Analysis of experimental mink enteritis virus infection in mink: in situ hybridization, serology, and histopathology.

Strand-specific hybridization probes were used in in situ hybridization studies to localize cells containing mink enteritis virus (MEV) virion DNA or MEV replicative-form DNA and mRNA. Following the experimental MEV infection of 3-month-old unvaccinated mink, a significant increase in serum antibodies to MEV was detected at postinfection day (PID) 6, 2 days after the onset of fecal shedding of virus. Prior to the appearance of virus in feces, viral DNA could be detected in the mesenteric lymph node and intestine. The largest percentage of cells positive for virion DNA was 10% and was detected in the intestine on PID 6. However, replication of the virus apparently peaked at PID 4. The number of MEV replicative-form DNA and mRNA molecules was found to be approximately 250,000 copies per infected lymph node cell or crypt epithelial cell. The localization, levels, and time course of viral replication have important implications for the pathogenesis of MEV-induced disease. The data presented on MEV are correlated with earlier results on the other mink parvovirus, Aleutian mink disease parvovirus, and a possible explanation for the remarkable differences in pathogenesis of disease caused by these two parvoviruses is discussed.     

The mechanism of action of nitro-heterocyclic antimicrobial drugs. Metabolic activation by micro-organisms.

Although the target of the antimicrobial drug 1-methyl-2-nitro-5-vinylimidazole (MEV) has been shown to be DNA (Goldstein et al., 1977) the drug was ineffective in cell-free systems because it was not activated. Both the rate of metabolic activation of MEV and its antibacterial activity were increased when bacteria were grown in limiting oxygen. Mutants of Escherichia coli which were conditionally resistant to nitroimidazoles and nitrofurans were defective in drug activation. The activities of these drugs against E. coli correlated with their rates of metabolism. The antimicrobial spectrum of the drugs appeared to be related to their reducibility by different species.     

Analysis of deoxyribonucleic acid from five nuclear polyhedrosis viruses infecting plusiine larvae (Lepidoptera: Noctuidae)

Deoxyribonucleic acid (DNA) from isolates of five nuclear polyhedrosis viruses (NPV) from lepidopterous hosts of the noctuid subfamily Plusiinae was analyzed by ion-exchange and paper chromatography. Viruses and production hosts were: Trichoplusia ni singly embedded virion type (SEV) from T. ni, Pseudoplusia includens SEV from P. includens, T. ni multiply embedded virion type (MEV) from T. ni, Autographa californica MEV from A. californica, A. californica MEV from T. ni, and Rachiplusia ou MEV from R. ou. Neither uracil nor 5-methyl cytosine was detected in the DNAs. Adenine:thymine (A:T) and guanine:cytosine (G:C) ratios were nearly constant for all the NPVs. AT:GC ratios for the SEVs were 1.60 and 1.57 and were clearly separable from those of the MEVs which ranged from 1.32 to 1.38. No differences in DNA composition within SEV or MEV groups were apparent.     

A mouse linkage testing stock possessing multiple copies of the endogenous ecotropic murine leukemia virus genome

A new linkage testing stock of the laboratory mouse has been constructed. The stock, designated MEV (multiple ecotropic provirus), was developed by inbreeding and selection beginning with the cross of strains C58/J and AKXD-14. Eleven different murine leukemia virus (MuLV) proviruses have been fixed in the MEV/1Ty strain. Nine of these can be uniquely identified by Southern blotting of PvuII-digested DNA and probing with a cloned fragment of the ecotropic viral genome. Two proviruses had been mapped previously to chromosome 7, while single proviruses had been mapped to chromosomes 2, 9, and 11. The mapping of six additional proviruses, derived from C58/J, to chromosomes 1, 3, 5, 10, 18, and 19 is described. Another C58/J provirus was mapped to chromosome 8 and proved to be identical to the previously mapped C58v-1 virus inducibility gene of strain C58/Lw. Three dominant visible markers, hammer-toe (Hm), steel (Sl), and caracul-J (CaJ), located on chromosomes 5, 10, and 15, respectively, have been introduced onto the MEV genetic background by repeated backcrosses to provide additional linkage markers. It is estimated that approximately 50% of the genome can be screened by scoring 50 fully informative gametes from a linkage cross of the MEV-Hm, -Sl, -CaJ stock for the combination of viral and visible markers. A strategy for efficiently mapping new recessive visible mutations by pooling tissues for DNA extraction from mutant homozygotes among F2 progeny is described. Ways of further improving the MEV stock are discussed. The location of the Myb proto-oncogene is defined relative to Sl and one of the C58/J proviruses on chromosome 10.     

