Isolation and partial characterization of equine herpesvirus type 1 in Czechia

Equine herpesvirus type 1 was determined as the etiological cause of an abortion storm in Czechia in 2003 after the virus strain was isolated from aborted fetus and identified by serological means and by PCR technique. Cloning and sequencing of the glycoprotein D confirmed the identity of the isolates and showed molecular relationships to known EHV-1 strains. Comparison of glycoprotein D sequences with corresponding sequence of EHV-1 reference strains (Kentucky-A and Ab1) revealed high nucleotide homology. The Czech isolate of EHV-1 virus does not differ significantly from the Ab1 strain regarding the glycoprotein D gene and does not bear the frameshift in the 3' terminus which occurs in the Kentucky-A strain.     

甘蓝型油菜线粒体功能未知基因 ORF 117的载体构建与转化

本研究克隆了甘蓝型油菜线粒体功能未知基因 ORF 117,构建了带有线粒体转运信号肽序列（TP ）的植物过表达载体35S ∷TP-ORF117、35S ∷TP-EGFP 和35S ∷TP-ORF117-EGFP ,转化野生型拟南芥并进行抗除草剂筛选,共获得纯合的转基因拟南芥株系62个。qPCR 表明,ORF 117在转基因材料中得到高效过表达。用转35S ∷TP-EGFP 、35S ∷TP-ORF117-EGFP 拟南芥制备原生质体,经线粒体特异染料染色,在激光共聚焦显微镜下做线粒体、GFP 共定位检测,GFP 蛋白和 ORF117-EGFP 融合蛋白被准确定位到了线粒体。     

Sequence analysis of the 4.7-kb BamHI-EcoRI fragment of the equine herpesvirus type-1 short unique region.

To localize gene that may encode immunogens potentially important for recombinant vaccine design, we have analysed a region of the equine herpesvirus type-1 (EHV-1) genome where a glycoprotein-encoding gene had previously been mapped. The 4707-bp BamHI-EcoRI fragment from the short unique region of the EHV-1 genome was sequenced. This sequence contains three entire open reading frames (ORFs), and portions of two more. ORF1 codes for 161 amino acids (aa), and represents the C terminus of a possible membrane-bound protein. ORF2 (424 aa) and ORF3 (550 aa) are potential glycoprotein-encoding genes; the predicted aa sequences contain possible signal sequences, N-linked glycosylation sites and transmembrane domains; they also show homology to the glycoproteins gI and gE of herpes simplex virus type-1 (HSV-1), and the related proteins of pseudorabies virus and varicella-zoster virus. The predicted aa sequence of ORF4 shares no homology with other known herpesvirus proteins, but the nucleotide sequence shows a high level of homology with the corresponding region of the EHV-4 genome. ORF5 may be related to US9 of HSV-1.     

The varicella-zoster virus (VZV) open reading frame 47 (ORF47) protein kinase is dispensable for viral replication and is not required for phosphorylation of ORF63 protein, the VZV homolog of herpes simplex virus ICP22.

To investigate the role of varicella-zoster virus (VZV) open reading frame 47 (ORF47) protein kinase during infection, a VZV mutant was generated in which two contiguous stop codons were introduced into ORF47, thus eliminating expression of the ORF47 kinase. ORF47 kinase was not essential for the growth of VZV in cultured cells, and the growth rate of the VZV mutant lacking ORF47 protein was indistinguishable from that of parental VZV. Nuclear extracts from cells infected with parental VZV contained several phosphorylated proteins which were not detected in extracts from cells infected with the ORF47 mutant. The herpes simplex virus type 1 (HSV-1) UL13 protein (the homolog of VZV ORF47 protein) is responsible for the posttranslational processing associated with phosphorylation of HSV-1 ICP22 (the homolog of VZV ORF63 protein). Immunoprecipitation of 32P-labeled proteins from cells infected with parental virus and those infected with ORF47 mutant virus yielded similar amounts of the VZV phosphoproteins encoded by ORF4, ORF62, ORF63, and ORF68 (VZV gE), and the electrophoretic migration of these proteins was not affected by the lack of ORF47 kinase. Therefore, while the VZV ORF47 protein is capable of phosphorylating several cellular or viral proteins, it is not required for phosphorylation of the ORF63 protein in virus-infected cells.     

