the total prevalence might be due to the missing samples for the individual testing. Our results show that pooled sample testing can yield a good approximation of the actual prevalence, at least for settings in Africa. The varying prevalence and inhomogeneous regional distribution of porcine HoV correspond to previous findings from Europe, China, and the United States in wild boar and domestic pigs ( 3 , 5 , 6 ). Overall, no general defined pig-breeding program is in place in Cameroon. Douala and Yaoundé are the main markets for pig trade. Yaoundé, the main town for pig purchase and slaughter, gets live pigs from northwestern (Bamenda), western, and northern Cameroon, and Douala receives pigs from northwestern (Bamenda), western, and southwestern Cameroon. To fully understand the observed regional prevalences, the presence of HoV needs to be investigated in detail in the southwest, west, and north, where intensive farming systems are in place and pig farming is of economic importance. Near full-length genome data were generated from 3 positive samples, and partial sequence information was retrieved for 8 additional samples (Figure) as described ( 3 ). The phylogenetic analysis showed a very close relation, with 98%–99% homology between the porcine HoV isolates from Cameroon, Europe, the United States, and China. In contrast to bovine HoV or PARV4 in humans and chimpanzees, only 1 porcine genotype has been observed worldwide, which might point to 1 common ancestor ( 8 , 9 ). Because the liver samples were taken from European landrace or cross-bred pigs, the common ancestor of HoV might have originated from Europe. Pigs chronically infected with HoV could have been imported from Europe to Cameroon during the extensive global industrial farming and pig trade in the last century. The detection of the European HEV genotype (gt) 3 instead of the “traditional” African gt1 and gt2 in these pigs supports this hypothesis ( 7 , 10 ). Nevertheless, the high grade of homology of porcine HoV found worldwide suggests a highly species-specific virus with a low mutation frequency. To study the circulating genotypes in Africa and to generate hypotheses on the influence of trade, researchers need to college data from other African countries. However, data on HoV in wild animals, such as warthogs and pot-bellied pigs, are limited and need to be addressed to elucidate the genomic intravariability and intervariability of this new family of parvoviruses. Figure Phylogenetic tree of near full-length (4.7 kbp) and partial sequences (open reading frame 2, 0.4–1.9 kbp) of porcine hokovirus (HoV)/partetraviruses (PtV) created by using MEGA5.1 (www.megasoftware.net) with the maximum-likelihood method (GTR+G+I) and bootstrap analysis of 500 resamplings. New sequences from Cameroon are shown in boldface. EU, Europe; CH, China; US, United States; Gt, genotype; PARV4, parvovirus 4. Scale bar indicates nucleotide substitutions per site.

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          Most cited references9

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          New DNA viruses identified in patients with acute viral infection syndrome.

          A sequence-independent PCR amplification method was used to identify viral nucleic acids in the plasma samples of 25 individuals presenting with symptoms of acute viral infection following high-risk behavior for human immunodeficiency virus type 1 transmission. GB virus C/hepatitis G virus was identified in three individuals and hepatitis B virus in one individual. Three previously undescribed DNA viruses were also detected, a parvovirus and two viruses related to TT virus (TTV). Nucleic acids in human plasma that were distantly related to bacterial sequences or with no detectable similarities to known sequences were also found. Nearly complete viral genome sequencing and phylogenetic analysis confirmed the presence of a new parvovirus distinct from known human and animal parvoviruses and of two related TTV-like viruses highly divergent from both the TTV and TTV-like minivirus groups. The detection of two previously undescribed viral species in a small group of individuals presenting acute viral syndrome with unknown etiology indicates that a rich yield of new human viruses may be readily identifiable using simple methods of sequence-independent nucleic acid amplification and limited sequencing.
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            A third genotype of the human parvovirus PARV4 in sub-Saharan Africa.

