Embryo Fibroblast Cultures Research Articles

EFFECTIVENESS OF CRYOPRESERVED CHICKEN EMBRYO FIBROBLAST CELLS FOR PROPAGATION OF DUCK ENTERITIS VIRUS AND FOWLPOX VIRUS

Primary chicken embryo fibroblast (CEF) cultures are excellent model systems for in-vitro virus propagation. However, the cumbersome preparation process of CEF makes cryopreservation highly essential. Cryopreservation in Liquid Nitrogen liquid phase (-196 °C) or vapour phase (-156 °C) are frequently used methods. In the present study, efficiency of cryopreserved primary CEF cells for the propagation of Duck plague virus and Fowl pox virus was evaluated. The cells were preserved for different time periods and different cooling methodologies were employed in order to assess the effects exerted by these variations. The efficiency of the cells was compared based on cell viability/ revival rate, ability to form monolayer and TCID50 of the propagated viruses. Cell count revealed viability of 75.60% and 68% post 2 weeks and 6 months of cryopreservation respectively following step freezing method. On the other hand, cells preserved using insulated cryo-containers exhibited 89% and 80% viability post 2 weeks and 6 months of cryopreservation respectively. Development of complete monolayer could be seen within 24 hours in all the batches irrespective of the cooling method used and the cryopreservation storage time. Infection with DPV and FPV yielded respective cytopathic effect from 48 hours onwards. CPE observed in the freshly prepared CEF and in those revived 2 weeks and 6 months post freezing, were similar in terms of typical characteristics and duration of appearance. From the findings of the present study it is evident that cryopreservation of CEF cells is a convenient and practical approach for virus propagation. Cryopreservation of primary cells offers a definite advantage over repeated culture preparation which is labour intensive, time consuming and dependent on the availability of embryonated eggs.

Retrospective Analysis of Experimental and Clinical Studies of Flakozid in Viral Hepatitis

Retrospective Analysis of Experimental and Clinical Studies of Flakozid in Viral Hepatitis

Effect of exogenous microflora of the hepatobiliary system on the biochemical composition of bile and lipid peroxidation processes of somatic cell membranes in chronic opisthorchiasis

It has been epidemiologically established that the rate of primary cholangiocarcinomas increases greatly in the ObIrtysh basin which is hyperendemic for helminth Opisthorchis felineus in comparison with other regions where the population is not infested. In this connection chronic opisthorchiasis is considered as facultative liver precancer. The study of cholangiocarcinogenesis mechanisms will allow developing pathogenetic approaches to prevention of this tumor. Aim of the study was to investigate the significance of microbiota of the hepatobiliary tract of chronic opisthorchiasis patients in changing of biochemical composition of bile and investigation of its biological influence on somatic cells membrane. Material and methods. Objects of research: bacteria; inbred mice infected by opisthorchis; samples of bile, cultures of human embryo fibroblasts and splenocytes of inbred mice in vitro; blood plasma; standard primary and secondary bile acids. We studied: species of bacteria colonizing bile ducts; quantitative and qualitative composition of bile acids, and level of diene conjugates and malondialdehyde in duct bile samples; biological activity of bile on processes of lipid peroxidation of cell membranes in vitro; influence of bile and lipid peroxidation products on cytomembrane permeability; activity of antioxidant systems of the body. Results and discussion. It was found out, that bile of patients with chronic opisthorchiasis in majority of cases (77.0 %) was infected by different species of bacteria. In 30.0 % of cases certain types of intestinal bacteria (Proteus vulgaris, P. mirabilis, Citrobacter freundii, Bacteroides alcaligues faecalis, Clostridium, Streptococcus faecalis, Escherichia coli) change biochemical composition of duct bile – deconjugated primary and secondary bile acids and also high level of total bile acids are detected. Bile of the above biochemical composition, against the background of depletion of antioxidant system, induces activation of cell membrane lipid peroxidation processes and significantly increases their permeability to toxic components of bile. These processes are promotor in cholangiocarcinogenesis.

