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1.
PLoS Pathog ; 17(1): e1009255, 2021 01.
Article in English | MEDLINE | ID: mdl-33508041

ABSTRACT

Cytomegalovirus (CMV) causes clinically important diseases in immune compromised and immune immature individuals. Based largely on work in the mouse model of murine (M)CMV, there is a consensus that myeloid cells are important for disseminating CMV from the site of infection. In theory, such dissemination should expose CMV to cell-mediated immunity and thus necessitate evasion of T cells and NK cells. However, this hypothesis remains untested. We constructed a recombinant MCMV encoding target sites for the hematopoietic specific miRNA miR-142-3p in the essential viral gene IE3. This virus disseminated poorly to the salivary gland following intranasal or footpad infections but not following intraperitoneal infection in C57BL/6 mice, demonstrating that dissemination by hematopoietic cells is essential for specific routes of infection. Remarkably, depletion of NK cells or T cells restored dissemination of this virus in C57BL/6 mice after intranasal infection, while dissemination occurred normally in BALB/c mice, which lack strong NK cell control of MCMV. These data show that cell-mediated immunity is responsible for restricting MCMV to hematopoietic cell-mediated dissemination. Infected hematopoietic cells avoided cell-mediated immunity via three immune evasion genes that modulate class I MHC and NKG2D ligands (m04, m06 and m152). MCMV lacking these 3 genes spread poorly to the salivary gland unless NK cells were depleted, but also failed to replicate persistently in either the nasal mucosa or salivary gland unless CD8+ T cells were depleted. Surprisingly, CD8+ T cells primed after intranasal infection required CD4+ T cell help to expand and become functional. Together, our data suggest that MCMV can use both hematopoietic cell-dependent and -independent means of dissemination after intranasal infection and that cell mediated immune responses restrict dissemination to infected hematopoietic cells, which are protected from NK cells during dissemination by viral immune evasion. In contrast, viral replication within mucosal tissues depends on evasion of T cells.


Subject(s)
Herpesviridae Infections/immunology , Immune Evasion , Immunity, Cellular , Muromegalovirus/immunology , Animals , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/virology , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/virology , Herpesviridae Infections/virology , Killer Cells, Natural/immunology , Killer Cells, Natural/virology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Muromegalovirus/genetics , Muromegalovirus/physiology , Virus Replication
2.
Expert Opin Drug Discov ; 16(4): 391-410, 2021 04.
Article in English | MEDLINE | ID: mdl-33232188

ABSTRACT

Introduction: Despite diverse treatment modalities and novel therapies, many cancers and patients are not effectively treated. Cancer immunotherapy has recently achieved breakthrough status yet is not effective in all cancer types or patients and can generate serious adverse effects. Oncolytic viruses (OVs) are a promising new therapeutic modality that harnesses virus biology and host interactions to treat cancer. OVs, genetically engineered or natural, preferentially replicate in and kill cancer cells, sparing normal cells/tissues, and mediating anti-tumor immunity.Areas covered: This review focuses on OVs as cancer therapeutic agents from a historical perspective, especially strategies to boost their immunotherapeutic activities. OVs offer a multifaceted platform, whose activities are modulated based on the parental virus and genetic alterations. In addition to direct viral effects, many OVs can be armed with therapeutic transgenes to also act as gene therapy vectors, and/or combined with other drugs or therapies.Expert opinion: OVs are an amazingly versatile and malleable class of cancer therapies. They tend to target cellular and host physiology as opposed to specific genetic alterations, which potentially enables broad responsiveness. The biological complexity of OVs have hindered their translation; however, the recent approval of talimogene laherparepvec (T-Vec) has invigorated the field.


Subject(s)
Immunotherapy/methods , Neoplasms/therapy , Oncolytic Virotherapy/methods , Animals , Antineoplastic Agents, Immunological/administration & dosage , Antineoplastic Agents, Immunological/pharmacology , Biological Products/administration & dosage , Biological Products/pharmacology , Herpesvirus 1, Human , Humans , Neoplasms/immunology , Oncolytic Viruses/immunology
3.
J Immunol ; 204(6): 1582-1591, 2020 03 15.
Article in English | MEDLINE | ID: mdl-32015010

ABSTRACT

NK cells play an important role in antiviral resistance. The integrin α2, which dimerizes with integrin ß1, distinguishes NK cells from innate lymphoid cells 1 and other leukocytes. Despite its use as an NK cell marker, little is known about the role of α2ß1 in NK cell biology. In this study, we show that in mice α2ß1 deficiency does not alter the balance of NK cell/ innate lymphoid cell 1 generation and slightly decreases the number of NK cells in the bone marrow and spleen without affecting NK cell maturation. NK cells deficient in α2ß1 had no impairment at entering or distributing within the draining lymph node of ectromelia virus (ECTV)-infected mice or at becoming effectors but proliferated poorly in response to ECTV and did not increase in numbers following infection with mouse CMV (MCMV). Still, α2ß1-deficient NK cells efficiently protected from lethal mousepox and controlled MCMV titers in the spleen. Thus, α2ß1 is required for optimal NK cell proliferation but is dispensable for protection against ECTV and MCMV, two well-established models of viral infection in which NK cells are known to be important.


