Targeted infection of endothelial cells by avian influenza virus A/FPV/Rostock/34 (H7N1) in chicken embryos

The tissue tropism and spread of infection of the highly pathogenic avian influenza virus A/FPV/Rostock/34 (H7N1) (FPV) were analyzed in 11-day-old chicken embryos. As shown by in situ hybridization, the virus caused generalized infection that was strictly confined to endothelial cells in all organs. Studies with reassortants of FPV and the apathogenic avian strain A/chick/Germany/N/49 (H10N7) revealed that endotheliotropism was linked to FPV hemagglutinin (HA). To further analyze the factors determining endotheliotropism, the HA-activating protease furin was cloned from chicken tissue. Ubiquitous expression of furin and other proprotein convertases in the chick embryo indicated that proteolytic activation of HA was not responsible for restriction of infection to the endothelium. To determine the expression of virus receptors in embryonic tissues, histochemical analysis of alpha2,3- and alpha2,6-linked neuraminic acid was carried out by lectin-binding assays. These receptors were found on endothelial cells and on several epithelial cells, but not on tissues surrounding endothelia. Finally, we analyzed the polarity of virus maturation in endothelial cells. Studies on cultured human endothelial cells employing confocal laser scanning microscopy revealed that HA is specifically targeted to the apical surface of these cells, and electron microscopy of embryonic tissues showed that virus maturation occurs also at the luminar side. Taken together, these observations indicate that endotheliotropism of FPV in the chicken embryo is determined, on one hand, by the high cleavability of HA, which mediates virus entry into the vascular system, and, on the other hand, by restricted receptor expression and polar budding, which prevent spread of infection into tissues surrounding endothelia.     

Expression of endogenous avian myeloblastosis virus information in different chicken cells.

Uninfected chicken cells were found to contain endogenous avian myeloblastosis virus (AMV)-specific information. Different tissues from chicken embryos and chickens expressed different amounts of the AMV-specific information. The endogenous AMV-related RNA was most abundant in bone marrow cells, which contained about 20 copies per cell. About 5 to 10 copies of AMV endogenous RNA per cell were found in embryonic yolk sac cells and bursa cells. The spleen, muscle, liver, and kidney cells of chickens and the fibroblasts of chicken embryos contained about two copies per cell. The amounts of AMV endogenous RNA in bone marrow, yolk sac, and bursa varied with age. From 19-day-old embryos to 2-week-old chickens, the bone marrow contained 20 copies of AMV RNA per cell. Bone marrow cells from 2-year-old chickens contained five copies per cell. Yolk sac cells of 10-day-old embryos and 1-day-old chickens were found to contain two copies per cell, whereas in 15- to 17-day-old embryos, these cells contained 5 to 10 copies. These results indicate that the level of endogenous AMV expression correlates with the development of granulopoiesis of the chicken hemopoietic system. The results of experiments on the thermostability of RNA-DNA hybrids indicated that the endogenous AMV RNA is closely related to viral AMV RNA. The expression of endogenous AMV information is independent of the activity of the chick helper factor. This endogenous AMV information is expressed as 20 to 21S RNA in both bone marrow and yolk sac cells.     

A recombinant Newcastle disease virus (NDV) expressing VP2 protein of infectious bursal disease virus (IBDV) protects against NDV and IBDV

Infectious bursal disease virus (IBDV) causes a highly immunosuppressive disease in chickens. Currently available, live IBDV vaccines can lead to generation of variant viruses. We have developed an alternative vaccine that will not create variant IBDV. By using the reverse genetics approach, we devised a recombinant Newcastle disease virus (NDV) vector from a commonly used vaccine strain LaSota to express the host-protective immunogen VP2 of a variant IBDV strain GLS-5. The gene encoding the VP2 protein of the IBDV was inserted into the most 3'-proximal locus of a full-length NDV cDNA for high-level expression. We successfully recovered the recombinant virus, rLaSota/VP2. The rLaSota/VP2 was genetically stable, at least up to 12 serial passages in chicken embryos, and was shown to express the VP2 protein. The VP2 protein was not incorporated into the virions of recombinant virus. Recombinant rLaSota/VP2 replicated to a titer similar to that of parental NDV strain LaSota in chicken embryos and cell cultures. To assess protective efficacy of the rLaSota/VP2, 2-day-old specific-pathogen-free chickens were vaccinated with the recombinant virus and challenged with a highly virulent NDV strain Texas GB or IBDV variant strain GLS-5 at 3 weeks postvaccination. Vaccination with rLaSota/VP2 generated antibody responses against both NDV and IBDV and provided 90% protection against NDV and IBDV. Booster immunization induced higher levels of antibody responses against both NDV and IBDV and conferred complete protection against both viruses. These results indicate that the recombinant NDV can be used as a vaccine vector for other avian pathogens.     