Serological comparison of polyhedron protein and virions from four nuclear polyhedrosis viruses of plusiine larvae (Lepidoptera: Noctuidae)

Purified polyhedron proteins and purified, ultrasonicated virions of four nuclear polyhedrosis viruses (NPVs), separable into two morphologic groups of singly and multiply embedded virion types (SEVs and MEVs), were investigated by immunodiffusion and immunoelectrophoresis. The four viruses were Pseudoplusia includens SEV, Trichoplusia ni SEV, T. ni MEV, and Autographa californica MEV. In immunodiffusion, SEV polyhedron proteins formed two precipitin bands with antiserum to SEV polyhedron proteins, while MEV polyhedron proteins formed only one. All four proteins formed one precipitin band with antiserum to MEV polyhedron protein, with a spur between SEV and MEV proteins. In immunoelectrophoresis, mobilities of SEV proteins were significantly different from those of MEVs. Precipitin arc patterns were similar to those in immunodiffusion when electrophoresis was carried out at 4 C; at room temperature, a single arc of precipitation formed with all four proteins. SEV virions formed five possibly identical precipitin bands in immunodiffusion with antiserum to SEV virions. MEV virions formed three possibly identical precipitin bands when reacted with antiserum to MEV virions. Little or no cross-reactions were observed between SEV and MEV virions or between virions and polyhedron proteins. In immunoelectrophoresis, SEV virions formed three precipitin arcs in reactions with SEV antisera and none with MEV antisera; MEV virions formed two arcs with MEV antisera and none with SEV antisera. When antisera were subjected to electrophoresis, five arcs were formed by SEVs and three by MEVs in homologous systems, and none were formed in heterologous systems.     

貂肠炎病毒的核苷酸序列和基因组结构

本试验测定了已克隆的貂肠炎病毒(MEV)复制型(RF)DNA的核苷酸序列,确定MEV基因组全长约为5064个核苷酸(nucleotides,nt),推测了3'端和5'端结构,在5'端非编码区有3个51 nt的重复。MEV基因组序列与犬细小病毒(CPV)、猫细小病毒(FPV)有很高的同源性,结构基因区的同源性分别达99.1%和99.9%,但在5'端非编码区有较大差异。MEV基因组结构与CPV和FPV基本一致,有两个大的开放阅读框架,分别编码688和722个氨基酸。在map unit(m.u.)3.7和m.u.39处有两个启动子,在m.u.97处有poly A位点。NS2、VP1和VP2的mRNA都发生剪接。     

Characterization of replicative form DNA of the autonomous parvovirus mink enteritis virus

Characterization of replicative form (RF) DNA of mink enteritis virus (MEV) was carried out. Most of the RF DNA were bound to terminal protein but some were free from the protein. The protein-free RF DNA increased about 7 times from 30 to 50 hr post-infection, while the DNA with protein increased less. The molecules of the replicative intermediate which were partially single-stranded DNA and bound to terminal protein were present. Two terminal conformations, "extended" and "turnaround," were observed in both ends of both terminal protein-bound and protein-free RF DNA. The 5' end labeling revealed that 5' ends of protein-free RF DNA were not blocked to phosphorylation by an amino acid or an oligopeptide which attaches to 5' ends of proteolytically deproteinized RF DNA. Restriction analysis of incomplete RF DNA which was partially double-stranded DNA showed that extended conformation was dominant in such incomplete RF molecules.     

