Primary Chicken Kidney Cells Research Articles

Adaptation of the infectious bronchitis virus H120 vaccine strain to Vero cell lines

Infectious bronchitis virus (IBV) has restricted cell and tissue tropism. IBVs, except the Beaudette strain, can infect and replicate in chicken embryos, primary chicken embryo kidneys, and primary chicken kidney cells, only. The limited viral cell tropism of IBV substantially hinders in vitro cell-based research on pathogenic mechanisms and vaccine development. Herein, the parental H120 vaccine strain was serially passaged for five generations in chicken embryos, 20 passages in CK cells and 80 passages in Vero cells. This passaging yielded a Vero cell-adapted strain designated HV80. To further understand viral evolution, serial assessments of infection, replication, and transmission in Vero cells were performed for the viruses obtained every tenth passage. The ability to form syncytia and the replication efficiency significantly after the 50th passage (strain HV50). HV80 also displayed tropism extension to DF-1, BHK-21, HEK-293 T, and HeLa cells. Whole genome sequencing of viruses from every tenth generation revealed a total of 19 amino acid point mutations in the viral genome by passage 80, nine of which occurred in the S gene. The second furin cleavage site appeared in viral evolution and may be associated with cell tropism extension of HV80.

The Genetic Stability, Replication Kinetics and Cytopathogenicity of Recombinant Avian Coronaviruses with a T16A or an A26F Mutation within the E Protein Is Cell-Type Dependent.

The envelope (E) protein of the avian coronavirus infectious bronchitis virus (IBV) is a small-membrane protein present in two forms during infection: a monomer and a pentameric ion channel. Each form has an independent role during replication; the monomer disrupts the secretory pathway, and the pentamer facilitates virion production. The presence of a T16A or A26F mutation within E exclusively generates the pentameric or monomeric form, respectively. We generated two recombinant IBVs (rIBVs) based on the apathogenic molecular clone Beau-R, containing either a T16A or A26F mutation, denoted as BeauR-T16A and BeauR-A26F. The replication and genetic stability of the rIBVs were assessed in several different cell types, including primary and continuous cells, ex vivo tracheal organ cultures (TOCs) and in ovo. Different replication profiles were observed between cell cultures of different origins. BeauR-A26F replicated to a lower level than Beau-R in Vero cells and in ovo but not in DF1, primary chicken kidney (CK) cells or TOCs. Genetic stability and cytopathic effects were found to differ depending on the cell system. The effect of the T16A and A26F mutations appear to be cell-type dependent, which, therefore, highlights the importance of cell type in the investigation of the IBV E protein.

Spectrophotometric microplate assay for titration and neutralization of avian nephritis virus based on the virus cytopathicity

IntroductionPlaque assay (PA) is a gold standard for virus titration and neutralization of various cytopathic viruses, including avian nephritis virus (ANV), the etiological agent associated with kidney disorders in chickens. In this study, as an alternative to the labor-intensive PA, we developed a spectrophotometric microplate assay (MA) for ANV titration and neutralization based on the virus cytopathicity to primary chicken kidney (CK) cells. MethodsCK cells were infected with ANV in the presence or absence of chicken serum in a 96-well microplate, and the virus-induced cytolysis was quantified by measurement of neutral red uptake using a spectrophotometer. The absorbance values obtained were subjected to a sigmoidal four-parameter logistic regression analysis for the virus titer determination and serum neutralization assessment. Accuracy and reliability of the serum neutralization MA in comparison to the standard PA was statistically evaluated. ResultsThe ANV-MA was capable of quantifying infectious virus titers based on a virus dose-dependent cytolysis of CK cells, and serum neutralization could be assessed as an inhibition of the virus-induced cytolysis accordingly. Statistical evaluation using a 2 × 2 contingency table and receiver-operating characteristic analyses showed 82 % sensitivity, 99 % specificity and 0.97 area under the curve, supporting an overall diagnostic accuracy of the neutralization MA. ConclusionThe newly developed MA using simplified experimental procedures in the microplate format and direct spectophotometric data readout is readily applicable to general laboratories for high-throughput screening of serum neutralization of ANV.