Construction of recombinant herpes simplex virus type I expressing green fluorescent protein without loss of any viral genes

For use in various applications in research on herpes simplex virus type 1, we attempted to generate recombinant HSV-1 expressing green fluorescent protein (GFP) without any loss of viral genes. Our results were as follows. (i) A recombinant HSV-1 (YK333), in which a GFP expression cassette driven by the Egr-1 promoter was inserted into the intergenic region between UL3 and UL4, was constructed. (ii) YK333 replicated as well as wild-type HSV-1 F strain in Vero cells. (iii) As one application of the recombinant YK333 for research on HSV-1, we developed a system to detect anti-herpetic activity, termed a fluorescence-based anti-viral assay. The 50% inhibitory concentration of ganciclovir for YK333 determined using our newly developed assay was comparable to that determined using a plaque reduction assay. YK333 will be a convenient tool for herpes simplex virus research, including such applications as monitoring of viral replication in vitro and in vivo, and rapid screening of potential anti-herpetic agents.     

Comparison of the complete DNA sequences of the Oka varicella vaccine and its parental virus

The DNA sequences of the Oka varicella vaccine virus (V-Oka) and its parental virus (P-Oka) were completed. Comparison of the sequences revealed 42 base substitutions, which led to 20 amino acid conversions and length differences in tandem repeat regions (R1, R3, and R4) and in an origin of DNA replication. Amino acid substitutions existed in open reading frames (ORFs) 6, 9A, 10, 21, 31, 39, 50, 52, 55, 59, 62, and 64. Of these, 15 base substitutions, leading to eight amino acid substitutions, were in the gene 62 region alone. Further DNA sequence analysis showed that these substitutions were specific for V-Oka and were not present in nine clinical isolates. The immediate-early gene 62 product (IE62) of P-Oka had stronger transactivational activity than the mutant IE62 contained in V-Oka in 293 and CV-1 cells. An infectious center assay of a plaque-purified clone (S7-01) from the V-Oka with 8 amino acid substitutions in ORF 62 showed smaller plaque formation and less-efficient virus-spreading activity than did P-Oka in human embryonic lung cells. Another clone (S-13) with only five substitutions in ORF 62 spread slightly faster than S7-01 but not as effectively as P-Oka. Moreover, transient luciferase assay in 293 cells showed that transactivational activities of IE62s of S7-01 and S7-13 were lower than that of P-Oka. Based on these results, it appears that amino acid substitutions in ORF 62 are responsible for virus growth and spreading from infected to uninfected cells. Furthermore, the Oka vaccine virus was completely distinguishable from P-Oka and 54 clinical isolates by seven restriction-enzyme fragment length polymorphisms that detected differences in the DNA sequence.     

Expression and function of the equine herpesvirus 1 virion-associated host shutoff homolog.

The ability of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively) to repress host cell protein synthesis early in infection has been studied extensively and found to involve the activities of the UL41 gene product, the virion-associated host shutoff (vhs) protein. To date, UL41 homologs have been identified in the genomes of three other alphaherpesviruses: equine herpesvirus 1 (EHV-1), varicella-zoster virus, and pseudorabies virus, but very little is known about the putative products of these homologous genes. Our earlier observations that no rapid early host protein shutoff occurred in EHV-1-infected cells led us to test EHV-1 vhs activity more thoroughly and to examine the expression and function of the EHV-1 UL41 homolog, ORF19. In the present study, the effects of EHV-1 and HSV-1 infections on cellular protein synthesis and mRNA degradation were compared at various multiplicities of infection in several cell types under an actinomycin D block. No virion-associated inhibition of cellular protein synthesis or vhs-induced cellular mRNA degradation was detected in cells infected with any of three EHV-1 strains (Ab4, KyA, and KyD) at multiplicities of infection at which HSV-1 strain F exhibited maximal vhs activity. However, further analyses revealed that (i) the EHV-1 vhs homolog gene, ORF19, was transcribed and translated into a 58-kDa protein in infected cells; (ii) the ORF19 protein was packaged into viral particles in amounts detectable in Western blots (immunoblots) with monoclonal antibodies; (iii) in cotransfection vhs activity assays, transiently-expressed ORF19 protein had intrinsic vhs activity comparable to that of wild-type HSV-1 vhs; and (iv) this intrinsic vhs activity was ablated by in vitro site-directed mutations in which either the functionally inactive HSV-1 vhs1 UL41 mutation (Thr at position 214 replaced by Ile [Thr-214-->Ile]) was recreated within ORF19 or two conserved residues within the putative poly(A) binding region of the ORF19 sequence were altered (Tyr-190, 192-->Phe). From these results we conclude that EHV-1's low vhs activity in infected cells is not a reflection of the ORF19 protein's intrinsic vhs activity but may be due instead to the amount of ORF19 protein associated with viral particles or to modulation of ORF19 protein's intrinsic activity by another viral component(s).     

