Association of aster yellows subgroup 16SrI-B phytoplasmas with a disease of Rehmannia glutinosa var. purpurea

A new phytoplasma disease of Rehmannia glutinosa var. purpurea was observed in the Czech Republic in 1998. Infected plants showing severely proliferating shoots, leaves reduced in size with vein clearing and chlorosis, shortened internodes and virescent petals died in advanced stages of the disease. Electron microscopy examination of the ultra-thin sections revealed the presence of numerous polymorphic bodies in phloem tissue of leaf midribs and petioles. The disease was successfully transmitted from infected plant via a dodder bridge into periwinkle ( Catharanthus roseus ). The phytoplasma aetiology of this disease was further confirmed by polymerase chain reaction (PCR) using universal primers R16F2/R16R2. Restriction fragment length polymorphism (RFLP) analysis of amplification products indicated the presence of aster yellows related phytoplasmas (16SrI-B) in naturally infected samples of R. glutinosa var . purpurea and in symptomatic periwinkle after dodder transmission of the agent. A comparison of the amplified sequence with 17 sequences available in the GenBank confirmed the classification of the phytoplasma in the subgroup 16SrI-B. This is the first report of natural occurrence of phytoplasma-associated disease in R. glutinosa var. purpurea.     

Phylogenetic Analysis of Field Isolates of Feline Calcivirus (FCV) in Japan by Sequencing Part of Its Capsid Gene

The molecular epidemiology of the infectious disease caused by feline calcivirus (FCV) in Japan was investigated by analysing the phylogenetic relationship among 21 Japanese field isolates, including the F4 strain, and 30 global isolates. Parts of the capsid gene (B–F) of the isolates were amplified by RT-PCR, and the amino acid sequences were compared with those from the global isolates. Thirty-seven and 14 out of a total of 51 isolates were clustered into two distinct genogroups, I and II respectively, by UPGMA and NJ analysis. Seven of the 21 Japanese isolates (33%) fell into group I together with 30 global isolates, while the other 14 Japanese isolates (67%) belonged to group II. The bootstrap repetition analysis of groups I and II formed by the NJ method gave a value of 99.0%. The 14 latter Japanese isolates were clearly separated from the isolates in group I, and they were different from any previously known FCV, forming a new genogroup, which implies that this lineage has been confined to Japan. Comparing the amino acid sequences shared by groups I and II, the amino acid at position 377 in B region was asparagine (Asn or Asp (NH₂)) in group I, while it was lysine (Lys) in all the strains in group II. Similarly, the amino acid at position 539 in the F region was alanine (Ala) or proline (Pro) in group I, while it was valine (Val) in group II; glycine (Gly) at position 557 in group I was serine (Ser) in Group II; and phenylalanine (Phe) or leucine (Leu) at position 566 in genogroup I was tyrosine (Tyr) in group II.     

A Universal `One-tube' RT-PCR Protocol for Amplifying Isolates of Bovine Viral Diarrhoea Virus

The increase in the knowledge of the genetic variability of BVDV and the identification of some of the genetic determinants of its pathogenicity require robust and practical tools for rapid molecular characterization of the various genotypes of this virus. This study was undertaken to develop a standard protocol for RT-PCR that allows the amplification of various parts of the genome of BVDV without the need for optimizing each individual reaction. The reaction set-up is very flexible because it consists of two pre-mixes. These are a master mix, with all the required reagents except the desired primers, which are the components of the second pre-mix and are therefore easily interchangeable between the different reactions. After adding any primer-containing pre-mix to the fixed master mix, a non-interrupted cycling protocol led to the generation of amplicons of up to 4 kbp in size in amounts sufficient for subsequent sequencing reactions. The method was applied to five different regions of the BVDV genome: (i) the well-known 5-UTR to differentiate genotypes I and II; (ii) the entire E2 gene, or an approximately 550 bp region within the E2 gene, in order to find the molecular equivalent of antigenic varieties; (iii) the entire structural protein coding region covering the Npro, capsid, E ^RNS, E1 and E2 genes; (iv) a 2.1 kbp region embracing the NS2/3 junction which is known to be cleaved in cytopathic biotypes of BVDV; and (v) the region covering the entire NS4B and NS5A/B genes. All six RT-PCRs were successfully applied using (i) primers with lengths of between 20 and 52 nucleotides, (ii) an aliquot of RNA extracted from either 10⁶ infected bovine embryonal lung cells or the same number of leukocytes from viraemic cattle, and (iii) all the genotype I and II strains of BVDV tested. The technique described was used to generate various Sindbis virus/BVDV recombinants. The correct processing of the amplicon-derived E2 glycoprotein of BVDV strain PT810 was demonstrated by its reaction with a monoclonal antibody in an immunofluorescence assay. Given the variety of RT-PCRs tested, we conclude that this universal protocol may be useful with other RNA viruses.     