水貂肠炎病毒山东株分离鉴定及生物学特性分析

【目的】本研究旨在研究水貂肠炎病毒(mink enteritis virus,MEV)的基因组遗传进化特征。【方法】对采自山东境内水貂养殖场的109份水貂腹泻样品进行MEV的分离和鉴定,利用血凝和血凝抑制试验、多步生长曲线绘制以及蛋白的三级结构模拟等,对分离毒株生物学特性进行分析,通过重叠PCR对分离株进行全基因扩增,使用MegAlign进行序列同源性比对分析,利用DNAMANV6对基因组5’末端和3’末端回文结构进行预测,应用MEGAV6进行遗传进化分析。【结果】共分离得到5株病毒,经电镜观察和间接免疫荧光试验鉴定为MEV毒株,分别命名为MUTQS-1-5,GenBank登录号分别为OK275645、OK275646、OK275647、OK275648和OK275649;各分离株5’-和3’-UTR分别由长回文序列组成,具有典型的细小病毒基因组末端的茎环样结构,NS1和VP2基因的推导氨基酸序列存在多个非同义突变位点,其中NS1蛋白的E/Q545V位氨基酸突变,以及VP2蛋白的F267Y、Y324I位氨基酸突变为首次在MEV上发现;生物学特性分析表明,上述突变并未明显改变病毒的血凝及...     

Low titers of measles antibody in mothers whose infants suffered from measles before eligible age for measles vaccination

Background

Mink enteritis virus (MEV) causes a highly contagious viral disease of mink with a worldwide distribution. MEV has a linear, single-stranded, negative-sense DNA with a genome length of approximately 5,000 bp. The VP2 protein is the major structural protein of the parvovirus encoded by the vp2 gene. VP2 is highly antigenic and plays important roles in determining viral host ranges and tissue tropisms. This study describes the bionomics and vp2 gene analysis of a mutated strain, MEV-DL, which was isolated recently in China and outlines its homologous relationships with other selected strains registered in Genbank.

Results

The MEV-DL strain can infect F81 cells with cytopathic effects. Pig erythrocytes were agglutinated by the MEV-DL strain. The generation of MEV-DL in F81 cells could infect mink within three months and cause a disease that was similar to that caused by wild-type MEV. A comparative analysis of the vp2 gene nucleotide (nt) sequence of MEV-DL showed that this was more than 99% homologous with other mink enteritis parvoviruses in Genbank. However, the nucleotide residues at positions 1,065 and 1,238 in the MEV-DL strain of the vp2 gene differed from those of all the other MEV strains described previously. It is noteworthy that the mutation at the nucleotide residues position 1,238 led to Asp/Gly replacement. This may lead to structural changes. A phylogenetic tree and sequence distance table were obtained, which showed that the MEV-DL and ZYL-1 strains had the closest inheritance distance.

Conclusions

A new variation of the vp2 gene exists in the MEV-DL strain, which may lead to structural changes of the VP2 protein. Phylogenetic analysis showed that MEV-DL may originate from the ZYL-1 strain in DaLian.     

Biochemical comparison of polyhedral protein from five nuclear polyhedrosis viruses infecting plusiine larvae (Lepidoptera: Noctuidae)

Polyhedral protein preparations from five nuclear polyhedrosis viruses isolated from four closely related host insects of the noctuid subfamily Plusiinae were characterized by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE), high voltage paper electrophoresis, and amino acid analysis. The viruses were Autographa california multiple-embedded virion type (MEV), Pseudoplusia includens singly embedded virion type (SEV), Rachiplusia ou MEV, Trichoplusia ni MEV, and T. ni SEV. Each was produced in its own host; A. californica MEV was also produced in T. ni larvae to determine possible host influence on polyhedral protein chemistry. Each test revealed minor, reproducible differences among most isolates. In SDS-PAGE, the major protein component ranged from 26,700 to 28,300 MW among the isolates. Differences were confined to minor protein bands or to band intensity. Peptide maps showed differences among most isolates in numbers of acidic and basic peptide spots, but all had an identical number of neutral spots. Migration patterns also differed among most isolates. The amino acid compositions of the six polyhedral inclusions were very similar, with aspartic and glutamic acids being the predominant residues. The greatest differences were found between the MEV and SEV groups, with lesser differences within each group. In all analyses, A. californica MEV produced in A. californica was indistinguishable from virus produced in T. ni.     