Antigenic Characterization of New Lineage II Insect-Specific Flaviviruses in Australian Mosquitoes and Identification of Host Restriction Factors.

We describe two new insect-specific flaviviruses (ISFs) isolated from mosquitoes in Australia, Binjari virus (BinJV) and Hidden Valley virus (HVV), that grow efficiently in mosquito cells but fail to replicate in a range of vertebrate cell lines. Phylogenetic analysis revealed that BinJV and HVV were closely related (90% amino acid sequence identity) and clustered with lineage II (dual-host affiliated) ISFs, including the Lammi and Nounané viruses. Using a panel of monoclonal antibodies prepared to BinJV viral proteins, we confirmed a close relationship between HVV and BinJV and revealed that they were antigenically quite divergent from other lineage II ISFs. We also constructed chimeric viruses between BinJV and the vertebrate-infecting West Nile virus (WNV) by swapping the structural genes (prM and E) to produce BinJ/WNVKUN-prME and WNVKUN/BinJV-prME. This allowed us to assess the role of different regions of the BinJV genome in vertebrate host restriction and revealed that while BinJV structural proteins facilitated entry to vertebrate cells, the process was inefficient. In contrast, the BinJV replicative components in wild-type BinJV and BinJ/WNVKUN-prME failed to initiate replication in a wide range of vertebrate cell lines at 37°C, including cells lacking components of the innate immune response. However, trace levels of replication of BinJ/WNVKUN-prME could be detected in some cultures of mouse embryo fibroblasts (MEFs) deficient in antiviral responses (IFNAR-/- MEFs or RNase L-/- MEFs) incubated at 34°C after inoculation. This suggests that BinJV replication in vertebrate cells is temperature sensitive and restricted at multiple stages of cellular infection, including inefficient cell entry and susceptibility to antiviral responses.IMPORTANCE The globally important flavivirus pathogens West Nile virus, Zika virus, dengue viruses, and yellow fever virus can infect mosquito vectors and be transmitted to humans and other vertebrate species in which they cause significant levels of disease and mortality. However, the subgroup of closely related flaviviruses, known as lineage II insect-specific flaviviruses (Lin II ISFs), only infect mosquitoes and cannot replicate in cells of vertebrate origin. Our data are the first to uncover the mechanisms that restrict the growth of Lin II ISFs in vertebrate cells and provides new insights into the evolution of these viruses and the mechanisms associated with host switching that may allow new mosquito-borne viral diseases to emerge. The new reagents generated in this study, including the first Lin II ISF-reactive monoclonal antibodies and Lin II ISF mutants and chimeric viruses, also provide new tools and approaches to enable further research advances in this field.

Activation of gga-miR-155 by reticuloendotheliosis virus T strain and its contribution to transformation.

The v-rel oncoprotein encoded by reticuloendotheliosis virus T strain (Rev-T) is a member of the rel/NF-κB family of transcription factors capable of transformation of primary chicken spleen and bone marrow cells. Rapid transformation of avian haematopoietic cells by v-rel occurs through a process of deregulation of multiple protein-encoding genes through its direct effect on their promoters. More recently, upregulation of oncogenic miR-155 and its precursor pre-miR-155 was demonstrated in both Rev-T-infected chicken embryo fibroblast cultures and Rev-T-induced B-cell lymphomas. Through electrophoresis mobility shift assay and reporter analysis on the gga-miR-155 promoter, we showed that the v-rel-induced miR-155 overexpression occurred by the direct binding to one of the putative NF-κB binding sites. Using the v-rel-induced transformation model on chicken embryonic splenocyte cultures, we could demonstrate a dynamic increase in miR-155 levels during the transformation. Transcriptome profiles of lymphoid cells transformed by v-rel showed upregulation of miR-155 accompanied by downregulation of a number of putative miR-155 targets such as Pu.1 and CEBPβ. We also showed that v-rel could rescue the suppression of miR-155 expression observed in Marek’s disease virus (MDV)-transformed cell lines, where its functional viral homologue MDV-miR-M4 is overexpressed. Demonstration of gene expression changes affecting major molecular pathways, including organismal injury and cancer in avian macrophages transfected with synthetic mature miR-155, underlines its potential direct role in transformation. Our study suggests that v-rel-induced transformation involves a complex set of events mediated by the direct activation of NF-κB targets, together with inhibitory effects on microRNA targets.

