Development of one-step real-time reverse transcription polymerase chain reaction assays for rapid detection of porcine group C rotaviruses.

Although the widespread occurrence of porcine group C rotaviruses (GCRV) is assumed, precise prevalence remains largely unknown because of the absence of reliable, specific, and rapid diagnostic methods. To detect and quantify porcine GCRV, the authors evaluated and optimized SYBR Green and TaqMan real-time reverse transcription polymerase chain reaction (RT-PCR) assays and applied them to 108 piglet fecal samples. Using serially diluted standard RNA transcripts of porcine GCRV VP6 gene, both SYBR Green and TaqMan real-time RT-PCR assays detected as few as 1 x 10(1) genome copies/microl (correlation coefficiency >0.99), whereas conventional RT-PCR detected 1.0 x 10(3) copies/microl. In addition, the conventional assay detected porcine GCRV in 24% (26/108) of fecal samples, whereas the detection rates of both SYBR Green and TaqMan assays were 72% (78 of 108) and 64% (70 of 108), respectively. The current study indicated that both real-time RT-PCR assays were reliable, specific, and rapid methods for the detection of porcine GCRV in porcine fecal samples.

Development of SYBR Green real-time RT-PCR for rapid detection, quantitation and diagnosis of unclassified bovine enteric calicivirus.

Unclassified bovine enteric calicivirus (BECV) is a newly recognized bovine enteric calicivirus that differs from bovine norovirus, and which causes diarrhea in the small intestines of calves. To date, methods such as real-time reverse transcription-polymerase chain reaction (RT-PCR) have not been developed for the rapid detection, quantitation and diagnosis of BECV. Presently, a BECV-specific SYBR Green real-time RT-PCR assay was evaluated and optimized. Diarrheic specimens (n=118) collected from 2004 to 2005 were subjected to RT-PCR, nested PCR and SYBR Green real-time RT-PCR. By conventional RT-PCR and nested PCR, 9 (7.6%) and 59 (50%) samples tested positive, respectively, whereas the SYBR Green assay detected BECV in 91 (77.1%) samples. Using BECV RNA standards generated by in vitro transcription, the SYBR Green real-time RT-PCR assay sensitively detected BECV RNA to 1.1 x 10(0)copies/microl (correlation coefficiency=0.98). The detection limits of the RT-PCR and nested PCR were 1.1 x 10(5) and 1.1 x 10(2)copies/microl, respectively. These results indicate that the SYBR Green real-time RT-PCR assay is more sensitive than conventional RT-PCR and nested PCR assays, and has potential as a reliable, reproducible, specific, sensitive and rapid tool for the detection, quantitation and diagnosis of unclassified BECV.

Genetic diversity of porcine circovirus type 2 in Korean pigs with postweaning multisystemic wasting syndrome during 2005-2007.

The comparison of nucleotide and deduced amino acid sequence was conducted with 9 porcine circovirus type 2 (PCV2) strains isolated from PCV2-infected pigs with postweaning multisystemic wasting syndrome (PMWS) and 50 tissue samples obtained from PCV2-infected 50 pigs with PMWS during 2005-2007. At amino acid positions 88-89 of the ORF2 gene, 50 Korean PCV2 had amino acids PR/L consistent with group 1 PCV2, whereas 9 Korean PCV2 contained amino acids KI, characteristic of group 2 PCV2. Phylogenetically, 47, 3, 2 and 7 Korean PCV2 belonged to subgroups 1A/B (79.7%), 1C (5.1%), 2D (3.4%) and 2E (11.9%), respectively. Although the predominant Korean PCV2 was subgroup 1A/B, subgroups 1C and 2E were still circulating and subgroup 2D PCV2 were found to be newly emerged in Korea.

Detection and molecular characterization of porcine group C rotaviruses in South Korea.