Subject(s)
Ectromelia, Infectious/immunology , Herpesviridae Infections/immunology , Integrin alpha2beta1/metabolism , Killer Cells, Natural/immunology , Animals , Cell Count , Cell Proliferation , Disease Models, Animal , Ectromelia virus/immunology , Ectromelia, Infectious/blood , Ectromelia, Infectious/virology , Female , Herpesviridae Infections/blood , Herpesviridae Infections/virology , Humans , Immunity, Innate , Integrin alpha2beta1/immunology , Killer Cells, Natural/metabolism , Male , Mice , Muromegalovirus/immunology , Virus Replication/immunology
4.
Med Microbiol Immunol ; 208(3-4): 457-468, 2019 Aug.
Article in English | MEDLINE | ID: mdl-30848361

ABSTRACT

Natural transmission of cytomegalovirus (CMV) has been difficult to observe. However, recent work using the mouse model of murine (M)CMV demonstrated that MCMV initially infects the nasal mucosa after transmission from mothers to pups. We found that intranasal (i.n.) inoculation of C57BL/6J mice resulted in reliable recovery of replicating virus from the nasal mucosa as assessed by plaque assay. After i.n. inoculation, CD8+ T-cell priming occurred in the mandibular, deep-cervical, and mediastinal lymph nodes within 3 days of infection. Although i.n. infection induced "memory inflation" of T cells specific for the M38316-323 epitope, there were no detectable CD8+ T-cell responses against the late-appearing IE3416-423 epitope, which contrasts with intraperitoneal (i.p.) infection. MCMV-specific T cells migrated into the nasal mucosa where they developed a tissue-resident memory (TRM) phenotype and this could occur independently of local virus infection or antigen. Strikingly however, virus replication was poorly controlled in the nasal mucosa and MCMV was detectable by plaque assay for at least 4 months after primary infection, making the nasal mucosa a second site for MCMV persistence. Unlike in the salivary glands, the persistence of MCMV in the nasal mucosa was not modulated by IL-10. Taken together, our data characterize the development of local and systemic T-cell responses after intranasal infection by MCMV and define the nasal mucosa, a natural site of viral entry, as a novel site of viral persistence.


Subject(s)
Cytomegalovirus Infections/immunology , Muromegalovirus/growth & development , Muromegalovirus/immunology , Nasal Mucosa/immunology , Nasal Mucosa/virology , T-Lymphocytes/immunology , Virus Replication , Animals , Disease Models, Animal , Immunity, Cellular , Mice, Inbred C57BL
5.
Virus Res ; 221: 38-46, 2016 08 02.
Article in English | MEDLINE | ID: mdl-27173788

ABSTRACT

Currently, murine cytomegalovirus (MCMV) infections have been studied extensively in inbred mice via intraperitoneal route with highly passaged strains. However, the question how a low-passage MCMV replicates in inbred mice via a natural route remained unanswered. Here, different inbred mice (BALB/c, C57BL/6 and NOD) were inoculated oronasally with a low-passage MCMV strain, HaNa1. Viral replication was evaluated by virus titration and quantitative real-time PCR, and antibody response was assessed by immunoperoxidase cell monolayer assay (IPMA). In BALB/c mice, virus persisted in nasal mucosa (from 3 dpi) and submandibular glands (from 7 dpi) until the end of experiment (49 dpi). In C57BL/6 mice, infectious virus was only detected in nasal mucosa from 3 dpi until 21 dpi; viral genome was still detectable in nasal mucosa until 49 dpi. Although infectious virus was not detected in submandibular glands of C57BL/6 mice, viral genome was detected from 7 dpi until 49 dpi. NOD mice appeared to be even more resistant with absence of any productive infection; viral genome was detected at low levels in nasal mucosa. We demonstrated that there was a strong correlation between on the one hand degree of productive replication and on the other hand the time of first appearance and titer of MCMV-specific IgG antibody. The deficiency of functional T and B cells and interleukin-2 (IL-2) common-γ chain (γc) did not increase the susceptibility to MCMV by the use of NOD.SCID and NSG mice. In addition, using monocytic cells from different inbred mice we found patterns of resistance similar to those seen in vivo, as assessed by viral antigen expression. Taken together, these results demonstrated that upon oronasal inoculation low-passage MCMV HaNa1 replication clearly differs between different inbred mice (BALB/c>C57BL/6>NOD); resistance in vivo to MCMV is partly due to less susceptibility of host target cells and is independent of T, B cells and γc signaling cytokine-dependent NK cell activities.