            PARV4 is a recently discovered human parvovirus widely distributed in injecting drug users in the USA and Europe, particularly in those co-infected with human immunodeficiency virus (HIV). Like parvovirus B19, PARV4 persists in previously exposed individuals. In bone marrow and lymphoid tissue, PARV4 sequences were detected in two sub-Saharan African study subjects with AIDS but without a reported history of parenteral exposure and who were uninfected with hepatitis C virus. PARV4 variants infecting these subjects were phylogenetically distinct from genotypes 1 and 2 (formerly PARV5) that were reported previously. Analysis of near-complete genome sequences demonstrated that they should be classified as a third (equidistant) PARV4 genotype. The availability of a further near-complete genome sequence of this novel genotype facilitated identification of conserved novel open reading frames embedded in the ORF2 coding sequence; one encoded a putative protein with identifiable homology to SAT proteins of members of the genus Parvovirus.
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              Hepatitis E Virus Genotype 3 Strains in Domestic Pigs, Cameroon

              To the Editor: Hepatitis E virus (HEV) is a positive-stranded, non-enveloped RNA virus of the family Hepeviridae that is considered to be the main causative agent of enterically transmitted acute hepatitis ( 1 ). HEV is classified into 4 genotypes ( 1 ). HEV genotypes 1 and 2 cause large waterborne epidemics of acute hepatitis in developing countries, especially in Africa and Asia ( 1 ). In contrast, HEV genotypes 3 and 4 are increasingly identified as causative agents of acute viral hepatitis in industrialized countries ( 1 ). Genotypes 1 and 2 are found only in humans, whereas genotypes 3 and 4 are associated with food-borne zoonotic transmission from domestic pigs, wild boar, and deer ( 1 ). In addition to these 4 genotypes, HEV-related viruses were detected in avian, rodent, and bat hosts, which formed novel genera within the family Hepeviridae ( 2 ). In Africa, HEV genotype 1 and 2 strains have been identified during HEV epidemics ( 3 – 5 ). An HEV genotype 3 strain was detected in 1 of 40 fecal samples from domestic pigs in Kinshasa, Democratic Republic of the Congo, and it was suggested that this strain was imported from Belgium to the Democratic Republic of the Congo by animal trade ( 6 ). Therefore, we investigated whether HEV strains of genotype 3 or 4 are circulating among domestic pigs in Cameroon. During February–March 2012, a total of 345 liver samples were collected from domestic pigs (age range 6 months–3 years) in abattoirs in Douala and Yaoundé, Cameroon, and in slaughter slaps (areas) in Bamenda, Cameroon. Pigs were mainly of the local breed. In addition, pigs originating from extensive cross-breeding (local X landrace and local X Duroc) were sampled. Liver samples were collected during post-mortem inspection. Viral RNA was extracted from liver samples by using the RTP DNA/RNA Virus Mini Kit II (STRATEC Molecular, Berlin, Germany) according to the manufacturer’s instructions. Extracted RNA was analyzed for HEV RNA by using 2 nested reverse transcription PCRs (RT-PCRs) specific for open reading frame 1 (OFR 1) and ORF 2 of HEV ( 7 , 8 ). Nested RT-PCRs and direct sequencing of amplicons were performed as described ( 9 ). RNA of HEV strain Hamburg-HB (GenBank accession no. JN986840) was used as a positive control for nested RT-PCRs. HEV RNA was detected in 2 samples from female pigs in Yaoundé (2/139) and 1 sample from a male pig in Bamenda (1/39). All 167 samples from Douala were negative for HEV RNA. The sample from Bamenda showed a positive result for the nested RT-PCR specific for HEV ORF 1. Genetic distances calculated with partial nucleotide sequences of ORF 1 (280 nt) and ORF 2 (373 nt) between strain Yaounde56 and the most closely related HEV genotype 3 strains from Japan (JSWINE150-Aom04R; GenBank accession no. AB221520) and Mongolia (swMN06-A1354; GenBank accession no. AB290105) were 90% and 91%, respectively. At the amino acid level, the partial RNA-dependent RNA polymerase sequence (ORF 1) and the partial capsid protein sequence (ORF 2) of strain Yaounde56 were identical to those of HEV genotype 3 strains HEV/Gt3/HSD40/2009 (GenBank accession no. AFO71833) from Germany and swJ12–1 (GenBank accession no. BAC66273) from Japan. Thus, all mutations were silent. In agreement with distance analysis, phylogenetic reconstruction using partial nucleotide sequences of ORF 2 (278 nt) showed a close relationship of strains Yaounde56 and Yaounde94 with HEV genotype 3 strains (Figure). However, the HEV strains from Cameroon do not cluster with the classified HEV genotype 3 subtype reference strains ( 10 ) in the phylogenetic tree (Figure). These strains cluster within a clade of subtype undefined strains and are most closely related to strain swMN06-A1354 from Mongolia (Figure). Figure Phylogenetic analysis of hepatitis E virus (HEV) strains, Cameroon. The Bayesian phylogenetic tree was constructed by using partial nucleotide sequence of open reading frame 2 (278 nt) of HEV. For each sequence used, the GenBank accession number, strain designation, source of isolation, country of isolation, and HEV subtype are shown. Multiple nucleotide sequence alignment was analyzed by using the Markov Chain Monte Carlo method implemented in the program MrBayes version 3.0 (http://mrbayes.sourceforge.net/) and applying the general time-reversible substitution model. Posterior probabilities are shown at the nodes of the tree to the right of the slash if >0.5. Bootstrap values calculated from 10,000 replicates are indicated at the nodes of the tree to the left of the slash. Alignment was analyzed by using the neighbor-joining method and resulted in same tree topology (not shown). Newly described HEV sequences are shown in boldface. Scale bar indicates evolutionary distance. UK, United Kingdom; USA, United States; DRC, Democratic Republic of Congo. Because the pig production cycle is shorter than that for cattle, pig production is a major economic activity in Cameroon. Most pigs in Cameroon are local raised, and extensive cross-breeding is used. The infection rate of pigs with HEV genotype 3 strains from Cameroon is lower than that of pigs from Europe. Thus, HEV genotype 3 seems to have an extensive distribution that includes Africa. Future studies should investigate how HEV genotype 3 strains contribute to sporadic HEV cases in Cameroon.
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                Author and article information