Small RNA-directed epigenetic programming of embryonic stem cell cardiac differentiation

Microinjection of small noncoding RNAs in one-cell embryos was reported in several instances to result in transcriptional activation of target genes. To determine the molecular mechanisms involved and to explore whether such epigenetic regulations could play a role in early development, we used a cell culture system as close as possible to the embryonic state. We report efficient cardiac differentiation of embryonic stem (ES) cells induced by small non-coding RNAs with sequences of Cdk9, a key player in cardiomyocyte differentiation. Transfer of oligoribonucleotides representing parts of the Cdk9 mRNA into ES and mouse embryo fibroblast cultures resulted in upregulation of transcription. Dependency on Argonaute proteins and endogenous antisense transcripts indicated that the inducer oligoribonucleotides were processed by the RNAi machinery. Upregulation of Cdk9 expression resulted in increased efficiency of cardiac differentiation suggesting a potential tool for stem cell-based regenerative medicine.

Susceptibility of primary chicken intestinal epithelial cells for low pathogenic avian influenza virus and velogenic viscerotropic Newcastle disease virus

Avian influenza virus (AIV) and Newcastle disease virus (NDV) share a high tropism for the avian respiratory epithelium and may cause severe clinical disease associated with high mortality. Both viruses have different pathotypes, which may lead to differences in the severity of the disease. Respiratory epithelial cells were shown to be the primary target cells for infection and replication. Nevertheless, intestinal epithelial cells (IECs) were also suggested as target cells for both viruses in avian species. Most studies on AIV and NDV focused on the respiratory tract, while information regarding the virus-host interaction at the intestinal epithelial cell interface is lacking. We established a primary chicken IEC culture model. Primary chicken embryo fibroblast cultures (CEFs) were used for comparison. IECs and CEFs were infected with a low infectious dose (LID; multiplicity of infection, MOI, of 0.01) or high infectious dose (HID, MOI of 1), of low pathogenic AIV (LPAIV) H9N2 or velogenic viscerotropic NDV (vvNDV) Herts 33/56. Virus replication, mRNA expression pattern of the type I and type III interferon (IFN) and related genes IFIT5 (interferon-induced protein with tetratricopeptide repeats 5) and ISG12 (interferon stimulated gene 12) were investigated at four, 16, and 24h post infection (hpi). The results suggest high susceptibility of primary chicken IECs for these AIV and NDV strains. Replication rates and expression pattern of IFNs as well as related genes differed between the infecting viruses as well as cell culture systems. Both viruses induced an IFN λ-increase of more than 30-fold in IECs, while IFN-α and IFN-β mRNA expression was either downregulated or only slightly increased with up to 10fold changes for the latter at 24h post LPAIV-infection. These results suggest a possible role of IFN λ in the control of viruses at the gut epithelial surface. LPAIV induced upregulation of IFIT5 as well as ISG12 expression in a dose and time dependent manner, while vvNDV infection only led to slight upregulation of IFIT5 and downregulation of ISG12, indicating differences in the down-stream regulation of the antiviral immune response between investigated viruses. Overall, our data demonstrate that IECs are a suitable model to investigate selected parameters of virus-host interaction for AIV and NDV and may be used to study other strains as well as other host species.