Subject(s)
Disease Susceptibility , Muromegalovirus/growth & development , Muromegalovirus/immunology , Virus Replication , Animals , Antibodies, Viral/blood , Antigens, Viral/analysis , Leukocytes, Mononuclear/virology , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Nasal Mucosa/virology , Real-Time Polymerase Chain Reaction , Submandibular Gland/virology , Time Factors , Viral Load
6.
Virus Res ; 217: 47-54, 2016 06 02.
Article in English | MEDLINE | ID: mdl-26945848

ABSTRACT

Murine cytomegalovirus (MCMV) infection in mice is a commonly used animal model for studying human cytomegalovirus (HCMV) infections. In our previous studies, a mouse model based on an oronasal MCMV infection was set up for mimicking a natural infection, and the spleen was hypothesized to regulate viremia and virus dissemination to distal organs such as submandibular glands. Here, the role of the spleen during an MCMV infection was investigated by the comparison of intact and splenectomized Balb/c mice. Both highly passaged MCMV Smith and low passaged MCMV HaNa1 were used. Various samples were collected at 7, 14, and 21 days post inoculation (dpi) for analyses by virus isolation/titration, co-cultivation and qPCR. The results showed that for both virus strains, 1) cell-associated virus in PBMC (determined by co-cultivation) was detected in intact mice but not in splenectomized mice; 2) the mean viral DNA load in PBMC of splenectomized mice was 4.4-(HaNa1)/2.7-(Smith) fold lower at the peak viremia (7dpi) in contrast to that of intact mice; and 3) infectious virus in the submandibular glands was detected later in splenectomized mice (14dpi) than in intact mice (7dpi). Moreover, the average virus titers in submandibular glands of splenectomized mice were 10-(HaNa1)/7.9-(Smith) fold lower at 14dpi and 1.7-(HaNa1)/2.1-(Smith) fold lower at 21dpi compared with that of intact mice. Upon inoculation with MCMV Smith, infectious virus was found in the kidneys and liver of intact mice, but not in splenectomized mice. Taken together, all these data clearly demonstrate that virus dissemination to distant organs is reduced in splenectomized mice, further confirming the importance of the spleen as a viremia booming site for a natural MCMV infection.


Subject(s)
Herpesviridae Infections/virology , Muromegalovirus/physiology , Spleen/virology , Viremia/virology , Animals , Cells, Cultured , Female , Herpesviridae Infections/blood , Leukocytes, Mononuclear/virology , Mice, Inbred BALB C , Mouth/virology , Nose/virology , Splenectomy
7.
Virus Res ; 215: 114-20, 2016 Apr 02.
Article in English | MEDLINE | ID: mdl-26732487

ABSTRACT

Cytomegaloviruses may infect mammals via oronasal route. However, up till now it remains unclear how this exposure leads to a general infection and shedding. To address this issue, BALB/c female mice were oronasally inoculated with either the highly passaged murine cytomegalovirus (MCMV) Smith or the low passaged MCMV HaNa1. Virus titration showed a productive virus replication of both strains in the nasal mucosa from 1 dpi until the end of the experiment (14 dpi), in lungs from 5 until 14 dpi, and in submandibular glands from 7 until 14 dpi. In contrast to MCMV HaNa1, MCMV Smith also established a low level productive infection in abdominal organs (spleen, liver and kidneys) from 5 dpi (spleen), 7 dpi (liver), and 10 dpi (kidneys) until the end of the experiment. Co-culture showed that for both strains, cell-associated virus was detected in a non-infectious form in nasopharynx-associated lymphoid tissues (NALT) from 1 until 14 dpi, in submandibular lymph nodes from 3 until 5 dpi, in deep cervical lymph nodes from 3 until 14 dpi, in mediastinal lymph nodes from 7 until 14 dpi, in spleen from 5 until at least 10 dpi and in the peripheral blood mononuclear cells (PBMC) at 7 and 10 dpi. The present study shows that upon oronasal exposure, MCMV first enters the nasal mucosa and NALT, from where the virus disseminates to the spleen possibly via the draining lymphatic system and blood; a subsequent cell-associated viremia transports MCMV to submandibular glands and for MCMV Smith also to liver and kidneys, where a second productive replication starts.