                Journal
                Emerg Infect Dis
                Emerging Infect. Dis
                EID
                Emerging Infectious Diseases
                Centers for Disease Control and Prevention
                1080-6040
                1080-6059
                December 2013
                : 19
                : 12
                : 2060-2062
                Affiliations
                [1]Robert Koch Institute, Berlin, Germany (C. Adlhoch, M. Ulrich, A. Löwa, H. Ellerbrok);
                [2]GenExpress, Berlin (M. Kaiser);
                [3]Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany (N.G. Schwarz, J. Ehlers, S. Poppert, J. Schmidt-Chanasit);
                [4] Institute of Agricultural Research for Development, Ngaoundere, Cameroon (M.T. Kingsley, A.M. Daniel);
                [5]Fundação Oswaldo Cruz, Rio de Janeiro, Brazil (V.S. de Paula)
                Author notes
                Address for correspondence: Cornelia Adlhoch, European Centre for Disease Prevention and Control, Surveillance and Response Support Unit, Tomtebodavägen 11 A, SE-171 83 Stockholm, Sweden; email: cornelia.adlhoch@ 123456ecdc.europa.eu
                Article
                13-0891
                10.3201/eid1912.130891
                3840879
                24274159
                60db73a0-486a-41ce-ab87-e7c1a93602bd
                History
                Categories
                Letters to the Editor
                Letter

                Infectious disease & Microbiology
                partetravirus,pigs,parv4,porcine hokovirus,cameroon,viruses,phov
                Infectious disease & Microbiology
                partetravirus, pigs, parv4, porcine hokovirus, cameroon, viruses, phov

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