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.

Genetic Characteristics and Immunogenicity of Pandemic H1N1 Influenza Virus Isolate from Pig in Korea

A pandemic influenza A (H1N1) virus strain was isolated from a pig farm in Korea in December 2009. The strain was propagated in and isolated from both the Madin-Darby canine kidney cell line and embryonated eggs. The partial and complete sequences of the strain were identical to those of A/California/04/2009, with >99% sequence similarity in the HA, NA, M, NS, NP, PA, PB1, and PB2 genes. The isolated strain was inactivated and used to prepare a swine influenza vaccine. This trial vaccine, containing the new isolate that has high sequence similarity with the pandemic influenza A (H1N1) virus, resulted in seroconversion in Guinea pigs and piglets. This strain could therefore be a potential vaccine candidate for swine influenza control in commercial farms.

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.

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.

Simultaneous detection and subtyping of porcine endogenous retroviruses proviral DNA using the dual priming oligonucleotide system

The purpose of this study was to develop a multiplex PCR that can detect porcine endogenous retrovirus (PERV) proviral genes (pol, envA, envB, envC) and porcine mitochondrial DNA, using a dual priming oligonucleotide (DPO) system. The primer specifically detected the PERV proviral genes pol, envA, envB, envC, and porcine mitochondrial DNA only in samples of pig origin. The sensitivity of the primer was demonstrated by simultaneous amplification of all 5 target genes in as little as 10 pg of pig DNA containing PERV proviral genes and mitochondrial DNA. The multiplex PCR, when applied to field samples, simultaneously and successfully amplified PERV proviral genes from liver, blood and hair root samples. Thus, the multiplex PCR developed in the current study using DPO-based primers is a rapid, sensitive and specific assay for the detection and subtyping of PERV proviral genes.

Comparison of the age-related porcine endogenous retrovirus (PERV) expression using duplex RT-PCR

Porcine endogenous retroviruses (PERVs) are members of family Retroviridae, genus Gamma retrovirus, and transmitted by both horizontally and vertically like other endogenous retroviruses (ERVs). PERV was initially described in the 1970s having inserted its gene in the host genome of different pig breeds, and three classes, PERV-A, PERV-B, and PERV-C are known. The therapeutic use of living cells, tissues, and organs from animals called xenotransplantation might relieve the limited supply of allografts in the treatment of organ dysfunction. Because of ethical considerations, compatible organ sizes, and physiology, the pig has been regarded as an alternative source for xenotransplantation. Sensitive duplex reverse transcription-polymerase chain reaction protocols for simultaneously detecting PERV gag mRNA and porcine glyceraldehydes 3-phosphate dehydrogenase mRNA in one tube was established. To compare the age-related PERV expression patterns of the lung, liver, spleen, kidney, heart, and pancreas in commercial pigs, 20 pigs from four age groups (5 heads each in 10 days-, 40 days-, 70 days-, and 110 days-old, respectively) were used in this study. The expression patterns of PERV were statistically different among age groups in lung, liver, and kidney (ANOVA, p<0.05). These data may support in the selection of appropriate donor pigs expressing low levels of PERV mRNA.

Genetic analysis of ORF5 of recent Korean porcine reproductive and respiratory syndrome viruses (PRRSVs) in viremic sera collected from MLV-vaccinating or non-vaccinating farms

The 23 open reading frame (ORF) 5 sequences of Korean type II porcine reproductive and respiratory syndrome virus (PRRSV) were collected from viremic sera from the (modified live vaccine) MLV-vaccinating and non-vaccinating farms from 2007 to 2008. The samples were phylogenetically analyzed with previous ORF5 sequences, including type I Korean PRRSV, and previously reported or collected sequences from 1997 to 2008. A MN184-like subgroup of type II Korean PRRSV was newly identified in the viremic sera collected from 2007 to 2008. And of the type I PRRSVs, one subgroup had 87.2~88.9% similarity with the Lelystad virus, showing a close relationship with the 27~2003 strain of Spain. The maximum parsimony tree of type II PRRSV from 1997 to 2008 showed that they had evolved to four lineages, subgroups 1, 2, 3 and 4. Most of the recently collected type II PRRSVs belonged to subgroup 4 (48%). The region of three B-cell epitopes and two T-cell epitopes of ORF5 amino acids sequences was considerably different from the MLV in subgroups 3 and 4. In conclusion, the existence of type I PRRSV, which was genetically different from Lelystad virus (Prototype of type I PRRSV), and heterologous type II PRRSVs of viremic pigs detected even in the MLV-vaccinating farms indicated the need for new vaccine approaches for the control of PRRSV in Korea.

Comparison of an enzyme-linked immunosorbent assay with serum neutralization test for serodiagnosis of porcine epidemic diarrhea virus infection

An indirect porcine epidemic diarrhea (PED) virus (PEDV) enzyme-linked immunosorbent assay (ELISA) was compared with the serum neutralization (SN) test by testing 46 samples from experimentally infected sows, 73 samples from naive sows, and 1, 024 field sow samples from 48 commercial swine farms of undefined PED status. The SN test and the ELISA were performed using PEDV, KPEDV-9 strain. Viral proteins as a coating antigen of PEDV ELISA were extracted from the cytoplasm of PEDV-infected Vero cells using a non-ionic detergent, Triton X-100, and a simple protocol of PEDV ELISA was followed. The presence of antibodies in these experimental samples was confirmed by SN and ELISA in which the sensitivity of the ELISA was 89.1%, and the corresponding specificity was 94.5%. On testing 1, 024 field samples, an overall agreement of 84.2% was generated between the SN and ELISA. This study demonstrates that the PEDV ELISA is a useful serodiagnostic screening test at herd level for detecting swine antibodies against PEDV.