The composition of liver chromatin proteins from embryos, chicks and adult chickens

The chemical composition of chromatin from the livers of 12-, 15- and 19-day-old embryos, of 1-day-old chicks and of adult chickens was analysed. The process of embryonic development is accompanied by an increase in non-histone chromatin proteins and chromatin RNA, as well as in the phosphorus content of chromatin phosphoproteins. The amount of these components decreases in the livers of 1-day-old chicks and adults. Two-dimensional polyacrylamide gel electrophoresis of acid-soluble chromatin proteins showed an increase in the amount of the H1 histone in 19-day-old embryos and adult chickens. Non-histone proteins of embryo liver chromatin showed a high content of the fraction of Mr of about 40 000; this was not the case for adult chickens. The non-histone protein fraction of Mr of about 120 000, characteristic of adult chicken liver proteins, was not found in the livers of 12- and 15-day-old embryos. Non-histone chromatin proteins isolated from the livers of animals of different age exhibited also quantitative differences.     

Fowlpox virus encodes a Bcl-2 homologue that protects cells from apoptotic death through interaction with the proapoptotic protein Bak

Poxviruses are renowned for encoding numerous immunomodulatory proteins capable of undermining potent immune defenses. One effective barrier against infection is apoptosis, a process controlled at the mitochondria by pro- and antiapoptotic members of the highly conserved Bcl-2 family of proteins. Although poxviruses are known to encode an array of effective inhibitors of apoptosis, members of the Avipoxvirus genus, which includes fowlpox virus, encode proteins with Bcl-2 homology. Here, we show that FPV039, a fowlpox virus protein with limited Bcl-2 homology, inhibited apoptosis in response to a variety of cytotoxic stimuli, including virus infection itself. Similar to other antiapoptotic Bcl-2 proteins, FPV039 localized predominantly to the mitochondria in both human and chicken cells and protected human cells from tumor necrosis factor alpha-induced loss of the mitochondrial membrane potential. In addition, coimmunoprecipitation revealed that FPV039 interacted constitutively with the proapoptotic Bcl-2 protein, Bak, in both human and chicken cells. Concordantly, FPV039 also inhibited apoptosis induced by the transient overexpression of Bak. To confirm these results in the context of virus infection, we generated a recombinant vaccinia virus lacking F1L, the endogenous apoptotic inhibitor in vaccinia virus, and expressing FPV039. In the context of vaccinia virus infection, FPV039 retained the ability to localize to the mitochondria and interacted with Bak. Moreover, FPV039 prevented the activation of Bak and protected infected cells from apoptosis induced by staurosporine and virus infection. Together, our data indicate that FPV039 is a functional Bcl-2 homologue that inhibits apoptosis by neutralizing the proapoptotic Bcl-2 family member Bak.     

利用Cre-loxP自动敲除H5亚型禽流感病毒血凝素重组鸡痘病毒中的报告基因

研究去除重组鸡痘病毒中的报告基因,构建一株只含目的基因的重组毒。将H5亚型AIV的HA基因作为靶基因,两侧含loxP序列的GFP表达盒插入鸡痘病毒重组臂基因构建了转移质粒载体,将其与脂质体混合转染CEF细胞,获得了表达H5和GFP的鸡痘病毒重组体。通过二次转染,利用Cre酶自动敲除重组病毒中的GFP基因,最终获得了只含H5血凝素基因表达盒的重组鸡痘病毒。免疫荧光和病毒滴度测定结果表明,经过连续传代后重组病毒仍然稳定复制并表达H5血凝素。用10⁵PFU和2×10⁵PFU rFPV H5免疫SPF鸡,28d后,免疫组鸡抗体平均滴度(HI)分别达到4log 2和4.5log 2,结果表明,H5HA基因重组病毒能刺激鸡群产生较高特异抗体。     