Subject(s)
Animal Structures/virology , Cytomegalovirus Infections/pathology , Cytomegalovirus Infections/virology , Muromegalovirus/pathogenicity , Viremia , Animals , Cytomegalovirus Infections/physiopathology , Disease Models, Animal , Female , Mice, Inbred BALB C , Muromegalovirus/physiology , Virus Replication
8.
Virus Res ; 211: 96-102, 2016 Jan 04.
Article in English | MEDLINE | ID: mdl-26474525

ABSTRACT

In healthy individuals, naturally acquired infections of human cytomegalovirus (HCMV) are generally asymptomatic. Animal models mimicking the natural primary HCMV infections in infants and adults are scarce. Here, neonatal and adult BALB/c mice were inoculated oronasally with a Belgian isolate HaNa1 of murine cytomegalovirus (MCMV). None of the mice showed clinical symptoms. In neonatal mice, a typical systemic infection occurred. In adult mice, viral replication was restricted to the nasal mucosa and submandibular glands. Infectious virus was not detected in trachea, oral mucosa, pharynx, esophagus, small intestines of both neonatal and adult mice at all time points. Nose was demonstrated to be the entry site. Double immunofluorescence staining showed that in nose infected cells were olfactory neurons and sustentacular cells in olfactory epithelium and were macrophages and dendritic cells in nasopharynx-associated lymphoid tissues (NALT). Neonatal and adult mice developed similar antibody response pattern, though former magnitude was lower. In summary, we have established intranasal (without anesthesia) infections of neonatal and adult mice with murine CMV HaNa1 strain, which mimic the range and extent of virus replication during natural primary HCMV infections in healthy infants and adults. These findings might bring new insights in the pathogenesis of natural primary CMV infections.


Subject(s)
Cytomegalovirus Infections/virology , Cytomegalovirus/physiology , Cytomegalovirus/pathogenicity , Nose/virology , Animal Structures/virology , Animals , Cytomegalovirus/genetics , Disease Models, Animal , Female , Humans , Male , Mice , Mice, Inbred BALB C , Virulence , Virus Replication
9.
Vet Res ; 46: 94, 2015 Sep 04.
Article in English | MEDLINE | ID: mdl-26340811

ABSTRACT

Murine cytomegalovirus (MCMV) Smith strain is widely used in mouse models to study HCMV infections. Due to high serial passages, MCMV Smith has acquired genetic and biological changes. Therefore, a low passaged strain would be more relevant to develop mouse models. Here, the pathogenesis of an infection with MCMV Smith was compared with that of an infection with a low passaged Belgian MCMV isolate HaNa1 in BALB/c adult mice following oronasal inoculation with either a low (10(4) TCID50/mouse) or high (10(6) TCID50/mouse) inoculation dose. Both strains were mainly replicating in nasal mucosa and submandibular glands for one to two months. In nasal mucosa, MCMV was detected earlier and longer (1-49 days post inoculation (dpi)) and reached higher titers with the high inoculation dose compared to the low inoculation dose (14-35 dpi). In submandibular glands, a similar finding was observed (high dose: 7-49 dpi; low dose: 14-42 dpi). In lungs, both strains showed a restricted replication. In spleen, liver and kidneys, only the Smith strain established a productive infection. The infected cells were identified as olfactory neurons and sustentacular cells in olfactory epithelium, macrophages and dendritic cells in NALT, acinar cells in submandibular glands, and macrophages and epithelial cells in lungs for both strains. Antibody analysis demonstrated for both strains that IgG2a was the main detectable antibody subclass. Overall, our results show that significant phenotypic differences exist between the two strains. MCMV HaNa1 has been shown to be interesting for use in mouse models in order to get better insights for HCMV infections in immunocompetent humans.


Subject(s)
Herpesviridae Infections/virology , Muromegalovirus/physiology , Animals , Disease Models, Animal , Female , Herpesviridae Infections/genetics , Mice , Mice, Inbred BALB C , Muromegalovirus/genetics , Specific Pathogen-Free Organisms , Tissue Distribution
10.
Virol J ; 9: 93, 2012 May 14.
Article in English | MEDLINE | ID: mdl-22584005

ABSTRACT

BACKGROUND: MicroRNAs (miRNAs) are a group of short (~22 nt) noncoding RNAs that specifically regulate gene expression at the post-transcriptional level. miRNA precursors (pre-miRNAs), which are imperfect stem loop structures of ~70 nt, are processed into mature miRNAs by cellular RNases III. To date, thousands of miRNAs have been identified in different organisms. Several viruses have been reported to encode miRNAs. FINDINGS: Here, we extended the analysis of miRNA-encoding potential to the Anatid herpesvirus 1 (AHV-1). Using computational approaches, we found that AHV-1 putatively encodes 12 mature miRNAs. We then compared the 12 mature miRNAs candidates with the all known miRNAs of the herpesvirus family. Interestingly, the "seed sequences" (nt 2 to 8) of 2 miRNAs were predicted to have the high conservation in position and/or sequence with the 2 miRNAs of Marek's disease virus type 1 (MDV-1). Additionally, we searched the targets from viral mRNAs. CONCLUSIONS: Using computational approaches, we found that AHV-1 putatively encodes 12 mature miRNAs and 2 miRNAs have the high conservation with the 2 miRNAs of MDV-1. The result suggested that AHV-1 and MDV-1 should have closed evolutionary relation, which provides a valuable evidence of classification of AHV-1. Additionally, seven viral gene targets were found, which suggested that AHV-1 miRNAs could affect its own gene expression.