Generation of an influenza A virus vector expressing biologically active human interleukin-2 from the NS gene segment

Engineering of the influenza A virus NS1 protein became an attractive approach to the development of influenza vaccine vectors since it can tolerate large inserts of foreign proteins. However, influenza virus vectors expressing long foreign sequences from the NS1 open reading frame (ORF) are usually replication deficient in animals due to the abrogation of their NS1 protein function. In this study, we describe a bicistronic expression strategy based on the insertion of an overlapping UAAUG stop-start codon cassette into the NS gene, allowing the reinitiation of translation of a foreign sequence. Although the expression level of green fluorescent protein (GFP) from the newly created reading frame was significantly lower than that obtained previously from an influenza virus vector expressing GFP from the NS1 ORF, the bicistronic vector appeared to be replication competent in mice and showed outstanding genetic stability. All viral isolates derived from mouse lungs at 10 days postinfection were still capable of expressing GFP in infected cells. Utilizing this bicistronic approach, we constructed another recombinant influenza virus, allowing the secretion of biologically active human interleukin-2 (IL-2). Although this virus also replicated to high titers in mouse lungs, it did not display any mortality rate in infected animals, in contrast to control viruses. Moreover, the IL-2-expressing virus showed an enhanced CD8+ response to viral antigens in mice after a single intranasal immunization. These results indicate that influenza viruses could be engineered for the expression of biologically active molecules such as cytokines for immune modulation purposes.     

Subcellular localization of a germiantion-specific cortex-lytic enzyme, SleB, of Bacilli during sporulation

The subcellular localization of a germination-specific cortex-lytic enzyme, SleB, of Bacillus subtilis during sporulation was observed by using fusions of N-terminal region of SleB to the green fluorescent protein (GFP). A fusion with a putative peptidoglycan-binding motif (SleB1-108-GFP) formed a fluorescent ring around the forespore of the wild type strain, as expected from the known location of the intact SleB in the dormant spore. SleB1-108-GFP formed a similar fluorescent ring around the forespore of the gerE mutant which has a severe defect in the coat structure, and of the cwlD mutant which lacks a muramic delta-lactam unique to the spore peptidoglycan (cortex), whereas the fusion could not attach to the spore of the cwlDgerE mutant. By contrast, a fusion without the motif (SleB1-45-GFP) could not be recruited around the forespore of the gerE mutant though it appeared to be accumulated on the outside of the spore of the wild type strain. Since SleB was shown to degrade only the cortex with muramic delta-lactam, these results suggested that a proper localization of SleB requires a strict interaction between the motif of the enzyme and the delta-lactam structure of the cortex, not the formation of normal coat layer.     

The equine herpesvirus 1 Us2 homolog encodes a nonessential membrane-associated virion component

Experiments were conducted to analyze the equine herpesvirus 1 (EHV-1) gene 68 product which is encoded by the EHV-1 Us2 homolog. An antiserum directed against the amino-terminal 206 amino acids of the EHV-1 Us2 protein specifically detected a protein with an Mr of 34,000 in cells infected with EHV-1 strain RacL11. EHV-1 strain Ab4 encodes a 44,000-Mr Us2 protein, whereas vaccine strain RacH, a high-passage derivative of RacL11, encodes a 31,000-Mr Us2 polypeptide. Irrespective of its size, the Us2 protein was incorporated into virions. The EHV-1 Us2 protein localized to membrane and nuclear fractions of RacL11-infected cells and to the envelope fraction of purified virions. To monitor intracellular trafficking of the protein, the green fluorescent protein (GFP) was fused to the carboxy terminus of the EHV-1 Us2 protein or to a truncated Us2 protein lacking a stretch of 16 hydrophobic amino acids at the extreme amino terminus. Both fusion proteins were detected at the plasma membrane and accumulated in the vicinity of nuclei of transfected cells. However, trafficking of either GFP fusion protein through the secretory pathway could not be demonstrated, and the EHV-1 Us2 protein lacked detectable N- and O-linked carbohydrates. Consistent with the presence of the Us2 protein in the viral envelope and plasma membrane of infected cells, a Us2-negative RacL11 mutant (L11DeltaUs2) exhibited delayed penetration kinetics and produced smaller plaques compared with either wild-type RacL11 or a Us2-repaired virus. After infection of BALB/c mice with L11DeltaUs2, reduced pathogenicity compared with the parental RacL11 virus and the repaired virus was observed. It is concluded that the EHV-1 Us2 protein modulates virus entry and cell-to-cell spread and appears to support sustained EHV-1 replication in vivo.     