鸡痘病毒复制非必需区的选择对重组鸡痘病毒免疫效力的影响

母源抗体的干扰是重组鸡痘病毒(FPV)活载体疫苗至今未能得到大规模推广应用的主要原因,而选择适当的FPV复制非必需区可能是解决这一问题的方法之一。根据已发表的美国致病株FPV的基因组设计两对引物,用PCR方法扩增FPV假定复制非必需区的两个侧翼区FPV1和FPV2 ,利用此假定复制非必需区构建FPV表达载体pP12LS及表达ZJ1株新城疫病毒(NDV)F基因的转移载体pP12LSF。pP12LSF与2 82E4株FPV共转染鸡胚成纤维细胞(CEF) ,经数轮蓝斑筛选得到纯化的重组病毒rFPV_FSC。重组FPV在CEF上连续传2 0代仍具有良好的遗传稳定性。对重组FPV进行免疫效力试验,在SPF鸡上,重组病毒rFPV_FSC和与之仅有复制非必需区差异的rFPV_FSB均能抵抗NDV强毒的攻击,提供10 0 %的保护。但在有母源抗体的商品鸡上,rFPV_FSC与rFPV_FSB的免疫效力却有显著差异,保护率分别为10 0 %和6 1 5 4 % ,rFPV_FSC的免疫效力与NDV常规油苗相当。试验结果表明,母源抗体对重组FPV的免疫效力有一定的影响,而选择合适的FPV复制非必需区是克服母源抗体并提高重组FPV免疫效力的有效策略之一     

Fowlpox virus recombinants expressing the envelope glycoprotein of an avian reticuloendotheliosis retrovirus induce neutralizing antibodies and reduce viremia in chickens.

Eight stable fowlpox virus (FPV) recombinants which express the envelope glycoprotein of the spleen necrosis virus (SNV) strain of reticuloendotheliosis virus (REV), an avian retrovirus, were constructed. These recombinants differ in the genomic location of the inserted genes, in the orientation of the insert relative to flanking viral sequences, and in the promoter used to drive expression of the env gene. Of these variables, promoter strength seems to be the most crucial. The P7.5 promoter of vaccinia virus, which is commonly used in the construction of both vaccinia virus and FPV recombinants, resulted in lower levels of expression of the envelope antigen in infected chicken cells compared with a strong synthetic promoter, as determined by immunofluorescence and enzyme-linked immunosorbent assay. Two peptides encoded by the env gene, the 21-kDa transmembrane peptide and a 62-kDa precursor, were detected by immunoprecipitation of labeled proteins from cells infected with recombinant FPVs, using monoclonal antibodies against REV. These peptides comigrated with those precipitated from REV-infected cells. One of the recombinants (f29R-SNenv) was used for vaccination of 1-day-old chickens. Vaccinated chicks developed neutralizing antibodies to SNV more rapidly than did unvaccinated controls following SNV challenge and were protected against both viremia and the SNV-induced runting syndrome.     

Adrenal inhibition of corticotropin-releasing hormone-induced thyrotropin release: a comparative study in pre- and posthatch chicks