Rederivation of MHV and MEV antibody positive mice by cross-fostering and use of the microisolator caging system

This study established the feasibility of rederiving numerous mouse hepatitis virus (MHV) and mouse encephalomyelitis virus (MEV) antibody positive strains of mice using cross fostering techniques and a new caging system, thus permitting introduction of virus antibody free mice into a barrier facility. Serologic status of dams within the nucleus breeding colony was determined, and all mice within the breeding colony were housed in individual Microisolator cages. Specific pathogen free (SPF) foster mothers purchased from a commercial source were determined to have no detectable serum antibody to 11 murine viruses including MEV and MHV. Pups delivered naturally from time pregnant dams were cross fostered onto the SPF foster dams. The procedure of cross fostering was conducted within a positive flow, HEPA-filtered, mass air displacement unit within 24 hours of parturition. The virus status of pups from 49 litters was monitored serologically at weaning and again at 6 weeks of age. All cross fostered litters were serologically negative for antibody to mouse hepatitis virus. Seven of 29 litters were negative for MEV antibody titer using this cross fostering technique. Those litters negative serologically to both MHV and MEV (at 3 and 6 weeks) were transferred to a barrier facility and held in isolation. All rederived mice transferred to the barrier facility remained negative for MHV and MEV when sampled at 12 weeks of age.     

Diet of wild boar Sus scrofa L. and crop damage in an intensive agroecosystem

The Middle Ebro Valley (MEV) is a semiarid area in northeast Iberia where the original riparian ecosystems are almost extinct and were replaced by intensive irrigated agricultural lands. To minimize crop damages and to understand the impact of wild boar on relict riparian ecosystems, a culling program was undertaken from 1994 until 2004. To assess the impact of wild boars, we analyzed stomach contents and surveyed crop damage. In the MEV, wild boars feed mainly on crops, particularly, maize. Other elements of the diet that are of agricultural origin include wheat, barley, and alfalfa, which are the alternatives to maize in the period between harvest and seeding, which is the basis of seasonal changes in diet. Results indicate that wild boar actively selected maize crops and consumed wheat in proportion to its abundance; barley and alfalfa fields were damaged less than expected based on their abundance. In the MEV, the wild boar population is limited by the availability of shelter areas found in the scarce riparian ecosystems, which do not provide important food items for this population. We conclude that in the region of this study, wild boars are not a significant threat to the flora and fauna of riparian ecosystems, although as these habitats are restored and areas are protected, the carrying capacity for wild boars might increase.     

Efficacy of insect viruses propagated in vivo and in vitro

Laboratory bioassay and field trials demonstrated that the Autographa multiple-embedded (MEV) nucleopolyhedrosis virus (NPV) and Trichoplusia ME-NPV produced in cell culture was as effective as that produced in larvae. No difference in activity between the Autographa MEV, Trichoplusia MEV, and Trichoplusia SEV NPV was detected.     

Selection of Antiviral Peptides Against Mink Enteritis Virus Using a Phage Display Peptide Library

Mink enteritis virus (MEV) causes high morbidity and mortality in mink worldwide, and there are no effective treatments. This study used a phage display library to find specific peptides capable of binding MEV and preventing its replication in F81 cells. After three rounds of biopanning, the phage enrichment was 117 times higher than that after the first round. Twelve phage clones that showed threefold higher MEV-binding affinity than controls were selected by ELISA. Following sequence analyses, the peptides RLNNRARIILRA and LAHKSRLYERHM were synthesized and used for antiviral experiments. MTT assays demonstrated that both peptides increased cell viability by >20 % at 100 μg/ml when pre-incubated with MEV. However, no effect was seen if the peptides were added 2 h after viral inoculation of cells, indicating that the antiviral activity is due to inhibition of viral attachment to the cell surface.     