Subject(s)
Mardivirus/genetics , MicroRNAs/genetics , RNA, Viral/genetics , Base Sequence , Computational Biology/methods , Conserved Sequence , Genome, Viral , Molecular Sequence Data
11.
Virol J ; 8: 266, 2011 Jun 01.
Article in English | MEDLINE | ID: mdl-21631934

ABSTRACT

BACKGROUND: Real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR) has become the benchmark for detection and quantification of target gene expression level and been utilized increasingly in detection of viral load and therapy monitoring. The dynamic transcription variation of duck enteritis virus UL55 gene during the life cycle of duck enteritis virus in infected cells has not been reported yet. RESULTS: The newly identified duck enteritis virus UL55 gene was amplified and cloned into pMD18-T vector after digestion to generate a recombinant plasmid pMD18-T/UL55 for the establishment of qRT-PCR as standard DNA. The results of agarose gel electrophoresis and melting curve analysis demonstrated the primers we designed for qRT-PCR were specific and available. We used ß-actin as a reference gene for normalization and established two standard curves based on pMD18-T/UL55 and pMD18-T/ß-actin successfully. Based on that, the transcriptional analysis of DEV UL55 gene was performed, and the result suggested the expression of UL55 mRNA was at a low level from 0 to 8 h post-infection(p.i.), then accumulated quickly since 12 h p.i. and peaked at 36 h p.i., it can be detected till 60 h p.i.. Nucleic acid inhibition test was carried out for analyzing a temporal regulation condition of DEV UL55 gene, result revealed that it was sensitive to ganciclovir. Synthesis procedures of DEV UL55 gene can be inhibited by ganciclovir. CONCLUSIONS: The method we established in this paper can provide quantitative values reflecting the amounts of measured mRNA in samples. It's available for detection and quantification, also can be used in DEV diagnosis. The DEV UL55 gene was produced most abundantly during the late phase of replication in DEV-infected cells and the transcription of it depended on the synthesized DNA. DEV UL55 gene is a γ2 gene which occurs last and have a strict requirement for viral DNA synthesis.


Subject(s)
Gene Expression Profiling , Mardivirus/physiology , Reverse Transcriptase Polymerase Chain Reaction/methods , Viral Proteins/biosynthesis , Virology/methods , Actins/genetics , Animals , Cells, Cultured , DNA Primers/genetics , DNA, Viral/genetics , Ducks , Fibroblasts/virology , Mardivirus/genetics , Molecular Sequence Data , Reference Standards , Reverse Transcriptase Polymerase Chain Reaction/standards , Sequence Analysis, DNA , Viral Proteins/genetics
12.
Virol J ; 8: 241, 2011 May 19.
Article in English | MEDLINE | ID: mdl-21595918

ABSTRACT

BACKGROUND: At present, alphaherpesviruses gI gene and its encoding protein have been extensively studied. It is likely that gI protein and its homolog play similar roles in virions direct cell-to-cell spread of alphaherpesviruses. But, little is known about the characteristics of DEV gI gene. In this study, we expressed and presented the basic properties of the DEV gI protein. RESULTS: The special 1221-bp fragment containing complete open reading frame(ORF) of duck enteritis virus(DEV) gI gene was extracted from plasmid pMD18-T-gI, and then cloned into prokaryotic expression vector pET-32a(+), resulting in pET-32a(+)-gI. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pET-32a(+)-gI was transformed into E.coli BL21(DE3) competent cells for overexpression. DEV gI gene was successfully expressed by the addition of isopropyl-ß-D-thiogalactopyranoside(IPTG). SDS-PAGE showed that the recombinant protein His6-tagged gI molecular weight was about 61 kDa. Subsequently, the expressed product was applied to generate specific antibody against gI protein. The specificity of the rabbit immuneserum was confirmed by its ability to react with the recombinant protein His6-tagged gI. In addition, real time-PCR was used to determine the the levels of the mRNA transcripts of gI gene, the results showed that the DEV gI gene was transcribed most abundantly during the late phase of infection. Furthermore, indirect immunofluorescence(IIF) was established to study the gI protein expression and localization in DEV-infected duck embryo fibroblasts (DEFs), the results confirmed that the protein was expressed and located in the cytoplasm of the infected cells, intensively. CONCLUSIONS: The recombinant prokaryotic expression vector of DEV gI gene was constructed successfully. The gI protein was successfully expressed by E.coli BL21(DE3) and maintained its antigenicity very well. The basic information of the transcription and intracellular localization of gI gene were presented, that would be helpful to assess the possible role of DEV gI gene. The research will provide useful clues for further functional analysis of DEV gI gene.