Recent evidence indicates that corticotropin-releasing hormone (CRH) acts as a potent stimulator of thyrotropin (TSH) release in the chicken. In this study adrenal and thyroidal feedback mechanisms were studied. Administration of corticosterone 30 min prior to an ovine CRH (oCRH) challenge diminished the in vivo sensitivity of thyrotrophs to oCRH in 19-day-old chicken embryos (E19) (20 micrograms corticosterone; 2 micrograms oCRH) but not in 8-day-old chickens (C8) (40 micrograms corticosterone; 4 micrograms oCRH). At both ages studied, corticosterone (0.01 and 1 microM) did not alter the in vitro TSH response to oCRH (100 nM) indicating that an indirect mechanism is involved at the embryonic stage which is no longer present in posthatch chickens. In vitro, 3,5,3'-triiodothyronine (T3) pretreatment (0.01 and 1 microM) resulted at both ages studied in a dose-dependent drop in the in vitro oCRH-induced TSH release. As recorded previously, corticosterone treatment provoked a rise in plasma T3 in embryonic but not in posthatch chickens. The presence of an indirect adrenal feedback mechanism in chicken embryos may therefore be linked to the increase in plasma T3 which will alter the sensitivity of thyrotrophs to hypothalamic releasing factors. In conclusion, corticosterone does not directly modulate the responsiveness of thyrotrophs to CRH, but its feedback mechanism may be dependent on the evoked increase in plasma T3 which is only present in embryonic chickens. Corticosterone may in this regard play an essential role during embryonic development by coordinating thyroidal feedback mechanisms at the level of the chicken pituitary.     

NS1 Protein of Influenza A Virus Down-Regulates Apoptosis

Wild-type (WT) influenza A/PR/8/34 virus and its variant lacking the NS1 gene (delNS1) have been compared for their ability to mediate apoptosis in cultured cells and chicken embryos. Cell morphology, fragmentation of chromatin DNA, and caspase-dependent cleavage of the viral NP protein have been used as markers for apoptosis. Another marker was caspase cleavage of the viral M2 protein, which was also found to occur in an apoptosis-specific manner. In interferon (IFN)-competent host systems, such as MDCK cells, chicken fibroblasts, and 7-day-old chicken embryos, delNS1 virus induced apoptosis more rapidly and more efficiently than WT virus. As a consequence, delNS1 virus was also more lethal for chicken embryos than WT virus. In IFN-deficient Vero cells, however, apoptosis was delayed and developed with similar intensity after infection with both viruses. Taken together, these data indicate that the IFN antagonistic NS1 protein of influenza A viruses has IFN-dependent antiapoptotic potential.     

Characterization of poxviruses from forest birds in Hawaii

Two strains of avian pox viruses were isolated from cutaneous lesions in Hawaiian crows (Corvus hawaiiensis) examined in 1994 and a third from a biopsy obtained in 1992 from an infected bird of the Apapane species (Himatione sanguinea) by inoculation of the chorioallantoic membranes (CAM) of developing chicken embryos. The resulting proliferative CAM lesions contained eosinophilic cytoplasmic inclusion bodies characteristic of pox virus infection. The pathogenicity of these three viruses in domestic chickens was mild as evidenced by the development of relatively minor lesions of short duration at the sites of inoculation. Their virulence in this host was similar to that of a fowlpox virus (FPV) vaccine strain and contrasted greatly with the ability of two field strains of FPV to produce extensive proliferative lesions. One of the Hawaiian crow pox virus isolates as well as the one originating from the Apapane species could be propagated in two secondary avian cell lines, QT-35 and LMH. A comparison of the restriction fragment length polymorphisms (RFLP) of the genomes of the two cell line-adapted viruses, generated by EcoRI digestion, revealed a limited degree of similarity. Moreover, neither profile was comparable to those of the two field isolates of FPV, which were almost indistinguishable from each other. Thus, based on the genetic distinctness of the two Hawaiian bird viruses, they appear to represent different strains of avipoxvirus.     

PRODUCTION OF GERMLINE CHIMERIC CHICKENS BY TRANSFER OF CULTURED PRIMORDIAL GERM CELLS

Primordial germ cells (PGCs) from stage 27 (5.5-day-old) Korean native ogol chicken embryonic germinal ridges were cultured in vitro for 5 days. As in in vivo culture, these cultured PGCs were expected to have already passed beyond the migration stage. Approximately 200 of these PGCs were transferred into 2.5-day-old white leghorn embryonic blood stream, and then the recipient embryos were incubated until hatching. The rate of hatching was 58.8% in the manipulated eggs. Six out of 60 recipients were identified as germline chimeric chickens by their feather colour. The frequency of germline transmission of donor PGCs was 1.3–3.1% regardless of sex. The stage 27 PGCs will be very useful for collecting large numbers of PGCs, handling of exogenous DNA transfection during culture, and for the production of desired transgenic chickens.     

