Chicken 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

Inhibitory Activity of Illicium verum Extracts against Avian Viruses.

This study aimed at screening the inhibitory activity of Illicium verum extracts against avian reovirus, infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), and infectious laryngotracheitis virus (ILTV). The cytotoxic and antiviral actions of 3 extracts, absolute methanol (100MOH), 50% methanol (50MOH), and aqueous extracts (WA.), were evaluated by MTT assay. The Illicium verum extracts were added to the cultured chick embryo fibroblast (CEF) with tested viruses in three attacks, preinoculation, postinoculation, and simultaneous inoculation. The three extracts showed antiviral inhibitory activity against all tested viruses during simultaneous inoculation and preinoculation except 100MOH and 50MOH that showed no effect against IBDV, thereby suggesting that the extracts have a preventive effect on CEF against viruses. During postinoculation, the extracts exhibited inhibitory effects against NDV and avian reovirus, while no effect against IBDV recorded and only the 100MOH showed an inhibitory effect against ILTV. The initial results of this study suggest that Illicium verum may be a candidate for a natural alternative source for antiviral agents.

Explore the potential effect of natural herbals to resist Newcastle Disease Virus

In this study, we examined the dose-dependent effects of the formula on Newcastle disease virus (NDV). In in-vitro test, the formula within safety concentration scope and NDV were added into cultured chick embryo fibroblast in 3 modes, and the cellular A570 values were determined by MTT (3-(4, 5-dimethyithiazol-2-yl)-2, 5-diphenyltetrazolium bromide) method. In in-vivo test, we examined the expression of interferon-induced transmembrane protein 3 (IFITM3) and Interferons (IFNs) in NDV-infected chickens. The results showed that the highest virus inhibitory rates of the formula at optimal concentration group were the highest (15.625 mg/mL) in post-adding and simultaneous-adding drug and virus modes, whereas medium concentration (7.813 mg/mL) showed the highest virus inhibitory rates in pre-adding drug mode. In vivo, the formula significantly upregulated the expression of IFITM3 in NDV-infected chickens at 3-D post-infection. However, the levels of IFNs were significantly downregulated. On days 5 and 7 post-infection, the levels of IFNs quickly upregulated. Moreover, the formula can significantly upregulate the antibody to resist the NDV compared with model control group on days 5 and 7 post-infection. In animals treated with the formula, the survival rate was nearly 37% higher at 7 d post-infection. We also found that the formula had a significantly stronger effect than a single herb on upregulating the expression of IFITM3. It confirmed that the formula could significantly inhibit the infectivity of NDV to chick embryo fibroblast. Also, the formula could significantly upregulated IFITM3 expression and inhibited virus replication in NDV-infected chickens. During the early stage of infection, IFNs were consumed to stimulate IFITM3 to inhibit virus replication, whereas during later stages of the infection, the formula upregulated the levels of IFNs and their antibodies to maintain a high level of immunity.

Integration of ALV into CTDSPL and CTDSPL2 genes in B-cell lymphomas promotes cell immortalization, migration and survival.

Avian leukosis virus induces tumors in chickens by integrating into the genome and altering expression of nearby genes. Thus, ALV can be used as an insertional mutagenesis tool to identify novel genes involved in tumorigenesis. Deep sequencing analysis of viral integration sites has identified CTDSPL and CTDSPL2 as common integration sites in ALV-induced B-cell lymphomas, suggesting a potential role in driving oncogenesis. We show that in tumors with integrations in these genes, the viral promoter is driving the expression of a truncated fusion transcript. Overexpression in cultured chick embryo fibroblasts reveals that CTDSPL and CTDSPL2 have oncogenic properties, including promoting cell migration. We also show that CTDSPL2 has a previously uncharacterized role in protecting cells from apoptosis induced by oxidative stress. Further, the truncated viral fusion transcripts of both CTDSPL and CTDSPL2 promote immortalization in primary cell culture.

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.

Recombinant Marek's Disease Virus as a Vector-Based Vaccine against Avian Leukosis Virus Subgroup J in Chicken.

Avian leukosis virus subgroup J (ALV-J) is an immunosuppressive virus that causes considerable economic losses to the chicken industry in China. However, there is currently no effective vaccine to prevent ALV-J infection. In order to reduce the losses caused by ALV-J, we constructed two effective ALV-J vaccines by inserting the ALV-J (strain JL093-1) env or gag+env genes into the US2 gene of the Marek’s disease herpesviruses (MDV) by transfection of overlapping fosmid DNAs, creating two recombinant MDVs, rMDV/ALV-gag+env and rMDV/ALV-env. Analysis of cultured chicken embryo fibroblasts infected with the rMDVs revealed that Env and Gag were successfully expressed and that there was no difference in growth kinetics in cells infected with rMDVs compared with that of cells infected with the parent MDV. Chickens vaccinated with either rMDV revealed that positive serum antibodies were induced. Both rMDVs also effectively reduced the rate of positive viremia in chicken flocks challenged with ALV-J. The protective effect provided by rMDV/ALV-env inoculation was slightly stronger than that provided by rMDV/ALV-gag+env. This represents the first study where a potential rMDV vaccine, expressing ALV-J antigenic genes, has been shown to be effective in the prevention of ALV-J. Our study also opens new avenues for the control of MDV and ALV-J co-infection.