Evaluation and validation of reference gene stability during Marek’s disease virus (MDV) infection

Quantitative RT-PCR (qRT-PCR) is widely used in the study of relative gene expression in general, and has been used in the field of Marek’s disease (MD) research to measure transcriptional responses to infection and/or vaccination. Studies in the past have either employed cellular β-actin (BACT) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal reference genes, although the stability of their expression in the context of Marek’s disease virus (MDV) infection has never been investigated. In the present study, we compared the stability of five reference genes (BACT, 28S RNA, 18S RNA, GAPDH, Peptidyl-prolyl-isomerase B [PPIB], a.k.a. cyclophilin B) as standard internal controls in chicken embryo fibroblast (CEFs) cultures infected with either MD vaccine or oncogenic MDV1 viruses. We further extend these analyses to reference gene stability in spleen lymphomas induced by infection of commercial broiler chickens with a very virulent plus MDV1 (vv+ TK-2a virus). Two excel based algorithms, (Bestkeeper and Normfinder) were employed to compare reference gene stability. Bestkeeper and Normfinder analysis of reference gene stability in virus- and mock-infected cells, showed that 28S RNA and PPIB displayed higher stability in CEF infections with either oncogenic or vaccine viruses. In addition, both Bestkeeper and Normfinder determined 28S RNA and PPIB to be the most stably-expressed reference genes in vivo in vv+ TK-2a-induced spleen lymphomas. Furthermore, Bestkeeper and Normfinder analyses both determined BACT to be the least stable reference gene during MDV infection of CEF with oncogenic viruses, vaccine viruses, as well as in vv+ TK-2a-induced spleen lymphomas.

Far Upstream Element Binding Protein Plays a Crucial Role in Embryonic Development, Hematopoiesis, and Stabilizing Myc Expression Levels

The transcription factor far upstream element binding protein (FBP) binds and activates the MYC promoter when far upstream element is via TFIIH helicase activity early in the transcription cycle. The fundamental biology and pathology of FBP are complex. In some tumors FBP seems pro-oncogenic, whereas in others it is a tumor suppressor. We generated an FBP knockout (Fubp1(-/-)) mouse to study FBP deficiency. FBP is embryo lethal from embryonic day 10.5 to birth. A spectrum of pathology is associated with FBP loss; besides cerebral hyperplasia and pulmonary hypoplasia, pale livers, hypoplastic spleen, thymus, and bone marrow, cardiac hypertrophy, placental distress, and small size were all indicative of anemia. Immunophenotyping of hematopoietic cells in wild-type versus knockout livers revealed irregular trilineage anemia, with deficits in colony formation. Despite normal numbers of hematopoietic stem cells, transplantation of Fubp1(-/-) hematopoietic stem cells into irradiated mice entirely failed to reconstitute hematopoiesis. In competitive transplantation assays against wild-type donor bone marrow, Fubp1(-/-) hematopoietic stem cells functioned only sporadically at a low level. Although cultures of wild-type mouse embryo fibroblasts set Myc levels precisely, Myc levels of mouse varied wildly between fibroblasts harvested from different Fubp1(-/-) embryos, suggesting that FBP contributes to Myc set point fixation. FBP helps to hold multiple physiologic processes to close tolerances, at least in part by constraining Myc expression.

Role of the poultry red mite (Dermanyssus gallinae) in the transmission of avian influenza A virus