Comparison of 2-day-old and 14-day-old chicken colonization models for Campylobacter jejuni

In this study, we compared two types of chicken infection models for Campylobacter jejuni in terms of infectious dose required to colonize the chickens and the susceptibility of chickens of different ages to persistent colonization by C. jejuni. In one model, chickens at day 2 posthatching were used, and in the other, 14-day-old chickens were used. The minimum C. jejuni cell number required to colonize 14-day-old chickens was 5 x 10(4) cells, and that for 2-day-old chickens was 5 x 10(3). The ability of various C. jejuni strains to colonize the chicken gastrointestinal tract was the same in both models.     

A chicken embryo model for the maintenance and amplification of Cryptosporidium parvum and Cryptosporidium baileyi oocysts

Cryptosporidium is a genus of apicomplexan parasites that inhabit the respiratory and gastrointestinal tracts of vertebrates. Research of these parasites is limited by a lack of model hosts. This study aimed to determine the extent to which infection at the embryo stage can enhance the propagation of Cryptosporidium oocysts in chickens. Nine-day-old chicken embryos and one-day-old chickens were experimentally infected with different doses of Cryptosporidium baileyi and Cryptosporidium parvum oocysts. Post hatching, all chickens had demonstrable infections, and the infection dose had no effect on the course of infection. Chickens infected as embryos shed oocysts immediately after hatching and shed significantly more oocysts over the course of the infection than chickens infected as one-day-olds. In chickens infected as embryos, C. baileyi was found in all organs except the brain whereas, C. parvum was only found in the gastrointestinal tract and trachea. In chickens infected as one-day-olds, C. baileyi was only found in the gastrointestinal tract and trachea. Chickens infected as embryos with C. baileyi died within 16 days of hatching. All other chickens cleared the infection. Infection of chickens as embryos could be used as an effective and simple model for the propagation of C. baileyi and C. parvum.     

表达新城疫病毒F48E8株融合蛋白基因的重组鸡痘病毒及其免？…

将将城疫病毒（ＮＤＶ）Ｆ４８Ｅ８株融合蛋白基因导入鸡痘病毒（ＦＰＶ）插入载体ｐＥＧＦ１１７５－１的Ｐ７．５启动子下游,得到转移载体ｐＦＧ１１７５－１重组质粒。采用脂质体转染技术,将该质粒转染ＦＰＶ２８２Ｅ株感染的鸡胚成纤维细胞（ＣＥＦ）。,经过多次蓝斑筛选纯化,获稳定的重组病毒ｒＦＰＶ－ＮＤＦ。间接免疫荧光试验表明,ｒＦＰＶ－ＮＤＦ感染的ＣＥＦ中表达了ＮＤＶ的融合蛋白。用ｒＦＰＶ－ＮＤＦ免疫的ＳＦ     

Cultivation of Cryptosporidium baileyi: studies with cell cultures, avian embryos, and pathogenicity of chicken embryo-passaged oocysts

Sporozoites of Cryptosporidium baileyi did not undergo development in primary cell cultures from either avian or mammalian hosts, or in mammalian cell lines. Oocysts of C. baileyi produced infections resulting in complete development to sporulated oocysts in chicken embryos and embryos of 8 other avian species examined. Inoculation of 4 X 10(5) oocysts was not pathogenic for avian embryos as evidenced by the lack of gross lesions or death. Oocysts obtained after C. baileyi had been passaged 10 times (first experiment) or 20 times (second experiment) in chicken embryos still caused clinical respiratory disease and gross airsacculitis when inoculated intratracheally into 2-day-old broiler chickens. Oocysts that had been passaged 10 times in chicken embryos were similarly pathogenic for 4-day-old turkeys after intratracheal inoculation.     