Subject(s)
Glycoproteins/metabolism , Mardivirus/physiology , Viral Structural Proteins/metabolism , Animals , Cells, Cultured , Cytoplasm/chemistry , Ducks , Fibroblasts/virology , Fluorescent Antibody Technique, Indirect , Gene Expression Profiling , Glycoproteins/genetics , Mardivirus/genetics , Reverse Transcriptase Polymerase Chain Reaction , Viral Structural Proteins/genetics
13.
Virol J ; 8: 256, 2011 May 24.
Article in English | MEDLINE | ID: mdl-21609474

ABSTRACT

BACKGROUND: Characteration of the newly identified duck enteritis virus UL55 gene product has not been reported yet. Knowledge of the protein UL55 can provide useful insights about its function. RESULTS: The newly identified duck enteritis virus UL55 gene was about 561 bp, it was amplified and digested for construction of a recombinant plasmid pET32a(+)/UL55 for expression in Escherichia coli. SDS-PAGE analysis revealed the recombinant protein UL55(pUL55) was overexpressed in Escherichia coli BL21 host cells after induction by 0.2 mM IPTG at 37°C for 4 h and aggregated as inclusion bodies. The denatured protein about 40 KDa named pUL55 was purified by washing five times, and used to immune rabbits for preparation of polyclonal antibody. The prepared polyclonal antibody against pUL55 was detected and determined by Agar immundiffusion and Neutralization test. The results of Wstern blotting assay and intracellular analysis revealed that pUL55 was expressed most abundantly during the late phase of replication and mainly distributed in cytoplasm in duck enteritis virus infected cells. CONCLUSIONS: In this study, the duck enteritis virus UL55 protein was successfully expressed in prokaryotic expression system. Besides, we have prepared the polyclonal antibody against recombinant prtein UL55, and characterized some properties of the duck enteritis virus UL55 protein for the first time. The research will be useful for further functional analysis of this gene.


Subject(s)
Gene Expression Profiling , Mardivirus/physiology , Viral Proteins/biosynthesis , Virus Replication , Animals , Cells, Cultured , Cytoplasm/chemistry , Ducks , Fibroblasts/chemistry , Fibroblasts/virology , Mardivirus/genetics , Microscopy, Fluorescence , Viral Proteins/genetics
14.
Virol J ; 8: 235, 2011 May 17.
Article in English | MEDLINE | ID: mdl-21586146

ABSTRACT

BACKGROUND: Most of the previous research work had focused on the epidemiology and prevention of duck enteritis virus (DEV). Whilst with the development of protocols in molecular biology, nowadays more and more information about the genes of DEV was reported. But little information about DEV UL53 gene and glycoprotein K(gK) was known except our reported data. RESULTS: In our paper, the fluorescent quantitative real-time PCR(FQ-RT-PCR) assay and nucleic acid inhibition test were used to study the transcription characteristic of the DEV UL53 gene. Except detecting the mRNA of DEV UL53 gene, the product gK encoded by UL53 gene was detected through the expression kinetics of UL53 gene by the purified rabbit anti-UL53 protein polyclonal antibodies. Western-blotting and indirect immunofluorescence assays were used to detect gK. From the results of these experiments, the UL53 gene and gK were respectively identified as a late gene and a really late protein. On the other hand, the indirect immunofluorescence assay provided another information that the intracellular localization of DEV gK was mainly distributed in cytoplasm. CONCLUSIONS: By way of conclusions, we conceded that DEV UL53 gene is a really late gene, which is coincident with properties of UL53 homologs from other herpesvirus, such as ILTV(Infectious Laryngotracheitis virus) and HSV-1(Herpes simplex virus type 1). The properties of intracellular localization about gK protein provided a foundation for further functional analysis and further studies will be focused on constructing of the UL53 gene DEV mutant.


Subject(s)
Ducks , Gene Expression Profiling , Mardivirus/physiology , Poultry Diseases/virology , Transcription, Genetic , Viral Proteins/metabolism , Virus Replication , Animals , Blotting, Western , Cells, Cultured , Fibroblasts/virology , Mardivirus/genetics , Microscopy, Fluorescence , Viral Proteins/genetics
15.
Virol J ; 8: 82, 2011 Feb 24.
Article in English | MEDLINE | ID: mdl-21349183