Exogenous nitric oxide stimulates early egress of Eimeria tenella sporozoites from primary chicken kidney cells in vitro.

Egress plays a vital role in the life cycle of apicomplexan parasites including Eimeria tenella, which has been attracting attention from various research groups. Many recent studies have focused on early egress induced by immune molecules to develop a new method of apicomplexan parasite elimination. In this study, we investigated whether nitric oxide (NO), an immune molecule produced by different types of cells in response to cytokine stimulation, could induce early egress of eimerian sporozoites in vitro. Eimeria tenella sporozoites were extracted and cultured in primary chicken kidney cells. The number of sporozoites egressed from infected cells was analyzed by flow cytometry after treatment with NO released by sodium nitroferricyanide (II) dihydrate. The results showed that exogenous NO stimulated the rapid egress of E. tenella sporozoites from primary chicken kidney cells before replication of the parasite. We also found that egress was dependent on intra-parasitic calcium ion (Ca2+) levels and no damage occurred to host cells after egress. The virulence of egressed sporozoites was significantly lower than that of fresh sporozoites. The results of this study contribute to a novel field examining the interactions between apicomplexan parasites and their host cells, as well as that of the clearance of intracellular pathogens by the host immune system.

The V617I Substitution in Avian Coronavirus IBV Spike Protein Plays a Crucial Role in Adaptation to Primary Chicken Kidney Cells.

The naturally isolated avian coronavirus infectious bronchitis virus (IBV) generally cannot replicate in chicken kidney (CK) cells. To explore the molecular mechanism of IBV adapting to CK cells, a series of recombinant viruses were constructed by chimerizing the S genes of CK cell-adapted strain H120 and non-adapted strain IBYZ. The results showed that the S2 subunit determines the difference in cell tropism of the two strains. After comparing the amino acid sequences of S protein of CK cell-adapted strain YZ120, with its parental strain IBYZ, three amino acid substitutions, A138V, L581F, and V617I, were identified. Using YZ120 as the backbone, one or more of the above-mentioned substitutions were eliminated to verify the correlation between these sites and CK cell tropism. The results showed that the CK cell tropism of the YZ120 strain depends on the V617I substitution, the change of L581F promoted the adaptation in CK cells, and the change at 138 position was not directly related to the CK cell tropism. Further validation experiments also showed that V617I had a decisive role in the adaptation of IBV to CK cells, but other areas of the virus genome also affected the replication efficiency of the virus in CK cells.

Comparative Analysis of Gene Expression in Virulent and Attenuated Strains of Infectious Bronchitis Virus at Subcodon Resolution.

Like all coronaviruses, avian infectious bronchitis virus (IBV) possesses a long, single-stranded, positive-sense RNA genome (∼27 kb) and has a complex replication strategy that includes the production of a nested set of subgenomic mRNAs (sgmRNAs). Here, we used whole-transcriptome sequencing (RNASeq) and ribosome profiling (RiboSeq) to delineate gene expression in the IBV M41-CK and Beau-R strains at subcodon resolution. RNASeq facilitated a comparative analysis of viral RNA synthesis and revealed two novel transcription junction sites in the attenuated Beau-R strain, one of which would generate a sgmRNA encoding a ribosomally occupied open reading frame (dORF) located downstream of the nucleocapsid coding region. RiboSeq permitted quantification of the translational efficiency of virus gene expression and identified, for the first time, sites of ribosomal pausing on the genome. Quantification of reads flanking the programmed ribosomal frameshifting (PRF) signal at the genomic RNA ORF1a/ORF1b junction revealed that PRF in IBV is highly efficient (33 to 40%). Triplet phasing of RiboSeq data allowed precise determination of reading frames and revealed the translation of two ORFs (ORF4b and ORF4c on sgmRNA IR), which are widely conserved across IBV isolates. Analysis of differential gene expression in infected primary chick kidney cells indicated that the host cell response to IBV occurs primarily at the level of transcription, with global upregulation of immune-related mRNA transcripts following infection and comparatively modest changes in the translation efficiencies of host genes. Cellular genes and gene networks differentially expressed during virus infection were also identified, giving insights into the host cell response to IBV infection.IMPORTANCE IBV is a major avian pathogen and presents a substantial economic burden to the poultry industry. Improved vaccination strategies are urgently needed to curb the global spread of this virus, and the development of suitable vaccine candidates will be aided by an improved understanding of IBV molecular biology. Our high-resolution data have enabled a precise study of transcription and translation in cells infected with both pathogenic and attenuated forms of IBV and expand our understanding of gammacoronaviral gene expression. We demonstrate that gene expression shows considerable intraspecies variation, with single nucleotide polymorphisms being associated with altered production of sgmRNA transcripts, and our RiboSeq data sets enabled us to uncover novel ribosomally occupied ORFs in both strains. The numerous cellular genes and gene networks found to be differentially expressed during virus infection provide insights into the host cell response to IBV infection.