甜菜黑色焦枯病毒外壳蛋白与病毒致病性的关系

利用RT-PCR方法,构建获得了由T7RNA聚合酶启动子驱动的甜菜黑色焦枯病毒(BBSV)全长cDNA克隆pUBF52.摩擦接种苋色藜(Chenopodiumamaranticolor)后,体外转录产物可导致与野生病毒相同的枯斑症状,蛋白质印迹和RNA印迹检测也都证明了转录产物的侵染活性.构建了BBSVp24基因的原核表达载体pECP1,转化大肠杆菌BL21后的诱导表达产物能够与BBSV的抗血清呈现特异性反应,表明该基因编码产生BBSV的外壳蛋白(CP).以pUBF52为模板,分别构建了BBSVCP基因的移码突变体和不同程度的缺失突变体.侵染性检测表明,CP基因的移码突变对BBSV在苋色藜上所导致的枯斑症状及病毒RNA在寄主体内的积累基本没有影响,但CP基因的大部或完全缺失会使体内病毒RNA的积累水平大大降低,其中CP基因完全缺失的突变体转录物接种苋色藜后仅能够产生很轻的枯斑症状.将绿色荧光蛋白(GFP)基因和葡糖苷酸酶(GUS)基因分别与BBSVCP基因的5′端融合,构建了表达载体pBGFP和pBGUS.摩擦接种苋色藜叶片后可观察到GFP或GUS基因的表达,为探索利用BBSV作为外源蛋白的表达载体奠定了基础.     

A simple analysis system for the estimation of recombination efficiency using fluorescence-activated cell sorting

A simple and accurate analysis system for the estimation of recombination efficiency in vivo using fluorescence-activated cell sorting (FACS) was designed and was subsequently used to compare the efficiency of recombination related to different spacer mutants. F(3) and F(5) mutant sequences were used for Flpe-mediated cassette exchange, and m2 and lox2272 mutant sequences were used for Cre-mediated cassette exchange due to their high incompatibility with wild-type sequences. The incompatibilities with wild-type were almost the same between mutant sequences. However, the recombination efficiencies were different. F(3) and m2 could mediate more efficient recombination than F(5) and lox2272, respectively. These results are consistent with the fact that the sequence of spacer region affects not only the reactivity upon wild-type sequence but also the recombination efficiency. It was also confirmed that the recombination process was mediated in a site-specific manner through PCR analysis using different sizes of exchange cassettes. In this experiment, the feasibility of the FACS analysis system for the estimation of recombination efficiency was verified. This system should be readily applicable for estimating recombination efficiency of other various mutant candidates, which will contribute to more precise and efficient site-specific recombination.     

Molecular characterisation of equid alphaherpesvirus 1 strains isolated from aborted fetuses in Poland

Background

Equid alphaherpesvirus 1 (EHV-1) is one of the main infectious causative agents of abortion in mares and can also be associated with stillbirth, neonatal foal death, rhinopneumonitis in young horses and a neurological disorder called equine herpesvirus myeloencephalopathy (EHM). The neuropathogenicity of the virus was shown to be significantly higher in EHV-1 strains that carry a single nucleotide point (SNP) mutation in the ORF30, which encodes a catalytic subunit of viral DNA polymerase (ORF30 D₇₅₂). Another gene, ORF68 is frequently used for phylogenetic analysis of EHV-1.

Methods

27 EHV-1 strains isolated from aborted equine fetuses in Poland, collected between 1993 and 2017, were subjected to PCR targeting the open reading frames (ORFs) 30 and 68 of the EHV-1 genome. PCR products obtained were sequenced and SNPs were analyzed and compared to sequences available in GenBank.

Results

None of the analyzed sequences belonged to the ORF30 D₇₅₂neuropathogenic genotype: all EHV-1 belonged to the non-neuropathogenic variant N₇₅₂. On the basis of ORF68 sequences, the majority of EHV-1 sequences (76.9%) cannot be assigned to any of the known groups; only six sequences (23.1%) clustered within groups II and IV.

Conclusions

EHV-1 strains obtained from abortion cases belong to the non-neuropathogenic genotype. Many EHV-1 ORF68 sequences have similar SNPs to those already described in Poland, but a clear geographical distribution was not observed. A single particular ORF68 sequence type was observed in strains isolated from 2001 onwards.