The aim of this study was to investigate the role of the poultry red mite (Dermanyssus [D.] gallinae) in the horizontal transmission of avian influenza A virus (AIV) to chickens. This mite is the most common ectoparasite in poultry worldwide, and may play a role in the spread of infectious agents including AIV. Currently, the control of mites is difficult due to frequently developed resistance to many acaricides, their nocturnality and their ability to survive hidden without feeding for months. D. gallinae were collected in a commercial layer farm and housed in self-made fibreboard boxes. SPF chickens were intravenously infected with AIV strain A/turkey/Ontario/7732/1966 (H5N9). The viraemia in chickens was monitored and at an appropriate time point about 1000 mites were allowed to suck on the AIV infected chickens. Re-isolation of the virus from blood-filled mites was tried daily for 14 days using chicken embryo fibroblast cultures and embryonated chicken eggs. Subsequently, the virus containing mites were placed into boxes that contained naïve SPF chickens to enable virus transmission from mites to chickens. Possible transmission to the chickens was examined using clinical signs, serology, gross lesions, histopathology and immunohistochemistry. Chickens developed a dose-dependent viraemia one day after infection, therefore this day was chosen for the bloodmeal of the mites. AIV was detected in mites after bloodsucking on AIV-infected chickens over a 10-day period. Naïve SPF chickens were infected during bloodsucking of AIV carrying mites. AIV isolates in mites and in chickens were undistinguishable from the original AIV inoculum by RT-PCR. D. gallinae ingested AIV during bloodmeals on AIV infected chickens and are able to transmit AIV to SPF chickens. Therefore, mites serve as mechanical vector of AIV and may play a major role in the circulation of AIV within a facility or area although the life span of infectious virus in the mite is limited. The proven transmission requires more than ever a systematic control of this ectoparasite in order to maintain poultry health and productivity. The demonstrated vector function of this mite is of great significance for poultry flocks all over the world.

Lamivudine Inhibits the Replication of ALV-J Associated Acutely Transforming Virus and its Helper Virus and Tumor Growth In vitro and In vivo.

To study the antiviral effects of lamivudine on avian leukosis virus subgroup J (ALV-J) and its inhibitory effect on the growth of fibrosarcomas caused by acute transforming avian leukosis virus, a series of experiments were performed in chicken embryo fibroblast cultures and 1-day-old chickens inoculated with an acutely transforming viral stock Fu-J (SDAU1005). This stock was prepared from an acutely fibrosarcoma of field cases in chicken farms and contained both the replication-defective virus Fu-J carrying v-fps oncogene and its helper virus ALV-J strain SDAU1005. The results from three different assays in cell cultures demonstrated the significant inhibitory effect of lamivudine on the replication of both SDAU1005 and Fu-J viruses. Furthermore, the effect was dose dependent in the concentration range of 1–4 μg/ml. In chicken experiments, lamivudine could decrease the viral loads of SDAU1005 and Fu-J in the plasma of inoculated chickens, delay the appearance of acute sarcomas, and decrease chicken mortality in the early stage. This model may be used to directly evaluate the inhibitory effects of lamivudine on such tumors and to understand the relationship between the replication-defective virus and its helper virus while also assessing tumor processes.

Duck tembusu virus and its envelope protein induce programmed cell death.

The cytopathic effect produced in cells infected with duck tembusu virus (DTMUV) suggests that this emerging virus may induce apoptosis in primary cultures of duck embryo fibroblasts (DEF). Here, we present evidence that DTMUV infection of cultured cells activates apoptosis and that the ability of DTMUV to induce apoptosis is not restricted to cell type because DTMUV-induced apoptosis in duck and mammalian host cells. We further investigated which viral components induce apoptosis in DTMUV-infected host cells. The major envelope glycoprotein (E) was investigated for its apoptotic activities in expressed cells. Transient expression of the E protein alone triggered apoptosis in DEF, Vero, and BHK cells. Expression of the E protein resulted in activation of caspase-3-like proteases in cultured cells. These results indicate that infection of cells with DTMUV or expression of DTMUV E protein alone induces apoptosis, providing the basis for future to define the molecules that play key roles in the fate of DTMUV-infected cells.

Whole-blood immunoassay for γH2AX as a radiation biodosimetry assay with minimal sample preparation

The current state of the art in high-throughput minimally invasive radiation biodosimetry involves the collection of samples in the field and analysis at a centralized facility. We have developed a simple biological immunoassay for radiation exposure that could extend this analysis out of the laboratory into the field. Such a forward placed assay would facilitate triage of a potentially exposed population. The phosphorylation and localization of the histone H2AX at double-stranded DNA breaks has already been proven to be an adequate surrogate assay for reporting DNA damage proportional to radiation dose. Here, we develop an assay for phosphorylated H2AX directed against minimally processed sample lysates. We conduct preliminary verification of H2AX phosphorylation using irradiated mouse embryo fibroblast cultures. Additional dosimetry is performed using human blood samples irradiated ex vivo. The assay reports H2AX phosphorylation in human blood samples in response to ionizing radiation over a range of 0-5Gy in a linear fashion, without requiring filtering, enrichment, or purification of the blood sample.