Generation of a recombinant GFP-tagged infectious bronchitis virus (IBV)

Infectious Bronchitis Virus (IBV) is a highly contagious gammacoronavirus which infects poultry. Using reverse genetics, enhanced green fluorescence protein (eGFP) has been inserted into a pathogenic strain of IBV, M41, in place of a newly identified open reading frame (ORF), ORF7. ORF7 has been identified in several IBV strains including M41 and apathogenic strain Beau-R. ORF7 is located immediately downstream of the N gene, preceding the 3’ UTR. This region has been chosen because we are interested in investigating whether a protein can be transcribed from the associated transcription regulatory sequence (TRS-B). M41 has a deletion at the 3’ end of the genome and does not encode all of ORF7, suggesting it is not essential for the viral replication and is not required for a pathogenic phenotype. The TRS-B, TAACA for ORF7 in Beau-R, is non-canonical and is located after the stop codon for the N gene. In M41 only part of the TRS-B is present, TAA, so nucleotides CA were added alongside the eGFP sequence. A number of viruses were successfully rescued. Growth kinetics in primary Chicken Kidney cells (CK) were comparable to the parent virus M41-K. The stability of the eGFP sequence has been assessed in CK cells and in embryonated eggs. The newly constructed TRS-B however was not utilised suggesting that it is not solely the TRS-B that controls the transcription of ORF 7.

Recombinant infectious bronchitis viruses expressing heterologous S1 subunits: potential for a new generation of vaccines that replicate in Vero cells.

The spike glycoprotein (S) of infectious bronchitis virus (IBV) comprises two subunits, S1 and S2. We have previously demonstrated that the S2 subunit of the avirulent Beau-R strain is responsible for its extended cellular tropism for Vero cells. Two recombinant infectious bronchitis viruses (rIBVs) have been generated; the immunogenic S1 subunit is derived from the IBV vaccine strain, H120, or the virulent field strain, QX, within the genetic background of Beau-R. The rIBVs BeauR-H120(S1) and BeauR-QX(S1) are capable of replicating in primary chicken kidney cell cultures and in Vero cells. These results demonstrate that rIBVs are able to express S1 subunits from genetically diverse strains of IBV, which will enable the rational design of a future generation of IBV vaccines that may be grown in Vero cells.

The S2 Subunit of Infectious Bronchitis Virus Beaudette Is a Determinant of Cellular Tropism.