Group C rotaviruses (GCRVs) cause acute diarrhea in humans and animals worldwide and the evidence for a possible zoonotic role of GCRVs has been recently provided. However, there is little evidence of porcine GCRV infections or of their genetic diversity in South Korea. We examined 137 diarrheic fecal specimens from 55 farms collected from six provinces. RT-PCR utilizing primer pairs specific for the GCRV VP6 gene detected GCRV-positive reactions in 36 (26.2%) diarrheic fecal samples. Of these, 17 samples (12.4%) tested positive for porcine GCRVs alone and 19 samples (13.8%) were also positive for other pathogens. Other enteric pathogens except for GCRV were detected in 64 feces samples (46.7%) and no enteric pathogens were evident in 37 feces samples (27.0%). Phylogenetic and sequence homology analyses of GCRV partial VP6 gene between 23 Korean and other known porcine GCRVs demonstrated that Korean strains belonged to the porcine lineage. Furthermore, one Korean porcine strain shared the highest nucleotide (89.7-89.0%) and deduced amino acid sequence (92.9-93.9%) identities with bovine GCRV strains and was placed in the bovine GCRV lineage indicative of bovine origin. In conclusion, porcine GCRV infections are widespread in piglets with diarrhea in South Korea. The infecting porcine GCRVs mostly belong to the porcine lineage with the exception of one bovine-like GCRV, which possibly originated from bovine GCRV due to interspecies transmission.

Molecular detection and characterization of unclassified bovine enteric caliciviruses in South Korea.

The unclassified bovine enteric calicivirus (BEC) is a new bovine enteric calicivirus that is different from bovine norovirus, and causes diarrhea and pathologies in the small intestine of calves. This virus includes Nebraska (NB)- and Newbury agent 1 (NA1)-like strains. The prevalence of this BEC and its genetic characterization has only been reported in the UK and the USA. This study examined the prevalence and genetic diversity of these BECs in diarrheic calves in South Korea. Among a total of 645 diarrheic fecal specimens obtained from 629 cattle herds, these unclassified BECs were detected in 59 (9.1%) diarrheic fecal samples from 57 herds (9.3%) by either RT-PCR or nested PCR. Sequence and phylogenetic analyses of the partial RdRp gene showed that all the Korean BECs clustered together and were closely related to the NB-like viruses (80.9-88.1% nucleotide and 84.5-98.4% amino acid) but not to the NA1-like viruses (75.8-78.4% nucleotide and 79.7-82.8% amino acid). Although these viruses could not be classified into NA1- and NB-like viruses from the sequence and phylogenetic data of the entire capsid gene, all the Korean BECs clustered together on a branch separate from the other known BECs. These results show that these BEC infections are endemic in diarrheic calves in South Korea. The infecting strains are genetically closer to the NB-like viruses but have a distinct evolutionary pathway.

Essential role for platelet-activating factor-induced NF-kappaB activation in macrophage-derived angiogenesis.

Activated monocyte-macrophages have been implicated in tumor angiogenesis via their capacity to produce many potent angiogenic factors. However, the mechanisms leading to production of these angiogenic factors in macrophages remain to be elucidated. In this study, we demonstrated by use of a mouse Matrigel implantation model that mouse peritoneal macrophages induce angiogenesis. mRNA expression and protein synthesis of macrophage-derived crucial angiogenic factors such as IL-1, TNF-alpha, basic fibroblast growth factor, and vascular endothelial growth factor (VEGF) were blocked by platelet-activating factor (PAF) receptor antagonists. It was also observed that inhibitors of NF-kappaB blocked macrophage production of these angiogenic factors. Gene expression and protein synthesis of the angiogenic factors cited above were also inhibited in IkappaBalpha-mutated macrophages. VEGF is the most potent angiogenic factor in macrophage-induced angiogenesis. PAF antagonists or NF-kappaB inhibitors also inhibit the capacity of conditioned medium from LPS-stimulated human peripheral blood monocytes to induce sprouting of porcine pulmonary arterial endothelial cells. These data indicate that PAF-induced NF-kappaB activation is a common upstream pathway leading to the production of crucial macrophage-derived angiogenic factors. This will provide an important clue for a better understanding of mechanisms involved in tumor angiogenesis.