A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to rapidly detect foot-and-mouth disease virus serotype C (FMDV C). By testing 10-fold serial dilutions of FMDV C samples, sensitivity of the FMDV C RT-LAMP was found to be 10 times higher than that of conventional reverse transcription-PCR (RT-PCR). No cross-reactivity with A, Asia 1, or O FMDV or swine vesicular disease virus (SVDV) indicated that FMDV C RT-LAMP may be an exciting novel method for detecting FMDV C.
Animals ; Foot-and-Mouth Disease/*diagnosis ; Foot-and-Mouth Disease Virus/genetics ; Nucleic Acid Amplification Techniques/*methods/veterinary ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Reverse Transcription/genetics ; Sensitivity and Specificity

A one step reverse transcription PCR (RT-PCR) combined nested PCR was set up to increase efficiency in the diagnosis of canine distemper virus (CDV) infection after developement of nested PCR. Two PCR primer sets were designed based on the sequence of nucleocapsid gene of CDV Onderstepoort strain. One-step RT-PCR with the outer primer pair was revealed to detect 10(2) PFU/ml. The sensitivity was increased hundredfold using the one-step RT-PCR combined with the nested PCR. Specificity of the PCR was also confirmed using other related canine virus and peripheral blood mononuclear cells (PBMC) and body secretes of healthy dogs. Of the 51 blood samples from dogs clinically suspected of CD, 45 samples were revealed as positive by one-step RT-PCR combined with nested PCR. However, only 15 samples were identified as positive with a single one step RT-PCR. Therefore approximately 60% increase in the efficiency of the diagnosis was observed by the combined method. These results suggested that one step RT-PCR combined with nested PCR could be a sensitive, specific, and practical method for diagnosis of CDV infection.
Animals ; Distemper Virus, Canine/genetics/*isolation & purification ; Dogs ; Polymerase Chain Reaction/*methods/*veterinary ; RNA, Viral/genetics/isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Vaccines, Attenuated ; Viral Vaccines

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.
Animals ; Endogenous Retroviruses/*metabolism ; Gene Expression Regulation, Viral/*physiology ; RNA, Messenger/genetics/metabolism ; RNA, Viral/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction/methods/*veterinary ; Sensitivity and Specificity ; Swine/*virology

One step TaqMan reverse transcription polymerase chain reaction (RT-PCR) using TaqMan probe was developed for detection of Japanese encephalitis virus (JEV). Real-time RT-PCR was optimized to quantify JEV using the detection system (Rotor Gene 2000 detector) and dual-labeled fluorogenic probes. The gene specific labeled fluorogenic probe for the 3' non-translated region (3' NTR) was used to detect JEV. When the specificity of the assay using specific JEV primers was evaluated by testing three different JEV strains, other swine viruses and bovine viral diarrhea virus, no cross-reactions were detected with non-JE reference viruses. A single tube TaqMan assay was shown to be 10-fold more sensitive than the conventional two-step RT-PCR method. Detection limits of two step and real-time RT-PCR for JEV were 112 TCID50 /ml and 11.2 TCID50 /ml, respectively. Quantification of JEV was accomplished by a standard curve plotting cycle threshold values (Ct ) versus infectivity titer. Real-time RT-PCR assay using single tube method could be used as a sensitive diagnostic test, and supplied the results in real time for detection and quantification of JEV. We could detect JEV RNA genome in plasma samples of pigs inoculated with KV1899 strain at 2 days post inoculation, but couldn't in 41 fetus samples. This assay was sensitive, specific, rapid and quantitative for the detection of JEV from laboratory and field samples.
Animals ; DNA Primers/chemistry ; DNA Probes/chemistry ; Encephalitis Virus, Japanese/genetics/*isolation&purification ; Encephalitis, Japanese/diagnosis/*veterinary/virology ; RNA, Viral/analysis ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction/methods/*veterinary ; Sensitivity and Specificity ; Swine ; Swine Diseases/*diagnosis/virology ; *Taq Polymerase

Porcine reproductive and respiratory syndrome virus(PRRSV)0, porcine circovirus type 2(PCV-2) and porcine parvovirus (PPV)0 infections were investigated as possible causes of the postweaning multisystemic wasting syndrome(PMWS). Specific primers for RT-PCR and PCR were designed for the differential detection of PRRSV, PCV-2 and PPV. Using PCR, these viruses were detected in homogenized tissue samples from pigs that had respiratory of reproductive problems in the time period between 1998 and 2000; the overall prevalences were: PRRSV 31.4%, PCV-2 46.5%, and PPV 8.1%. PCV-2 was also detected in aborted fetal tissues.
Aborted Fetus/virology ; Animals ; Base Sequence ; Circoviridae Infections/diagnosis/epidemiology/*veterinary ; Circovirus/genetics/isolation&purification ; DNA Primers ; Diagnosis, Differential ; Korea/epidemiology ; Parvoviridae Infections/diagnosis/epidemiology/*veterinary ; Parvovirus, Porcine/genetics/isolation&purification ; Polymerase Chain Reaction/methods/veterinary ; Porcine Reproductive and Respiratory Syndrome/diagnosis/*epidemiology ; Porcine respiratory and reproductive syndrome virus/genetics/isolation & purification ; Prevalence ; Respiratory Tract Infections/veterinary/virology ; Reverse Transcriptase Polymerase Chain Reaction/methods/veterinary ; Sequence Homology ; Swine ; Swine Diseases/diagnosis/*epidemiology ; Wasting Syndrome/*veterinary/virology