Prevalence of bovine herpesvirus-4 infection in cats in Central Michigan

The gammaherpesvirus bovine herpesvirus-4 (BHV-4) has been isolated from a wide variety of animals, including lions and domestic cats. Although BHV-4 antibodies have been detected in normal cats and cats with urinary disorders, the epidemiology and pathogenic role of BHV-4 in cats is unknown. The purpose of this study was to determine the prevalence of BHV-4 antibodies and viral nucleic acid in a population of free-roaming cats. Plasma and peripheral blood leukocyte samples were collected from 52 male and 52 female free-roaming cats impounded at a regional animal control facility in Central Michigan. Plasma concentrations of BHV-4 antibodies were measured with an indirect fluorescent antibody test. Peripheral blood leukocyte DNA was isolated, and a 2-stage polymerase chain reaction with heminested primers delineating a conserved portion of the BHV-4 glycoprotein B gene homologue was used to amplify BHV-4-specific DNA sequences. BHV-4 antibodies were detected in 38 (73%) male and 23 (44%) female cats. Seropositive cats were significantly more likely to be male than female (odds ratio = 3.22; P = .007). Cell-associated viremia was detected in 17 (33%) male and 11 (21%) female cats. Of the 61 seropositive cats, 23 (38%) had a detectable viremia; only 5 (12%) seronegative cats had detectable viremia. Seropositive cats were significantly more likely to be viremic than seronegative cats (OR = 4.30: P = .009). Our results suggest that BHV-4 infection may be more widespread in certain cat populations than previously reported. Furthermore, many cats seropositive for BHV-4 antibodies have a concurrent cell-associated viremia.     

Effect of polymorphisms linked to LEP gene on its expression on adipose tissues in beef cattle

In cattle, genetic markers at the leptin (LEP) gene and at those linked to the gene have been described as affecting calving interval (markers LEPSau3AI and IDVGA51), or daily weight gain (BMS1074 and BM1500). This work investigated the effect of these alleles on LEP mRNA levels in cattle subcutaneous and omental adipose tissues. A sample of 137 females of a Brangus‐Ibage beef cattle herd was analysed to evaluate the distribution of the polymorphisms; then, animals having at least one of the IDVGA51*181 (allele 181 at marker IDVGA51; six animals), LEPSau3AI*2 (four), BMS1074*151 (13), BM1500*135 (six) alleles and a control group composed of animals without any of these alleles (four animals) were submitted to surgery to obtain omental and subcutaneous adipose tissues. Leptin mRNA expression was quantified by TaqMan RT‐PCR, using 18S rRNA as internal control and adjusted for the effect of body condition score, through regression analysis. Omental fat had LEP gene expression 33% lower than the subcutaneous tissue. Carriers of IDVGA*181 and BMS1074*151 showed subcutaneous fat leptin mRNA levels higher than the controls. Leptin controls feed intake and coordinates reproduction; therefore, animals with higher LEP gene expression will probably have lower daily weight gain than others with similar forage offer and nutritional condition and probably will also have longer calving interval.     

