Porcine circovirus type 3 (PCV3) was detected in Shandong, China. One hundred and thirty-two of 222 (59.46%) samples were PCV3 positive, while 52 of 132 (39.39%) samples were co-infected with PCV2. There were no clinical signs of infection in either multiparous sows or live-born infants. Two strains of PCV3 were indentified from natural stillborn foetuses. Phylogenetic analysis showed the two strains of PCV3 are 96% identical to the known PCV3/Pig/USA (KX778720.1, KX966193.1 and KX898030.1) and closely related to Barbel Circovirus. Further studies of the epidemiology of PCV3 and the co-infection with PCV2 are needed.
Several biochemical properties and the sequence of the fusion glycoprotein (F) have indicated that turkey rhinotracheitis virus (TRTV) is a pneumovirus, subfamily Pneumovirinae of the Paramyxoviridae family. As TRTV was known to generate polycistronic mRNAs, cDNA was generated from TRTV strain UK/3BV/85-infected Vero cell mRNAs using an oligonucleotide primer corresponding to a region of the F gene. Sequencing of four cDNAs revealed that the gene adjacent to the beginning (3' end) of the F gene was that for the matrix (M) protein, i.e., that TRTV had the partial gene order 3'-M-F-5'. This was unexpected as human respiratory syncytial (RS) virus, the type species of the genus Pneumovirus, has the partial gene order 3'-M-SH-G-F-5', where SH and G are the small hydrophobic protein and attachment glycoprotein, respectively. Instead TRTV resembled the Morbillivirus and Paramyxovirus genera of the Paramyxoviridae (subfamily Paramyxovirinae) which have the partial gene order 3'-M-F-5'. Two further oligonucleotides, one corresponding to a sequence near the end of the M gene and the other (oligo B) to a sequence near the beginning of the F gene, with their 5' ends spaced 300 nucleotides apart on the basis of the cDNA sequence, were used in a polymerase chain reaction (PCR) using genomic RNA as template. Only a PCR product of 0.3 kb was obtained. The same sized product was also obtained using these oligonucleotides and genomic RNA from three other TRTV strains (SA/91/78, UK/8544/85, and SA/2381/88) which had been grown in chicken tracheal organ cultures. In addition PCR was performed using genomic RNA from TRTV-3BV and SA/2381/88 with oligo B and another oligonucleotide near the 5' end of the gene upstream from M, spaced 1141 nucleotides apart on the basis of the sequence data. Only a 1.14-kb PCR product was obtained. Larger products would have been expected if another gene had been situated between M and F. The absence of such larger products, plus the demonstration that infected cells contained M-F dicistronic mRNAs, supported the conclusion that in the TRTV genome the M gene is adjacent to the F gene in the order 3'-M-F-5'. The 5' termini of the M and F mRNAs were confirmed by mRNA mapping. The TRTV M gene encoded a protein of 254 amino acids, very similar to that of RS virus (256 residues; 37% amino acid identity) but very different from that of the morbilliviruses and paramyxoviruses (approximately 350 residues).(ABSTRACT TRUNCATED AT 400 WORDS)
In this study, the co-infection of Torque teno sus virus (TTSuV) and porcine circovirus type 3 (PCV3) was reported. One hundred and ten of 132 (83.3%) PCV3-positive samples were co-infected with Torque teno sus virus 1 (TTSuV1). Ninety-four of 132 (71.2%) PCV3-positive samples were co-infected with Torque teno sus virus 2 (TTSuV2). Sixty-six of 132 (50.0%) of PCV3-positive samples were co-infected with both TTSuV1 and TTSuV2. There were no clinical signs of infection in pigs that were both PCV3-positive and PCV2-negative, in either multiparous sows or live-born infants. The high co-infection rate provides valuable information for the further study of the pathological correlation between PCV3 and TTSuVs.