Remodeling the isoprenoid pathway in tobacco by expressing the cytoplasmic mevalonate pathway in chloroplasts

Metabolic engineering to enhance production of isoprenoid metabolites for industrial and medical purposes is an important goal. The substrate for isoprenoid synthesis in plants is produced by the mevalonate pathway (MEV) in the cytosol and by the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in plastids. A multi-gene approach was employed to insert the entire cytosolic MEV pathway into the tobacco chloroplast genome. Molecular analysis confirmed the site-specific insertion of seven transgenes and homoplasmy. Functionality was demonstrated by unimpeded growth on fosmidomycin, which specifically inhibits the MEP pathway. Transplastomic plants containing the MEV pathway genes accumulated higher levels of mevalonate, carotenoids, squalene, sterols, and triacyglycerols than control plants. This is the first time an entire eukaryotic pathway with six enzymes has been transplastomically expressed in plants. Thus, we have developed an important tool to redirect metabolic fluxes in the isoprenoid biosynthesis pathway and a viable multigene strategy for engineering metabolism in plants.     

MicroRNA profile analysis of a feline kidney cell line before and after infection with mink enteritis virus

MicroRNAs (miRNAs) are small regulatory RNAs that play a significant role in eukaryotes by targeting mRNAs for cleavage or translational repression. Recent studies have also shown them to be associated with cellular changes following viral infection. Mink enteritis virus (MEV) is one of the most important viral pathogens in the mink industry. To study the involvement of miRNAs in the MEV infection process, we used Illumina's ultrahigh throughput approach to sequencing miRNA libraries from the feline kidney (F81) cell line before and after infection with MEV. Using this bioinformatics approach we identified 196 known mammalian miRNA orthologs belonging to 152 miRNA families in F81 cells. Additionally, 97 miRNA*s of these miRNAs were detected. As well as known miRNAs, 384 and 398 novel miRNA precursor candidates were identified in uninfected and MEV-infected F81 cells respectively that have not been reported in other mammals. In MEV-infected cells 3 miRNAs were significantly down-regulated and 4 up-regulated including 3 significantly. The majority (12 of 16) of randomly selected miRNA expression profiles by qRT-PCR were consistent with those identified by deep sequencing. A total of 88 miRNAs were predicted to target interferon-associated genes; 6 appear to target the 3′UTR of MEV-specific receptor transferring receptor mRNAs; and 8 to target the MEV mRNA coding region. No miRNAs coded by MEV itself were detected.     

Biochemical comparison of virion proteins from five nuclear polyhedrosis viruses infecting plusiine larvae (Lepidoptera: Noctuidae)

The virions of six isolates of five nuclear polyhedrosis viruses (NPV) infecting plusiine larvae (Lepidoptera: Noctuidae) were reproducibly separated by sucrose density gradient centrifugation and fractionation. Purity of the preparations was established by electron microscopy. Virion proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); each produced 12 distinct polypeptides ranging from 10,300 to 82,900 mw. Qualitative and quantitative differences were found between most of the polypeptide patterns. The singly embedded viron (SEV)-type isolates had two major components with mw in the range of 32,900–35,200; multiply embedded virion (MEV)-type isolates had a major component of ca. 12,500 mw. SEV isolates showed almost no within-group differences, while minor differences were found among the MEV banding patterns in both intensity and presence of certain bands. Capsids and envelopes from MEV had two to four polypeptides with mw between 10,800 and 26,900. The presence of more than one polypeptide and electron microscopy of sample composition suggested that the capsid and envelope are composed of several distinct proteins.