The spike (S) glycoprotein of the avian gammacoronavirus infectious bronchitis virus (IBV) is comprised of two subunits (S1 and S2), has a role in virulence in vivo, and is responsible for cellular tropism in vitro We have previously demonstrated that replacement of the S glycoprotein ectodomain from the avirulent Beaudette strain of IBV with the corresponding region from the virulent M41-CK strain resulted in a recombinant virus, BeauR-M41(S), with the in vitro cell tropism of M41-CK. The IBV Beaudette strain is able to replicate in both primary chick kidney cells and Vero cells, whereas the IBV M41-CK strain replicates in primary cells only. In order to investigate the region of the IBV S responsible for growth in Vero cells, we generated a series of recombinant IBVs expressing chimeric S glycoproteins, consisting of regions from the Beaudette and M41-CK S gene sequences, within the genomic background of Beaudette. The S2, but not the S1, subunit of the Beaudette S was found to confer the ability to grow in Vero cells. Various combinations of Beaudette-specific amino acids were introduced into the S2 subunit of M41 to determine the minimum requirement to confer tropism for growth in Vero cells. The ability of IBV to grow and produce infectious progeny virus in Vero cells was subsequently narrowed down to just 3 amino acids surrounding the S2' cleavage site. Conversely, swapping of the 3 Beaudette-associated amino acids with the corresponding ones from M41 was sufficient to abolish Beaudette growth in Vero cells.IMPORTANCE Infectious bronchitis remains a major problem in the global poultry industry, despite the existence of many different vaccines. IBV vaccines, both live attenuated and inactivated, are currently grown on embryonated hen's eggs, a cumbersome and expensive process due to the fact that most IBV strains do not grow in cultured cells. The reverse genetics system for IBV creates the opportunity for generating rationally designed and more effective vaccines. The observation that IBV Beaudette has the additional tropism for growth on Vero cells also invokes the possibility of generating IBV vaccines produced from cultured cells rather than by the use of embryonated eggs. The regions of the IBV Beaudette S glycoprotein involved in the determination of extended cellular tropism were identified in this study. This information will enable the rational design of a future generation of IBV vaccines that may be grown on Vero cells.

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Establishment of an in vitro chicken epithelial cell line model to investigate Eimeria tenella gamete development

BackgroundEimeria tenella infection leads to acute intestinal disorders responsible for important economic losses in poultry farming worldwide. The life-cycle of E. tenella is monoxenous with the chicken as the exclusive host; infection occurs in caecal epithelial cells. However, in vitro, the complete life-cycle of the parasite has only been propagated successfully in primary chicken kidney cells, which comprise undefined mixed cell populations; no cell line model has been able to consistently support the development of the sexual stages of the parasite. We therefore sought to develop a new model to study E. tenella gametogony in vitro using a recently characterised chicken cell line (CLEC-213) exhibiting an epithelial cell phenotype.MethodsCLEC-213 were infected with sporozoites from a precocious strain or with second generation merozoites (merozoites II) from wild type strains. Sexual stages of the parasite were determined both at the gene and protein levels.ResultsTo our knowledge, we show for the first time in CLEC-213, that sporozoites from a precocious strain of E. tenella were able to develop to gametes, as verified by measuring gene expression and by using antibodies to a microgamete-specific protein (EtFOA1: flagellar outer arm protein 1) and a macrogamete-specific protein (EtGAM-56), but oocysts were not observed. However, both gametes and oocysts were observed when cells were infected with merozoites II from wild type strains, demonstrating that completion of the final steps of the parasite cycle is possible in CLEC-213 cells.ConclusionThe epithelial cell line CLEC-213 constitutes a useful avian tool for studying Eimeria epithelial cell interactions and the effect of drugs on E. tenella invasion, merogony and gametogony.

Protection against Infectious Bronchitis Virus, a Corona Virus Infection, Using Ostrich Antibodies

