New genotype classification and molecular characterization of canine and feline parvoviruses

Background@#Canine parvovirus (CPV) and feline panleukopenia (FPV) cause severe intestinal disease and leukopenia. @*Objectives@#In Korea, there have been a few studies on Korean FPV and CPV-2 strains. We attempted to investigate several genetic properties of FPV and CPV-2. @*Methods@#Several FPV and CPV sequences from around world were analyzed by Bayesian phylo-geographical analysis. @*Results@#The parvoviruses strains were newly classified into FPV, CPV 2-I, CPV 2-II, and CPV 2-III genotypes. In the strains isolated in this study, Gigucheon, Rara and Jun belong to the FPV, while Rachi strain belong to CPV 2-III. With respect to CPV type 2, the new genotypes are inconsistent with the previous genotype classifications (CPV-2a, -2b, and -2c). The root of CPV-I strains were inferred to be originated from a USA strain, while the CPV-II and III were derived from Italy strains that originated in the USA. Based on VP2 protein analysis, CPV 2-I included CPV-2a-like isolates only, as differentiated by the change in residue S297A/N. Almost CPV-2a isolates were classified into CPV 2-III, and a large portion of CPV-2c isolates was classified into CPV 2-II. Two residue substitutions F267Y and Y324I of the VP2 protein were characterized in the isolates of CPV 2-III only. @*Conclusions@#We provided an updated insight on FPV and CPV-2 genotypes by molecular-basedand our findings demonstrate the genetic characterization according to the new genotypes.

Regulation of porcine endogenous retrovirus by dual LTR1+2 (Long Terminal Region) miRNA in primary porcine kidney cells

Porcine endogenous retroviruses (PERVs) integrate into germline DNA as proviral genome that enables vertical transmission from parents to their offspring. The provirus usually survives as part of the host genome rather than as an infectious agent, but may become pathogenic if it crosses species barriers. Therefore, replication-competent PERV should be controlled through selective breeding or knockout technologies. Two microRNAs (miRNAs), dual LTR1 and LTR2, were selected to inhibit the expression of PERV in primary porcine kidney cells. The inhibition efficiency of the miRNAs was compared based on their inhibition of different PERV regions, specifically long terminal repeats (LTRs), gag, pol, and env. Gene expression was quantified using real-time polymerase chain reaction and the C-type reverse transcriptase (RT) activity was determined. The messenger RNA (mRNA) expression of the PERV LTR and env regions was determined in HeLa cells co-cultured with primary porcine kidney cells. The mRNA expression of the LTR, gag, pol, and env regions of PERV was dramatically inhibited by dual miRNA from 24 to 144 h after transfection, with the highest inhibition observed for the LTR and pol regions at 120 h. Additionally, the RT activity of PERV in the co-culture experiment of porcine and human cells was reduced by 84.4% at the sixth passage. The dual LTR 1+2 miRNA efficiently silences PERV in primary porcine kidney cells.

Duplex nested reverse transcriptase polymerase chain reaction for simultaneous detection of type 2 porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 from tissue samples

There are high levels of co-incidence of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) in porcine tissue. This study established a duplex nested reverse transcriptase polymerase chain reaction (RT-PCR) method that targets the genomic RNA of type 2 PRRSV and the mRNA of PCV2 in infected tissues. The method amplified discriminative bands of 347 bp and 265 bp specific for type 2 PRRSV and PCV2, respectively. The limits of detection of the duplex nested RT-PCR were 10(1.5) TCID₅₀/mL for type 2 PRRSV and 10² infected cells/mL for PCV2. The kappa statistic, which measures agreement between methods, was 0.867, indicating a good level of agreement. This RNA-based duplex RT-PCR approach can be another way to detect type 2 PRRSV and PCV2 simultaneously and with improved convenience.

Quantification and genotyping of PCV2 DNA in the tissues of PCV2-infected conventional pigs with different clinical signs / 대한수의학회지

This study was focused on the genotyping and quantification of Porcine circovirus type 2 (PCV2) in thirty PCV2-positive pigs with different clinical symptoms (PCV2-infected without wasting, PCV2-infected with wasting, PCV2-infected with wasting and lymphoid depletion). The quantity of PCV2 DNA in diverse tissues was significantly differed among these groups. (One-way ANOVA test, p < 0.001) Interestingly, PCV2-DNA load in tissues of PCV2-infected pigs without wasting and PCV2-infected pigs with wasting and lymphoid depletion were not significantly differed (p = 0.38), while they were all significantly higher when compared with PCV2-infected pigs with wasting-only. PCV2 DNA quantity in tissues was significantly higher in PCV2a and 2b co-infected pigs compared to the PCV2b only-infected pigs (Wilcoxon test, p = 0.039). The PCV2a and 2b co-infected pigs had increased wasting and lymphoid depletion rate but it was not statistically significant. Therefore, this cross-sectional study suggested that PCV2 DNA load in tissues was diverse by clinical and histological findings. Furthermore, co-infection of PCV2a and 2b affected to the PCV2 DNA load in tissues with increased rate of wasting and lymphoid depletion.

Identification of two cytopathogenic agents, Mycoplasma hyorhinis and mammalian orthoreovirus 3 based on modified particle associated nucleic acids PCR / 대한수의학회지

Swine diseases could be caused by unrecognized or minor pathogens. In this study, two unknown cytopathogenic agents were isolated from swine, through cell culture. In order to identify these two cytopathogenic agent (designated CP129 and #2045-7), a particle associated nucleic acids PCR (PAN-PCR) from previous paper was used with simple modification. The cloning procedure was more specified in this study by adding cell control system. According to the modified PAN-PCR, two and four agents-specific DNA sequences were obtained from CP129 and #2045-7, respectively, and they were identified as Mycoplasma (M.) hyorhinis and Mammalian orthoreovirus by nucleotide BLAST. Since M. hyorhinis (CP129) was filterable and non-visible by microscope, this unusual virus-like nature of M. hyorhinis (CP129) was discussed. Especially, the reovirus (#2045-7) was a serotype 3 and a triple reassortant among three serotypes of reoviruses. It was grouped with recently reported reoviruses from disease cases (swine, human and feline), based on the genetic analysis of L1 and S1 partial sequences. In conclusion, two unknown cytopathogenic agents were successfully identified using modified PAN-PCR with cell control system and they were characterized in this study.