engleski

Presence of Torque teno virus sus virus in porcine circovirus type 2-associated diseases in Croatia

Torque teno virus (TTV) is a ubiquitous and species-specific virus that infects domestic pigs and wild boars. Four subtypes of viral species TTSuV1 (A, B, C and D) and two subtypes of TTSuV2 have been proposed. Whether these two species are involved in clinical cases of porcine circovirus type 2-associated disease (PCVD) is unclear. TTSuV can serve as a “trigger” or co-factor for porcine circovirus type 2 (PCV2) in the pathogenesis of postweaning multisystemic wasting syndrome (PMWS). TTSuV2 prevalence is much higher in PMWS-affected animals than in healthy domestic swine. In addition, co-infection of TTSuV1 and porcine reproductive and respiratory syndrome virus (PRRSV) in gnotobiotic pigs has been linked to porcine dermatitis nephropathy syndrome (PDNS). TTSuV1 in gnotobiotic pigs can produce interstitial pneumonia, transient thymic atrophy, membranous glomerulonephropathy and modest lymphocytic to histiocytic infiltrates in liver. Numerous studies have found TTSuV to be common in aborted fetuses and fetuses collected from slaughtered sows, suggesting that TTSuV is virus responsible for abortion occurrence. However, definitive evidence for this is lacking. This study investigated the presence of TTSuV in animals infected by PCV2 and affected by PCVDs other than PMWS and PDNS. Altogether 85 samples of PCV2-infected organs were collected from 63 fetuses, 19 pigs and 3 wild boars throughout Croatia. All the fetuses lacked specific lesions associated with PCV2 reproductive disorders, as well as PCV2 based on immunohistochemistry and in situ hybridization. However, they were shown to contain PCV2 genomic DNA through the PCR method described below. The three lymph nodes from wild boars affected by PMWS contained abundant PCV2 genome, based on in situ hybridization. Five samples of lungs showed proliferative and necrotizing pneumonia (PNP), which were analyzed by a double staining method involving in situ hybridization to detect PCV2 genome and immunohistochemistry to detect PRRSV antigen. Strong signal due to PCV2 genome was observed in alveolar macrophages, pneumocytes and epithelial cells, while strong signal due to PRRSV antigen was detected in alveolar macrophages. Of the remaining 22 samples, 14 were of pooled kidneys and lymph nodes from weaning pigs, in which in situ hybridization studies revealed strong signal due to PCV2 genome in the epithelial cells of kidney tubuli. The presence or absence of TTSuV1 or TTSuV2 in samples was determined using a specific, one-step PCR. TTSuV amplicons were purified and sequenced. Phylogenetic analysis of TTSuV1 and TTSuV2 was performed using a 218-nt partial sequence (TTSuV1) or 177-nt partial sequence (TTSuV2) of the non-coding region. A sequence matrix polymorphism was computed using MEGA5 and DnaSP (version 5). Reference TTSuV type sequences were used to assign every sequence to a type. In addition, amino acid sequence alignments were used to construct a minimum spanning tree using a median-joining (MJ) network algorithm implemented in the program Network (version 4.6.0.0.). TTSuV2 prevalence was high in animals affected by nephropathy and PNP, and prevalence of both TTSuV1 and TTSuV2 was high in wild boars affected by PMWS. TTSuV1 prevalence was low in animals affected by nephropathy and PNP, and prevalence of both TTSuV1 and TTSuV2 was low in animals with reproductive disorders. Sequencing of the Croatian samples uncovered three subtypes of TTSuV1 (A, C, D) and one subtype of TTSuV2 (A). MJ network analysis revealed significant genetic diversity among the Croatian isolates. Renal tissue from animals with nephropathy were morphologically similar to the so-called “Turkey egg kidney” characteristic of PDNS, and this morphology was associated with the presence of PCV2 genome, based on in situ hybridization. The role of PCV2 in PDNS is unclear, and recent studies suggest that the same lesions can arise during TTSuV and PRRSV co-infection. Nevertheless, our observations of highTTSuV2 prevalence in animals with nephropathy and the pathomorphological similarity between infected animals and “Turkey egg kidney”, coupled with the recent finding that the PDNS that causes Turkey egg kidney is associated with TTSuV infection, indicates the need to reevaluate the role of TTSuV2 in nephropathy. TTSuV2 was also found at relatively high prevalence in animals with PNP. Just as our results suggest TTSuV2 participation in pathogenesis of nephropathy, our observation of a high TTSuV2 prevalence in animals with PNP, coupled with the demonstration that TTSuV can produce interstitial pneumonia (Krakowka and Ellis 2008), indicates the need to re-evaluate the role of TTSuV2 in pathogenesis of PNP. PCR amplicons were sequenced to exclude possible cross-reaction with PCV2. Both TTSuV and PCV2 have a circular, single-stranded DNA genome, and they are even identical in one genomic region. The sequences of a non-coding region from our animals were compared with partial ORF1 and complete ORF2 sequences deposited in GenBank, since the partial overlap among these sequences ensured that results could be compared. Nevertheless, given that the sequences analyzed were non-coding and did not correspond to specific marker positions in ORF1 or ORF2 regions, more precise phylogenetic analysis of the complete TTSuV genome should be performed. The results of this study are inconsistent with the idea that TTSuV plays a role in pig reproductive disease. More importantly, they provide evidence from clinical cases that TTSuV2 may play a certain role in renal and pulmonary lesions when it co-infects with PCV2. The high genetic diversity between both Croatian genogroups of TTSuV reflects the diversity observed among TTSuV strains around the world.

TTSuV ; PCVDs ; Croatia

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