A highly pathogenic swine disease designated as 'porcine high fever disease (PHFD)' appeared recently in China. Porcine reproductive and respiratory syndrome virus (PRRSV) was identified as an agent associated with PHFD, and two discontiguous sequence deletions were identified as a genetic marker in the Nsp2 region of the viral genome. To examine PHFD in Shandong province, a total of 10 PRRSV isolates were recovered from pig herds that had never been vaccinated for PRRS. Sequence analysis of open reading frame 5 (ORF5) showed that the level of identity among the 10 isolates ranged between 88.2 and 99.2%. For the non-structural protein 2 (Nsp2) gene, three isolates shared high sequence identity with VR-2332, the prototype virus of the North American genotype, while the remaining seven isolates exhibited two discontiguous sequence deletions that were identical to those of PHFD: a one-amino-acid (phenylalanine) deletion at position 482 and a 29-amino-acid deletion at positions 533-561 of Nsp2. Experimental infection of pigs with SD-JN, which was one of the seven isolates containing such deletions, resulted in severe clinical symptoms characterized by red discoloration on the body and hemorrhages in the lungs, kidneys, and inguinal lymph nodes, accompanied by higher mortality and longer duration of viremia. These symptoms were similar to those of PHFD observed in the field. Our results show that VR2332-like PRRSV coexists with PHFD-associated atypical PRRSV in pig herds in the Shandong area, and different PRRSV isolates differ greatly in their pathogenesis and virulence in pigs.
In this study, a rapid and specific assay for the detection of porcine circovirus type 3 (PCV3) was established using loop-mediated isothermal amplification (LAMP). Four primers were specifically designed to amplify PCV3. The LAMP assay was effectively optimized to amplify PCV3 by water bath at 60°C for 60 min. The detection limit was approximately 1 × 10 copy in this LAMP assay. Compared to porcine circovirus type 2 (PCV2), both gE and gD genes of pseudorabies virus (PRV) and porcine parvovirus (PPV), the LAMP assay showed a high specific detection of PCV3. A visible detection method was developed using SYBR Green I to recognize the results rapidly. Based on the detection of 20 clinical tissue samples, the LAMP assay was more practical and convenient than classical PCR due to its simplicity, high sensitivity, rapidity, specificity, visibility and cost efficiency.
Field studies using the synthetic sex pheromone of Trichophysetis cretacea, a trinary blend of (Z)‐11‐hexadecenyl acetate (Z11‐16:OAc), (Z)‐11‐hexadecenal (Z11‐16:Ald) and (Z)‐11‐hexadecenol (Z11‐16:OH), were performed in Sichuan to determine operational parameters for detection and control, such as dispenser type, blend ratio, dosage, and trap type, height and density. Of three pheromone dispensers tested, grey halo‐butyl isoprene elastomeric septa were significantly more effective than polyvinyl chloride capillary tubing or silicone rubber septa. The ratio of the three components in the blend significantly affected moth catch. In the halo‐butyl isoprene septa, the most effective ratio was 5 : 2 : 1 Z11‐16:OAc:Z11‐16:Ald:Z11‐16:OH. Sticky wing traps caught significantly more moths than water, noctuid moth or cone funnel traps. The most effective height at which wing traps were hung was 20 cm above the jasmine plants. Optimum trap density was 45 traps per hectare. Addition of volatile jasmine compounds did not increase the attractiveness of the sex pheromone. A dosage of 50 μg Z11‐16:OAc per lure was most effective in the autumn weather conditions of Quanwei. These data provide sufficient information to develop effective protocols for using the T. cretacea pheromone to detect and monitor this pest in the jasmine fields.