Pathotyping of recent Indian field isolates of Marek's disease virus serotype 1.

A study was undertaken to assess the virulence of Marek's disease virus (MDV) serotype 1 field isolates obtained from poultry flocks of southern part of India. Five representative MDV serotype 1 strains were isolated from eighty-six blood samples collected from fifteen farms. Three out of five isolates which were free from avian leukosis virus (ALV) and reticuloendotheliosis virus (REV) were adapted in chicken embryo fibroblast (CEF) culture and designated as Ind/TN/11/01, Ind/KA/12/02 and Ind/TN/12/03. Pathotyping assay was conducted in two trials. In the first trial, non-vaccinated chickens were challenged (trial I), while in second trial, two types of vaccinated chickens along with non-vaccinated controls were challenged (trial II). Birds inoculated with field isolate Ind/TN/12/03 had very low body (75.34 ± 3.04 g 15 days post infection (dpi)) and bursa Fabricii weight (1.64 ± 0.06 at 15 dpi) when compared to those inoculated with the other two isolates (Ind/TN/11/01 and Ind/KA/12/02) and uninoculated controls (body weight 111.33 ± 1.30 g and bursa Fabricii weight 4.33 ± 0.11 15 dpi). Incidence of early mortality syndrome (53%) and lymphoma (86%) induced by Ind/TN/12/03 was comparable with very virulent strains published elsewhere. In protection test, the percentage of Marek's disease (MD) incidence induced by Ind/TN/12/03 was 57.5% and 25% in monovalent and bivalent vaccine inoculated birds respectively compared to uninoculated control (100%). Based on the above findings in pathotyping experimental trials with a supportive evidence of histopathological observations, isolate Ind/TN/12/03 was considered as very virulent MDV and other two isolates were considered as virulent MDVs.

Interactions between Marek's disease virus Rispens/CVI988 vaccine strain and adenovirus field strain in chicken embryo fibroblast (CEF) cultures.

The aim of the study was to determine the influence of adenovirus infection on the replication of Marek's disease virus vaccine strain Rispens/CVI988 during in vitro co-infection studies. Adenovirus field strain JN-5/10j was isolated from sick chickens. The study was conducted in chicken embryo fibroblast cultures (CEF). Monolayers of CEFs were infected with Rispens strain and field adenovirus strain JN-5/10j with different doses (10(1.0)-10(3.0) TCID50) in the following manner: a) simultaneously, b) first, infection with Rispens strain and after 24 h infection with adenovirus strain JN-5/10j and c) infection with adenovirus strain JN-5/10j 24 h before infection with Rispens strain. After 18, 24, 48, 72, and 96 h of incubation, the copy number of the pp38 gene of Rispens strain was determined using Real-time PCR. The results indicated that the Adenovirus infection before the infection with Rispens strain reduced the replication of the pp38 gene after 48 h by 2 log10.

Mixed Infections of Marek's Disease and Reticuloendotheliosis Viruses in Layer Flocks in Argentina

The presence of reticuloendotheliosis virus (REV) was examined in flocks affected with Marek's disease (MD). Sera were positive to REV antibodies by agar gel precipitation. However, these findings were not conclusive since fowlpox vaccines can have REV fragments or the whole genome inserted. Frozen sections from tumors were positive for MD virus (MDV) but negative for REV. Chicken embryo fibroblast (CEF) and chicken kidney cell (CKC) culture inoculated with buffy coat cells or blood from the affected birds were examined. Positive cells were shown for REV and MDV by fluorescent antibodies tests in CEF and CKC, respectively, indicating the presence of REV in Argentinean layer flocks. This is the first report of REV in Argentina and also in South America.