Role of PARP in endothelial cell apoptosis induced by angiotensin Ⅱ

AIM: To explore the role of poly-(ADP-ribose)polymerase (PARP) in the cultured endothelial cell apoptosis induced by angiotensin Ⅱ.METHODS: The cultured endothelial cells were treated with angiotensin Ⅱ at concentration of 1 μmol/L.The apoptosis of endothelial cells was assessed by TUNEL.Meanwhile,the activity of PARP and the content of nitric oxide (NO) were also measured.RESULTS: Angiotensin Ⅱ induced apoptosis in endothelial cells in a time-dependent manner.The content of NO begun to increase at 6 h (P<0.05),and peaked at 24 h.The activity of PARP also increased at 6 h (P<0.05),peaked at 12 h,and was lower than that in the control at 48 h (P<0.05).CONCLUSION: The cytotoxicity of NO has a relevant role in apoptosis of endothelial cells induced by angiotensin Ⅱ,and can increases the activity of PARP.     

套式反转录—聚合酶链反应检测恙螨及鼠肺内肾综合征出血热病毒RNA

选择覆盖肾综合征出血热病毒（ＨＦＲＳＶ）膜蛋白（ＭＰ）基因的保守区核苷酸序列合成２对引物,采用异硫氰酸胍一步抽提ＲＮＡ,建立了ＲＴ－ＰＣＲ检测鼠体恙螨及游离恙螨体内ＨＦＲＳＶ－ＲＮＡ的方法,扩增产物经凝胶电泳及斑点印迹杂交证实具有特异性。结果显示,ＨＦＲＳＶ抗原阳性鼠体恙螨５０只组、１０只组、游离螨５０只组,ＨＦＲＳＶ抗原阳性鼠肺１０００ｍｇ、５００ｍｇ组经ＲＴ－ＰＣＲ检测为阳性;ＨＦＲＳＶ抗原阳性鼠体恙螨５只组,ＨＦＲＳＶ抗原阴性鼠体恙螨５０只和１０只组,游离螨１０只和５只组,鼠肺ＨＦＲＳＶ抗原阳性１００ｍｇ组均未见有明显扩增带。进一步用套式反转录－聚合酶链反应（ＮｅｓｔｅｄＲＴ－ＰＣＲ）检测,在ＲＴ－ＰＣＲ未测出ＨＦＲＳＶ－ＲＮＡ的各组中均检测有ＨＦＲＳＶ－ＲＮＡ。结果表明ＮｅｓｔｅｄＲＴ－ＰＣＲ具有高特异、高敏感的特点,可用于检测恙螨体内微量ＨＦＲＳＶ－ＲＮＡ,为确认恙螨作为ＨＦＲＳＶ的传播媒介提供了分子生物学证据。     

小鹅瘟病毒荧光RPA恒温快速检测方法的建立与应用

旨在建立一种以重组酶聚合酶扩增技术（RPA）为基础的快速检测方法,用于小鹅瘟病毒的快速检测。小鹅瘟是一种常见的水禽传染病,严重危害我国养鹅业的健康发展。为了快速准确对小鹅瘟进行诊断,减少该病的危害,本研究以小鹅瘟病毒VP3基因保守片段为靶点,利用重组酶聚合酶扩增技术（RPA）建立了一种准确高效的小鹅瘟病毒RPA恒温快速检测方法,并对检测方法的灵敏度、特异性进行评价,并与传统PCR和传统RT-PCR方法进行比较。结果表明,该检测方法具有较高的灵敏度,可检测到10 copies/μL的病毒核酸;具有良好的特异性,只特异性地扩增鹅细小病毒,而与鹅副黏病毒、鹅源鸭瘟病毒、小鹅流行性感冒病毒、鹅副伤寒病毒、大肠杆菌和曲霉菌均未发生交叉反应;同时该方法重复性检测的变异系数低于6%,具有很好的重复性。阳性符合试验表明该检测方法与荧光定量PCR符合率为99%。该方法可以很好地应用于小鹅瘟的大规模临床样本检测,为小鹅瘟病毒的高通量检测和流行病学调查提供技术手段。