Tomato (Lycopersicon esculentum Mill.) is an important vegetable crop grown in Liaoning Province, China. In May 2012, dark brown, angular lesions were observed on lower, older leaves of 5-month-old tomato plants of cv. Bi Jiao in commercial greenhouses in Dawa County, which is administered by Panjin City in Liaoning Province. The irregularly shaped lesions varied in size from 1 to 7 mm in diameter. The necrotic lesions were usually surrounded by a yellow halo. Sporulation was rarely seen on the leaf surfaces. This contrasts with tomato leaf mold caused by Passalora fulva, in which the conidia develop as a velvety brown patch in lesions. By July 2012, the same disease was found in research greenhouses of Shenyang Agriculture University and Liaoning Academy of Agricultural Sciences. The incidence of symptoms was 30 to 40%. To identify the pathogen, leaf pieces (3 to 5 mm) with both infected and healthy portions were taken at the edge of lesions and surface-disinfected by placing them in 75% ethanol for 5 s, then transferred to a 0.1% aqueous mercuric chloride solution for 30 s and rinsed with sterilized water three times. The sections were placed on potato dextrose agar (PDA) at 25°C in the dark. Ten pure fungal cultures were obtained from single spores. For studies of microscopic morphology, isolates were grown on synthetic nutrient agar (SNA) in slide cultures. Conidia ranged in shape from subglobose or ovoid (2 to 4 × 2 to 3 μm) to subcylindrical (4.5 to 17.8 × 2.4 to 4.5 μm). Conidiophores were straight to slightly flexuous with typical nodes. The internal transcribed spacer (ITS) region from isolate PJ12-36 was amplified using primers ITS1 and ITS4 and sequenced (GenBank Accession No. KC137278). The 560-bp amplicons had 99% identity to C. oxysporum (JQ775499). On the basis of morphological characteristics (1) and nucleotide homology, the isolate was identified as C. oxysporum. Koch's postulates were fulfilled in the laboratory on fully expanded leaves of 5-week-old tomato plants ‘Moneymaker’ inoculated with C. oxysporum conidial suspensions (107 conidia ml–1). Eight seedlings were incubated in an illuminated incubator at 25°C for 8 to 10 days with 85% relative humidity. Characteristic lesions that developed on inoculated leaves were similar in appearance to those observed on diseased tomato plants in the greenhouse. C. oxysporum was reisolated from lesions and its identity was confirmed by morphological characteristics. C. oxysporum was previously reported as the causal agent of a leaf spot disease of pepper (2) and greenhouse tomato (4). To our knowledge, this is the first report of C. oxysporum causing disease on tomato foliage in China. It is noteworthy that C. oxysporum has led to a decline in pepper yield in northern regions of China (3). In addition, pathogenicity tests showed that the isolate W10-02 from pepper and the isolate PJ12-36 from tomato could each cause damage to the opposite host, producing symptoms similar to those observed on the host of origin. Studies are needed on strategies to manage C. oxysporum in crops, because its prevalence may cause yield loss both on tomato and pepper in northern regions of China. References: (1) K. Bensch et al. Stud. Mycol. 67:1, 2010. (2) A. M. Hammouda. Plant Dis. 76:536, 1992. (3) X. Y. Huang et al. Plant Dis. 96:1072, 2012. (4) J. S. Lamboy and H. R. Dillard. Plant Dis. 81:228, 1997.
ABSTRACT. The aim of this study was to develop an event-specific qualitative and real-time quantitative polymerase chain reaction (PCR) method for detection of herbicide-tolerance genetically modified (GM) soybean A2704-12. The event-specific PCR primers were designed, based on the 5'-flanking integration sequence in the soybean genome, to amplify the 239-bp target fragment. Employing the same event-specific primers, qualitative PCR and real-time quantitative PCR detection methods were successfully developed. The results showed that the A2704-12 event could be specifically distinguished from other GM soybean events. In the qualitative PCR assay, the limit of detection was 0.05%, and in the real-time quantitative PCR assay, the limit of detection was less than 0.01%. Moreover, our genomic DNA (gDNA) extraction protocol is highthroughput, safe, and low-cost. The event-specific PCR assay system is costefficient by using SYBR Green I in real-time PCR, and by using the same primers in both the qualitative and quantitative PCR assays. We therefore developed a high-throughput, low-cost, and event-specific qualitative and quantitative PCR detection method for GM soybean A2704-12. The method would be useful for market supervision and management of GM soybean A2704-12 due to its high specificity and sensitivity.
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