In chickens, infectious bronchitis (IB) is a major respiratory disease. The respiratory system is the primary multiplication site of IB virus (IBV), a coronavirus, after which the virus is distributed to other organs. Poultry farms sustain considerable economic damage due to IB outbreaks in flocks, since IB causes a severe reduction in weight gain in chicks. In the present study, we produced the ostrich IgY against IBV by immunizing female ostriches with the IB viral antigens. The resultant purified IgY showed a strong neutralizing activity against IBV infection of cultured primary chick kidney cells. The infectivity of IBV was markedly inhibited in the trachea of chicks when ostrich IgY was injected intra-muscularly into newly hatched chicks prior to viral inhalation challenge at two weeks of age. Furthermore, the infection was strongly blocked in the tracheae when IgY was injected into chicks at one day and one week of age, with viral inhalation performed at three weeks of age. These findings suggest that the injection of ostrich IgY can help protect young chicks from IBV infections. In south Asian and African countries, broiler chicks are sent to poultry market around 30 days of age, so it is important to prevent IB outbreaks in very young flocks. We strongly believe that ostrich IgY will be a powerful weapon against IB infection in poultry farms on a wide scale and also hope that these findings will aid in the development of antibody vaccines for new type corona viruses, SARS-CoV and MERS-CoV.

Isolation and purification of Gallid herpesvirus 2 strains currently distributed in Japan.

Gallid herpesvirus 2 (GaHV-2) causes malignant lymphomas in chickens (Marek’s disease, MD). Although MD is controlled through vaccination efforts, field isolates of GaHV-2 have increased in virulence worldwide and even cause MD in vaccinated chickens. GaHV-2 strains are classified into four categories (mild, virulent, very virulent and very virulent +) based on the virulence exhibited in experimental infection in unvaccinated or MD-vaccinated susceptible chickens. Although MD cases are sporadically reported in Japan, the recent field strains of GaHV-2 in Japan have not been characterized. During isolation of recent field strains by using primary chicken kidney cell cultures, a method classically used for GaHV-2 isolation, vaccine strains were simultaneously isolated. Therefore, it is necessary to separate vaccine strains to characterize the virulence and pathogenicity of the GaHV-2 strains currently distributed in Japan. In this study, we prepared cell suspensions from the spleens of MD-symptomatic chickens, inoculated day-old-chicks and isolated GaHV-2 strains by primary chicken kidney cell cultures at 2−3 weeks post inoculation. The isolated strains were passaged several times on chicken embryo fibroblast cells, and PCR analysis revealed that the isolated strains were not contaminated with vaccine strains. Moreover, the contaminant vaccine strains were completely removed by the purification of plaques observed in chicken kidney cells. These procedures are necessary to isolate GaHV-2 field strains from vaccine strains in order to carry out future studies to characterize these strains and glean insights into GaHV-2 virulence and pathogenicity.

Calcium-dependent microneme protein discharge and in vitro egress of Eimeria tenella sporozoites

Egress is a vital step in the endogenous development of apicomplexan parasites, as it assures the parasites exit from consumed host cells and entry into fresh ones. However, little information has previously been reported on this step of Eimeria spp. In this study, we investigated in vitro egress of Eimeria tenella sporozoites triggered by acetaldehyde. We found that addition of exogenous acetaldehyde induces egress of sporozoites from primary chicken kidney cells (PCKs) and stimulate secretion of E. tenella microneme 2 protein (EtMic 2). Moreover, by using cellular calcium inhibitors, we further proved that these processes were dependent on the intracellular calcium of the parasites. Our findings provide clues to the study of interaction between eimerian parasites and their hosts.

Aerosol Disinfection Capacity of Slightly Acidic Hypochlorous Acid Water Towards Newcastle Disease Virus in the Air: An In Vivo Experiment.