The targeted RNA recombination was attempted to substitute the membrane (M) protein gene and part of the nucleocapsid (N) protein gene of mouse hepatitis virus with the corresponding sequences from bovine coronavirus. Using a defective interfering (DI) RNA-like cDNA construct derived from pMH54, 690 nucleotides representing the entire M gene and the 5' most 915 nucleotides of the N gene of the mouse hepatitis virus Albany 4 mutant were attempted to be replaced. Upon infection of cells with Albany 4 followed by transfection with synthetic RNA transcribed from the DI-like cDNA construct, recombinant mouse hepatitis viruses as the large plaque forming phenotype were isolated by plaque assays at the non-permissive temperature of 391 degrees C. By RT-PCR and sequencing, those large plaque phenotypes were confirmed to have contained the thermostable phenotype marker derived from the transfected RNA, demonstrating that recombination occurred between the Albany 4 genomic RNA and the in vitro RNA transcripts. Further analysis of the recombinant viruses indicated that there combination had taken place within the region of 222 nucleotides between positions 916 and 1,137 of the N gene. This is the region immediately downstream of the replacement sequence and the start of the temperature resistant phenotype marker. The results suggest that the M and part of the N genes of bovine coronavirus may not be able to complement the function of those of mouse hepatitis virus. This study redirects our current approach of utilizing the MHV targeted RNA recombination as a means to study bovine coronavirus genetics towards the construction of an infectious cDNA clone.
Amino Acid Sequence ; Animals ; Base Sequence ; Cattle ; Cells, Cultured ; Coronavirus, Bovine/*genetics ; DNA, Complementary/genetics ; Gene Targeting/veterinary ; Genetic Vectors ; Mice ; Molecular Sequence Data ; Murine hepatitis virus/*genetics ; Nucleocapsid Proteins/*genetics ; Phenotype ; Plaque Assay/veterinary ; RNA, Viral/chemistry/*genetics/isolation&purification ; Reverse Transcriptase Polymerase Chain Reaction/methods/veterinary ; Sequence Homology, Amino Acid ; Transfection/veterinary ; Viral Matrix Proteins/*genetics

Japanese encephalitis (JE) is an important vector-borne viral disease of humans and horses in Asia. JE outbreaks occur regularly amongst humans in certain parts of India and sporadic cases occur among horses. In this study, JE seroprevalence and evidence of JE virus (JEV) infection among horses in Haryana (India) is described. Antibodies against JEV were detected in 67 out of 637 (10.5%) horses screened between 2006 and 2010. Two foals exhibiting neurological signs were positive for JEV RNA by RT-PCR; JEV was isolated from the serum of one of the foals collected on the second day of illness. This is the first report of JEV isolation from a horse in India. Furthermore, a pool of mosquitoes collected from the premises housing these foals was positive for JEV RNA by RT-PCR. Three structural genes, capsid (C), premembrane (prM), and envelope (E) of the isolated virus (JE/eq/India/H225/2009) spanning 2,500 nucleotides (from 134 to 2,633) were cloned and sequenced. BLAST results showed that these genes had a greater than 97% nucleotide sequence identity with different human JEV isolates from India. Phylogenetic analysis based on E- and C/prM genes indicated that the equine JEV isolate belonged to genotype III and was closely related to the Vellore group of JEV isolates from India.
Animals ; Antibodies, Monoclonal ; Cloning, Molecular ; Culex/virology ; Encephalitis Virus, Japanese/*genetics/*isolation & purification ; Encephalitis, Japanese/epidemiology/*veterinary/virology ; Enzyme-Linked Immunosorbent Assay/methods/veterinary ; Female ; Genes, Viral ; Genotype ; Horse Diseases/epidemiology/*virology ; Horses ; India/epidemiology ; RNA, Viral/genetics/isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Seroepidemiologic Studies