ABSTRACT

BACKGROUND: Previous studies have indicated that the VP19c protein and its homology play similar roles in capsid assembly of all Alphaherpesvirus subfamily. However, there is no report on the VP19c protein of duck enteritis virus (DEV). In this study, we expressed the DEV VP19c protein and presented its antigenic properties. Moreover, we developed polyclonal antibody against the VP19c protein and characterized it. METHODS: A recombinant VP19c (rVP19c) and N-terminal were expressed in Escherichia coli (E.coli) and purified by Ni²+-affinity chromatography. The antigenic properties of the recombinant protein were determined by Western blot and indirect enzyme-linked immunosorbent assay (ELISA). Furthermore, the polyclonal antibodies against the purified recombinant proteins were produced and the titer of polyclonal antibody was determined by ELISA analysis. Finally, the antibody was used to recognize the VP19c in the cells infected with DEV in the immunofluorescence assay. RESULTS: The N-terminally His-tagged rVP19c and rVP19c(N) were produced as inclusion bodies in E. coli strain BL21 (DE3) with molecular weight of about 66 and 46 kDa. Then the proteins were purified to reach the level of homogeneity. Western blot and ELISA analysis that the rVP19c seems to be structurally and antigenically very similar to native VP19c and the N-terminus of VP19c may contain most antigenic linear-epitopes. Furthermore, ELISA analysis demonstrated that the titer of polyclonal antibody was approximately 1:12800, and in the immunofluorescence assay, the antibody was able to recognize the VP19c in the cells infected with DEV. CONCLUSIONS: To our knowledge, this was the first report on basic properties of DEV VP19c protein. In the present study, we obtained a high-level expression of the recombinant VP19c protein as well as high titers of rabbit polyclonal antibody against to VP19c protein. The anti-rVP19c serum was able to detect the VP19c protein in DEV infected cells and the VP19c protein targeted to the nucleus as distinct punctate speckles. This specific polyclonal antibody provides a good tool for further studying structural and functional characterization of DEV VP19c.


Subject(s)
Capsid Proteins/biosynthesis , Capsid Proteins/immunology , Mardivirus/genetics , Mardivirus/immunology , Animals , Blotting, Western , Capsid Proteins/chemistry , Capsid Proteins/genetics , Cell Nucleus/chemistry , Chromatography, Affinity , Ducks , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Fibroblasts/virology , Gene Expression , Molecular Weight , Rabbits , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology
16.
Avian Dis ; 55(4): 626-32, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22312983

ABSTRACT

A recombinant UL55 protein (pUL55) of duck enteritis virus (DEV), produced in Escherichia coli, was tested for diagnostic applicability in an indirect enzyme-linked immunosorbent assay (1-ELISA) as a coating antigen. Serum dilutions of 1:6400 (0.025microg) are the maximum detection limits of the pUL55-ELISA, according to the determined cut-off value of 0.330. Antigenic cross-reactivity investigation in heterologous sera of ducks failed to provide evidence that other viruses of ducks could hamper the serodiagnosis of DEV, and the inhibition assay revealed that the specific binding of antigen and antibody can be inhibited by pUL55, both of which demonstrated a good specificity of the established pUL55-ELISA. This assay was further validated by comparison with a commercial 1-ELISA based on DEV (DEV-ELISA) and a neutralization test (NT). The results suggested that the sensitivity of pUL55-ELISA was almost as good as DEV-ELISA but was much higher than NT. The established pUL55-ELISA is a rapid, simple, sensitive, specific, and inexpensive serodiagnosis for detecting antibodies against DEV and has a potential to complement the traditional assays for serodiagnosis of DEV; it can be used as a diagnosis alternative candidate for serologic surveillance of DEV infection.


Subject(s)
Ducks , Enterovirus/isolation & purification , Enzyme-Linked Immunosorbent Assay/veterinary , Poultry Diseases/virology , Viral Proteins/isolation & purification , Animals , Enzyme-Linked Immunosorbent Assay/methods , Gene Expression Regulation, Viral , Neutralization Tests , Poultry Diseases/diagnosis , Sensitivity and Specificity , Serologic Tests
17.
Virol J ; 7: 241, 2010 Sep 17.
Article in English | MEDLINE | ID: mdl-20846457

ABSTRACT

Duck virus enteritis (DVE) is an acute, contagious herpesvirus infection of ducks, geese, and swans, which has produced significant economic losses in domestic and wild waterfowl. With the purpose of decreasing economic losses in the commercial duck industry, studying the unknown glycoprotein K (gK) of DEV may be a new method for preferably preventing and curing this disease. So this is the first time to product and purify the rabbit anti-tgK polyclonal antibody. Through the western blot and ELISA assay, the truncated glycoprotein K (tgK) has good antigenicity, also the antibody possesses high specificity and affinity. Meanwhile the rabbit anti-tgK polyclonal antibody has the potential to produce subunit vaccines and the functions of neutralizing DEV and anti-DEV infection because of its neutralization titer. Indirect immunofluorescent microscopy using the purified rabbit anti-tgK polyclonal antibody as diagnostic antibody was susceptive to detect a small quantity of antigen in tissues or cells. This approach also provides effective experimental technology for epidemiological investigation and retrospective diagnose of the preservative paraffin blocks.