Existence of bioaerosol contaminants in farms and outbreaks of some infectious organisms with the ability of transmission by air increase the need for enhancement of biosecurity, especially for the application of aerosol disinfectants. Here we selected slightly acidic hypochlorous acid water (SAHW) as a candidate and evaluated its virucidal efficacy toward a virus in the air. Three-day-old conventional chicks were challenged with 25 doses of Newcastle disease live vaccine (B1 strain) by spray with nebulizer (particle size <3 μm in diameter), while at the same time reverse osmosis water as the control and SAHW containing 50 or 100 parts per million (ppm) free available chlorine in pH 6 were sprayed on the treated chicks with other nebulizers. Exposed chicks were kept in separated cages in an isolator and observed for clinical signs. Oropharyngeal swab samples were collected from 2 to 5 days postexposure from each chick, and then the samples were titrated with primary chicken kidney cells to detect the virus. Cytopathic effects were observed, and a hemagglutination test was performed to confirm the result at 5 days postinoculation. Clinical signs (sneezing) were recorded, and the virus was isolated from the control and 50 ppm treatment groups, while no clinical signs were observed in and no virus was isolated from the 100 ppm treatment group. The virulent Newcastle disease virus (NDV) strain Sato, too, was immediately inactivated by SAHW containing 50 ppm chlorine in the aqueous phase. These data suggest that SAHW containing 100 ppm chlorine can be used for aerosol disinfection of NDV in farms.

Ethanol and isopropanol trigger rapid egress of intracellular Eimeria tenella sporozoites.

Egress from host cells is a vital step of the intracellular life cycle of apicomplexan parasites such as Toxoplasma gondii. This phenomenon has attracted attentions from many research groups. Previous studies have shown that ethanol could stimulate the release of microneme proteins by elevating intracellular Ca(2+) concentration of T. gondii, resulting in the parasite egress from host cells. However, little information about egress is known on Eimeria species, the causative agent of coccidiosis in poultry and livestock. In this report, we studied the effect of ethanol and isopropanol on the egress of eimerian parasites. Eimeria tenella sporozoites cultured in primary chicken kidney cells were treated with ethanol and isopropanol, then the egressed parasites were analyzed. Ethanol and isopropanol could induce the rapid egress of E. tenella sporozoites from host cells. No substantial damage was found in parasite-egressed host cells. Compared to the freshly isolated sporozoites, the re-invading ability and reproductivity of the egressed parasites significantly decreased by 43.4 and 44.1 % individually. We also found that fewer sporozoites egressed from host cells when the parasites developed for a longer time before the alcohol treatment. These results demonstrate an in vitro egress mode different from that of T. gondii, facilitating the deciphering of the mechanisms of egress of eimerian parasites.

Infectious Bronchitis Virus as a Vector for the Expression of Heterologous Genes

The avian coronavirus infectious bronchitis virus (IBV) is the causative agent of the respiratory disease infectious bronchitis of domestic fowl, and is controlled by routine vaccination. To explore the potential use of IBV as a vaccine vector a reverse genetics system was utilised to generate infectious recombinant IBVs (rIBVs) expressing the reporter genes enhanced green fluorescent protein (eGFP) or humanised Renilla luciferase (hRluc). Infectious rIBVs were obtained following the replacement of Gene 5 or the intergenic region (IR) with eGFP or hRluc, or the replacement of ORFs 3a and 3b with hRluc. The replacement of Gene 5 with an IBV codon-optimised version of the hRluc gene also resulted in successful rescue of infectious rIBV. Reporter gene expression was confirmed by fluorescence microscopy, or luciferase activity assays, for all successfully rescued rIBVs following infection of primary chick kidney (CK) cells. The genetic stability of rIBVs was analysed by serial passage on CK cells. Recombinant IBV stability varied depending on the genome region being replaced, with the reporter genes maintained up to at least passage 8 (P8) following replacement of Gene 5, P7 for replacement of the IR and P5 for replacement of ORFs 3a and 3b. Codon-optimisation of the hRluc gene, when replacing Gene 5, resulted in an increase in genome stability, with hRluc expression stable up to P10 compared to P8 for standard hRluc. Repeated passaging of rIBVs expressing hRluc at an MOI of 0.01 demonstrated an increase in stability, with hRluc expression stable up to at least P12 following the replacement of Gene 5. This study has demonstrated that heterologous genes can be incorporated into, and expressed from a range of IBV genome locations and that replacement of accessory Gene 5 offers a promising target for realising the potential of IBV as a vaccine vector for other avian pathogens.