Classical swine fever virus (CSFV) causes a highly contagious disease among swine that has an important economic impact worldwide. CSFV strain LOM is an attenuated virus of low virulent strain of Miyagi isolated from Japan in 1956. Eight DNA fragments representing the genome of the CSFV strain LOM were obtained by RT-PCR. These were used to determine the complete nucleotide sequence and construct a full-length cDNA clone which was called Flc-LOM. Sequence analysis of the recombinant clone (Flc-LOM) revealed the presence of eight mutations, resulting in two amino acid substitutions, when compared to the parental sequence. RNA transcripts of both LOM and Flc-LOM were directly infectious in PK-15 cells. The rescued Flc-LOM virus grew more slowly than the parental virus, LOM, in the cells. Intramuscular immunization with Flc-LOM was safe and highly immunogenic in pigs; no clinical signs or virus transmission to sentinel animals were observed after 35 days. CSFV-specific neutralizing antibodies were detected 14 days post-infection. After challenge with the virulent CSFV strain SW03, pigs immunized with Flc-LOM were shown to be fully protected. Thus, our newly established infectious clone of CSFV, Flc-LOM, could serve as a vaccine candidate.
Animals ; Antibodies, Viral/blood ; Base Sequence ; Cell Line ; Classical Swine Fever/immunology/*virology ; Classical swine fever virus/*genetics/immunology/pathogenicity ; Cloning, Molecular ; DNA, Complementary/genetics/immunology ; Immunization/methods/standards/veterinary ; Molecular Sequence Data ; Neutralization Tests/veterinary ; RNA, Viral/chemistry/genetics ; Recombinant Proteins/immunology ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Sequence Analysis, DNA ; Specific Pathogen-Free Organisms ; Swine ; Virulence

The aim of this study is to establish the method of loop-mediated indirect PCR assay for detection of Reproductive and Respiratory Syndrome Virus (PRRSV) infection and differentiation of highly pathogenic PRRSV (HP-PRRSV) and lowly pathogenic PRRSV (LP-PRRSV). Based on the alignments of ORF2 gene sequences and ORFla gene sequences of PRRSV Chinese isolates deposited in GenBank, two pairs of specific probes were designed and labeled to both ends of the soybean Lectin gene fragment by PCR, respectively. The probe-labeled soybean Lectin genes were used to be reporter genes for detection and differentiation of PRRSV. After one round strand displacement reaction, the reporter genes were amplified by reverse PCR. The specific PCR products were 193bp, 355bp for HP-PRRSV and 193bp, 442bp for LP-PRRSV, respectively. The method could detect 5. 6 TCID50/mL LP-PRRSV RNA and 18 TCIDs0/ mL HP-PRRSV RNA, and co-infection did not affect detection sensitivity. No amplification was observed with other porcine originated pathogens including CSFV, PPV, PRV, PCV2, ETEC and Haemophilus parasui. Twenty clinical samples were used for comparative testing with conventional PCR. Fourteen samples were found positive for PRRSV by the loop-mediated indirect PCR, of which 4 were LP-PRRSV, 9 HP-PRRSV and 1 LP/HP-PRRSV co-infection, consistent with the conventional PCR test results. In conclusion, the loop-mediated indirect PCR is a simple, rapid, sensitive and specific etiologic diagnosis tool, and suitable for the differential diagnosis of HP/LP-PRRSV, especially for identification of mixed infection of HP/LP-PRRSV.
Animals ; Coinfection ; veterinary ; DNA Primers ; genetics ; DNA, Complementary ; genetics ; Diagnosis, Differential ; Genes, Reporter ; Genetic Markers ; genetics ; Humans ; Porcine Reproductive and Respiratory Syndrome ; diagnosis ; virology ; Porcine respiratory and reproductive syndrome virus ; genetics ; isolation & purification ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; veterinary ; Sensitivity and Specificity ; Swine ; Time Factors ; Viral Proteins ; genetics

A simple, rapid and sensitive colorimetric Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) method was established to detect human influenza A H1N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the HA gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for one and half hour. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP assay was validated by cross-reaction with different swine and human influenza virus including human seasonal influenza A /H1N1 A /H3N2, influenza B and swine A /H1N1. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of human influenza A H1N1 HA gene. The assay was further evaluated with 30 clinical specimens with suspected pandemic influenza A H1N1 virus infection in parallel with RT-PCR detection and 26 clinical specimens with seasonal influenza virus infection. Our results showed that the RT-LAMP was able to achieve a sensitivity of 60 RNA copies with high specificity, and detection rate was comparable to that of the RT-PCR with the clinical samples of pandemic influenza A H1N1 infection. The RT-LAMP reaction with HNB could also be measured at 650nm in a microplate reader for quantitative analysis. Thus, we concluded that this colorimetric RT-LAMP assay had potential for the rapid screening of the human influenza A H1N1 virus infection in National influenza monitoring network laboratories and sentinel hospitals of provincial and municipal region in China.
Animals ; Colorimetry ; methods ; DNA Primers ; genetics ; Electrophoresis, Agar Gel ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; Influenza A Virus, H3N2 Subtype ; genetics ; Influenza, Human ; diagnosis ; virology ; Naphthalenesulfonates ; chemistry ; Nucleic Acid Amplification Techniques ; methods ; Orthomyxoviridae Infections ; diagnosis ; veterinary ; virology ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Swine ; Swine Diseases ; diagnosis ; virology ; Temperature