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.

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.

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.

Construction of recombinant Marek's disease virus (MDV) lacking the meq oncogene and co-expressing AIV-H9N2 HA and NA genes under control of exogenous promoters

To develop a recombinant Marek's disease virus (rMDV1) co-expressing the hemagglutinin gene (HA) and neuramidinase gene (NA) from a low pathogenic avian influenza virus (LPAIV) H9N2 strain and lacking the meq oncogene that shares homology with the Jun/Fos family of transcriptional factors, a wild strain of MDV GX0101 was used as parental virus, the HA and NA genes co-expression cassette under control of the CMV and SV40 early promoters was inserted at two meq sites of GX0101 to form a new meq knock-out mutant MDV (MZC12HA/NA) through homologous recombination. MZC12HA/NA was reconstituted by transfection of recombinant BAC–MDV DNA into the secondary chicken embryo fibroblast (CEF) cells. Highly purified MZC12HA/NA was obtained after four rounds of plaque purification and proliferation. In vitro growth properties of recombinant virus were also inspected and concluded that the MZC12HA/NA had the same growth kinetics in CEF cultures as its parental wild type virus GX0101. Southern blot indicated that co-expression cassette was successfully inserted at two copies sites of meq gene, so two meq genes were knocked-out completely. RT-qPCR showed transcription and expression levels of the HA and NA genes were both significantly higher than that of GX0101 own pp38 gene. Indirect fluorescence antibody (IFA) test, and Western blot analyses indicated that HA and NA genes were co-expressed simultaneously under control of the different promoters but meq genes were not. These results herald a new and effective recombinant meq-deleted MDV-based AIV-H9N2 vaccine may be useful in protecting chickens from very virulent MDV and H9N2 challenges.

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.

Zea mays leaf extracts inhibits etoposide-induced apoptosis in primary chick embryo fibroblasts

Chemotherapy drugs act on normal cells as well as cancer cells. Therefore, in the present study, the extent of cell death induced by etoposide-induced oxidative stress and the role of Zea mays leaf extracts were followed in primary cultured chick embryo fibroblasts (normal cells). Various apoptosis related parameters like cell viability, morphological changes, nuclear changes and apoptotic index were characterized. SRB and MTT assays were used to quantify the extent of cell death in the group exposed to etoposide, plant extracts and their combination. The treatment with etoposide exhibited cytotoxity in the primary chick embryo fibroblasts cells. The number of apoptotic cells increased in the oxidant treated groups. When administered along with etoposide, the leaf extracts resulted in a significantly decreased number of apoptotic cells. The maximum inhibition of etoposide-induced apoptosis was exhibited by the methanolic extract followed by the aqueous and chloroform extracts. Zea mays leaf extracts reduced the toxicity of etoposide in the normal cells and the leaf extracts, by themselves, did not cause any damage to normal cells when treated alone. These results indicate that the Zea mays leaves can render protection to chick embryo fibroblasts against etoposide-induced cell death.

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.

Zea mays leaf extracts protects primary chick embryo fibroblast cells from apoptosis induced by hydrogen peroxide in vitro

BackgroundIt is known that excessive oxidative damage, when unchecked, can result in cell death. Therefore, in the present study, the extent of cell death induced by H2O2-induced oxidative stress in primary cultured chick embryo fibroblasts and its protection by Zea mays leaf extracts was followed. MethodsVarious apoptosis related parameters like cell viability, morphological changes, nuclear changes and apoptotic index were characterized. SRB and MTT assays were used to quantify the extent of cell death in the group exposed to H2O2, plant extracts and their combination. Results and discussionThe treatment with hydrogen peroxide exhibited cytotoxicity in the primary chick embryo fibroblasts cells. The number of apoptotic cells increased in the oxidant treated groups. When administered along with H2O2, the leaf extracts resulted in a significantly decreased number of apoptotic cells. The maximum inhibition of H2O2-induced apoptosis was exhibited by the methanolic extract followed by the aqueous and chloroform extracts. ConclusionThese results indicate that the Zea mays leaves can render protection to chick embryo fibroblasts against H2O2-induced cell death.

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.

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.

Novel microRNAs encoded by duck enteritis virus

Duck enteritis virus (DEV) is an important herpesvirus pathogen associated with acute, highly contagious lethal disease in waterfowls. Using a deep sequencing approach on RNA from infected chicken embryo fibroblast cultures, we identified several novel DEV-encoded micro (mi)RNAs. Unlike most mardivirus-encoded miRNAs, DEV-encoded miRNAs mapped mostly to the unique long region of the genome. The precursors of DEV miR-D18 and miR-D19 overlapped with each other, suggesting similarities to miRNA-offset RNAs, although only the DEV-miR-D18-3p was functional in reporter assays. Identification of these novel miRNAs will add to the growing list of virus-encoded miRNAs enabling the exploration of their roles in pathogenesis.