Visualizing the autophagy pathway in avian cells and its application to studying infectious bronchitis virus

Autophagy is a highly conserved cellular response to starvation that leads to the degradation of organelles and long-lived proteins in lysosomes and is important for cellular homeostasis, tissue development and as a defense against aggregated proteins, damaged organelles and infectious agents. Although autophagy has been studied in many animal species, reagents to study autophagy in avian systems are lacking. Microtubule-associated protein 1 light chain 3 (MAP1LC3/LC3) is an important marker for autophagy and is used to follow autophagosome formation. Here we report the cloning of avian LC3 paralogs A, B and C from the domestic chicken, Gallus gallus domesticus, and the production of replication-deficient, recombinant adenovirus vectors expressing these avian LC3s tagged with EGFP and FLAG-mCherry. An additional recombinant adenovirus expressing EGFP-tagged LC3B containing a G120A mutation was also generated. These vectors can be used as tools to visualize autophagosome formation and fusion with endosomes/lysosomes in avian cells and provide a valuable resource for studying autophagy in avian cells. We have used them to study autophagy during replication of infectious bronchitis virus (IBV). IBV induced autophagic signaling in mammalian Vero cells but not primary avian chick kidney cells or the avian DF1 cell line. Furthermore, induction or inhibition of autophagy did not affect IBV replication, suggesting that classical autophagy may not be important for virus replication. However, expression of IBV nonstructural protein 6 alone did induce autophagic signaling in avian cells, as seen previously in mammalian cells. This may suggest that IBV can inhibit or control autophagy in avian cells, although IBV did not appear to inhibit autophagy induced by starvation or rapamycin treatment.

Biology of fowl adenovirus type 1 infection of heterologous cells

The JM1/1 strain of fowl adenovirus (FAV) serotype 1 isolated from gizzard erosion was used to investigate the biology of FAV in homologous (susceptible) and heterologous cells. The FAV JM1/1 strain is capable of efficient multiplication in primary chicken kidney (CK) cells, but not in Crandell-Rees feline kidney (CRFK) cells or Vero cells. FAV adsorption in heterologous cells was slightly higher than in CK cells. An early gene encoding a DNA-binding protein and a late gene encoding the hexon protein were expressed in CK cells. Only the early gene was expressed in Vero cells. Neither of these genes was expressed in CRFK cells. These results suggest that the virus was unable to multiply effectively due to suppression of viral gene expression in the heterologous cells used in this study.

Enhanced Egress of Intracellular Eimeria tenella Sporozoites by Splenic Lymphocytes from Coccidian-Infected Chickens

Egress, which describes the mechanism that some intracellular parasites use to exit from parasitophorous vacuoles and host cells, plays a very important role in the parasite life cycle and is central to Eimeria propagation and pathogenesis. Despite the importance of egress in the intracellular parasite's life cycle, very little information is known on this process compared to other steps, e.g., invasion. The present study was conducted to investigate the interplay between the host adaptive immune system and Eimeria egression. Splenic lymphocytes or soluble immune factors were incubated with parasite-infected host cells for 3 or 5 h, and the percentage of egress was calculated according to an established formula. Viability of egressed parasites and host cells was tested using trypan blue exclusion and annexin V and propidium iodide staining, respectively. We found that premature egression of sporozoites from Eimeria tenella-infected primary chicken kidney cells or from chicken peripheral blood mononuclear cells occurred when the cells were cocultured in vitro with spleen lymphocytes from E. tenella-infected chickens but not when they were cocultured with splenocytes from uninfected chickens. Eimeria-specific antibodies and cytokines (gamma interferon [IFN-γ], interleukin-2 [IL-2], and IL-15), derived from E. tenella-primed B and T lymphocytes, respectively, were capable of promoting premature egress of sporozoites from infected host cells. Both egressed parasites and host cells were viable, although the latter showed reduced reinvasion ability. These results suggest a novel, immune-mediated mechanism that the host exploits to interrupt the normal Eimeria life cycle in vivo and thereby block the release of mature parasites into the environment.