Subject(s)
Antibodies, Viral/isolation & purification , Ducks/virology , Glycoproteins/immunology , Herpesviridae/immunology , Viral Envelope Proteins/immunology , Animals , Antigens, Viral/analysis , Fluorescent Antibody Technique, Indirect/methods , Herpesvirus Vaccines/immunology , Neutralization Tests , Rabbits , Vaccines, Subunit/immunology
18.
Virol J ; 7: 168, 2010 Jul 21.
Article in English | MEDLINE | ID: mdl-20663161

ABSTRACT

BACKGROUND: Duck viral enteritis, which is caused by duck enteritis virus (DEV), causes significant economic losses in domestic and wild waterfowls because of the high mortality and low egg production rates. With the purpose of eliminating this disease and decreasing economic loss in the commercial duck industry, researching on glycoprotein K (gK) of DEV may be a new kind of method for preventing and curing this disease. Because glycoproteins project from the virus envelope as spikes and are directly involved in the host immune system and elicitation of the host immune responses, and also play an important role in mediating infection of target cells, the entry into cell for free virus and the maturation or egress of virus. The gK is one of the major envelope glycoproteins of DEV. However, little information correlated with gK is known, such as antigenic and functional characterization. RESULTS: Bioinformatic predictions revealed that the expression of the full-length gK gene (fgK) in a prokaryotic system is difficult because of the presence of suboptimal exon and transmembrane domains at the C-terminal. In this study, we found that the fgK gene might not be expressed in a prokaryotic system in accordance with the bioinformatic predictions. Further, we successfully used bioinformatics tools to guide the prokaryotic expression of the gK gene by designing a novel truncated gK gene (tgK). These findings indicated that bioinformatics provides theoretical data for target gene expression and saves time for our research. The recombinant tgK protein (tgK) was expressed and purified by immobilized metal affinity chromatography (IMAC). Western blotting and indirect enzyme-linked immunosorbent assay (ELISA) showed that the tgK possessed antigenic characteristics similar to native DEV-gK. CONCLUSIONS: In this work, the DEV-tgK was expressed successfully in prokaryotic system for the first time, which will provide usefull information for prokaryotic expression of alphaherpesvirus gK homologs, and the recombinant truncated gK possessed antigenic characteristics similar to native DEV gK. Because of the good reactionogenicity, specificity and sensitivity, the purified tgK could be useful for developing a sensitive serum diagnostic kit to monitor DEV outbreaks.


Subject(s)
Alphaherpesvirinae/isolation & purification , Antigens, Viral/genetics , Bird Diseases/diagnosis , Bird Diseases/virology , Herpesviridae Infections/veterinary , Viral Envelope Proteins/genetics , Virology/methods , Alphaherpesvirinae/genetics , Animals , Antibodies, Viral/blood , Antigens, Viral/immunology , Antigens, Viral/isolation & purification , Blotting, Western , Chromatography, Affinity , Cloning, Molecular , Computational Biology/methods , DNA, Viral/chemistry , DNA, Viral/genetics , Ducks , Enzyme-Linked Immunosorbent Assay , Gene Expression , Glycoproteins/genetics , Glycoproteins/immunology , Glycoproteins/isolation & purification , Herpesviridae Infections/diagnosis , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Sensitivity and Specificity , Sequence Analysis, DNA , Viral Envelope Proteins/immunology , Viral Envelope Proteins/isolation & purification
19.
Virol J ; 7: 162, 2010 Jul 17.
Article in English | MEDLINE | ID: mdl-20637115

ABSTRACT

Knowledge of the intracellular location of a protein can provide useful insights into its function. Bioinformatic studies have predicted that the DEV pUL38 mainly targets the cytoplasm and nucleus. In this study, we obtained anti-pUL38 polyclonal sera. These antibodies were functional in western blotting and immunofluorescence in DEV-infected duck embryo fibroblasts (DEFs). pUL38 was expressed as a 51-kDa protein from 8 h post-infection onward, initially showing a diffuse distribution throughout the cytoplasm, and later in the nucleus. Furthermore, pUL38 was found in purified virus. These results provide the first evidence of the kinetics of expression and intracellular localization of DEV pUL38.


Subject(s)
Enteritis/veterinary , Mardivirus/genetics , Marek Disease/virology , Poultry Diseases/virology , Viral Structural Proteins/genetics , Viral Structural Proteins/metabolism , Animals , Cell Line , Ducks , Enteritis/metabolism , Enteritis/virology , Gene Expression Regulation, Viral , Mardivirus/metabolism , Marek Disease/metabolism , Molecular Weight , Poultry Diseases/metabolism , Protein Transport , Viral Structural Proteins/chemistry
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