Isolation of Mouse Embryo Fibroblasts.

Preparation of primary cultures of embryo fibroblasts from genetically engineered mouse strains can provide a valuable resource for analyzing the consequences of genetic alterations at the cellular level. Mouse embryo fibroblasts (MEFs) have been particularly useful in cancer research, as they have facilitated the identification of the genetic changes that allow cells to overcome senescence and proliferate indefinitely in culture. The immortalized MEFs can then acquire additional mutations that lead to anchorage-independent growth and the ability to form tumors in mice. Recently we developed an MEF model system for analysis of the role of the tumor suppressor gene DLC1 in cellular transformation (Qian et al., 2012). In this communication we describe a protocol for the isolation of MEFs from day 13.5-day 14.5 mouse embryos. The MEFs obtained by this procedure are suitable for use in biochemical assays and for further genetic manipulations.

Effect of Fowl Adenovirus (FAdV-7) Infection on the Replication of Turkey Herpesvirus FC126 in Chicken Embryo Fibroblast Cultures

Abstract The aim of the study was to determine the influence of simultaneous infection of chicken embryo fibroblasts (CEF) with different doses of adenovirus field strain serotype 7 (FAdV-7 JN-5/10j) and turkey herpesvirus strain FC126 (FC126 HVT) on replication of the herpesvirus in in vitro cultures. Three experiments were performed: simultaneous infection of CEF with adenovirus and HVT; inoculation of CEF culture with adenovirus, followed by infection with HVT after 24 h; and inoculation of CEF with HVT, followed by the infection with adenovirus 24 h later. In order to detect the presence of HVT and adenovirus strains in CEF culture, SORF 1 and hexon genes were determined, respectively. The infection with adenovirus lowered replication of FC126 HVT in chicken embryo fibroblast.

Replication and Adaptive Mutations of Low Pathogenic Avian Influenza Viruses in Tracheal Organ Cultures of Different Avian Species

Transmission of avian influenza viruses (AIV) between different avian species may require genome mutations that allow efficient virus replication in a new species and could increase virulence. To study the role of domestic poultry in the evolution of AIV we compared replication of low pathogenic (LP) AIV of subtypes H9N2, H7N7 and H6N8 in tracheal organ cultures (TOC) and primary embryo fibroblast cultures of chicken, turkey, Pekin duck and homing pigeon. Virus strain-dependent and avian species-related differences between LPAIV were observed in growth kinetics and induction of ciliostasis in TOC. In particular, our data demonstrate high susceptibility to LPAIV of turkey TOC contrasted with low susceptibility of homing pigeon TOC. Serial virus passages in the cells of heterologous host species resulted in adaptive mutations in the AIV genome, especially in the receptor-binding site and protease cleavage site of the hemagglutinin. Our data highlight differences in susceptibility of different birds to AIV viruses and emphasizes potential role of poultry in the emergence of new virus variants.

Recombinant UL30 antigen-based single serum dilution ELISA for detection of duck viral enteritis

A recombinant UL30 antigen-based single serum dilution enzyme linked immunosorbent assay (ELISA) was developed to measure specific antibody in the sera of ducks against duck enteritis virus (DEV). The partial UL30 gene of DEV was cloned, expressed, purified and tested for its diagnostic use by designing a single serum dilution enzyme linked immuno-sorbent assay (ELISA). A total of 226 duck sera samples were tested using the assay. A linear relationship was found between the predicted antibody titres at a single working dilution of 1:100 and the corresponding serum titres observed as determined by the standard serial dilution method. Regression analysis was used to determine a standard curve from which an equation was derived which demonstrated this correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay proved to be specific, sensitive and accurate as compared to the virus neutralization test with a specificity, sensitivity and accuracy being 96%, 95% and 95% respectively.