网状内皮增生病病毒感染SPF鸡对疫苗免疫反应的抑制作用

我国鸡群中网状内皮增生病病毒(REV)感染已相当普遍,但对其造成的实际危害却不太清楚。本研究结果表明,1日龄SPF鸡感染REV会显著抑制对新城疫病毒(NDV)和禽流感病毒(AIV,H5和H9)疫苗的免疫反应。1周龄用相应灭活疫苗免疫后3周、4周和5周,REV感染组对不同病毒疫苗免疫后的HI效价显著低于对照组。高剂量REV感染组的抑制作用大于低剂量感染组,但统计学差异不显著。REV感染可造成中枢免疫器官萎缩,REV感染组的胸腺、法氏囊与体重比显著低于对照组。本研究证明了,REV早期感染会干扰鸡群对NDV、AIV的免疫效果,特别是会严重干扰对AIV疫苗的免疫效果。     

Apoptotic response of chicken embryonic fibroblast cells to infectious bursal disease virus infections reflects viral pathogenicity

Infectious bursal disease virus (IBDV) induces immunodeficiency in young chickens and apoptosis in chicken embryos. To understand the relation between the viral pathogenesis and the induction of cell death, chicken embryonic fibroblast (CEF) cells were infected with IBDV intermediate (im) and very virulent (vv) strains at different MOIs. The cell viability and DNA fragmentation were evaluated in infected cells. The cellular apoptotic pathway involve was investigated by determining the activities of caspase cascade. The imIBDV strain was replicated well in CEF cells and shown higher viral titers than vvIBDV. Apoptosis changes were observed only in vvIBDV-infected CEF cells at higher MOI 48 h post infection. Efflux of cytochrome c suggests that the intrinsic pathway of the apoptotic process induced by vvIBDV infection independently of virus replication. Prediction of caspase substrates cleavage sites revealed that different IBDV strains have conserved cleavage motif pattern for VP2 and VP5 viral proteins. These findings suggest the pathogenicity of IBDV strains might be involved in the induction of apoptosis in host cells.     

Induced liver tumour deaths by subgroup A Rous sarcoma virus in chicks inoculated via chorioallantoic membrane, a genetic marker

A study was made using two strains of light breed (White Leghorn strains, A and B) and four heavy beeds (Rhode Island Red, New Hampshire, Australorp, Columbian) to evaluate the breed difference in survival potential of chicks that were infected as 11-day-old embryos via chorioallantoic membranes (CAMs) with a subgroup A Rous sarcoma virus. Of the 1185 chicks hatched over multiple hatch-replicates, 845 chicks died rapidly of a fibrosarcomatous liver tumour (LT) with a peak mortality about 74% attained by the second week, post-hatch, in the heavy breeds and more than 90% by the second week in the light breed. The breeds did not differ in induced LT mortality when the chicks hatched from eggs that had at least 25 pock counts on CAMs, apparently genetically susceptible, i.e. 25 biologically active virus particles were enough to induce an unpreventable fatal LT. However, low pock-count on CAMs did not act as a pointer for predicting genetic resistance to infection because about 23% of chicks developed from eggs that had no pocks on CAMs, apparently genetically resistant, also died of LT, requiring further studies.     

Changes in the cellular localization of estrogen receptor alpha in the growing and regressing ovaries of Gallus domesticus during development

In this work, the presence of estrogen receptor alpha (ER-α) was determined in different cell subpopulations in the left growing and right regressing ovaries of Gallus domesticus from 13-day-old chicken embryos to one-month-old chickens by immunohistochemistry. Results revealed positive ER-α immunostaining in both ovaries during development, but the percentage, staining intensity, and cellular distribution of ER-α immunostaining changes according to whether it is the left or right ovary and with the animal’s age. In the left ovary, the ER-α was localized in the nuclei of the germinal epithelium and in germ cells of the ovarian cortex, as well as in the interstitial cells, undifferentiated cells, and epithelial cells of the lacunar channels of the ovarian medulla in all ages. In contrast, in the right ovary from 13-day-old chicken embryos to one-week-old chickens, only the epithelial cells of lacunar channels were ER-α immunoreactive, but in the right ovary of one-month-old chickens both the epithelial cells of lacunar channels and the interstitial cells presented ER-α. These results demonstrate differential expression of ER-α in both chicken ovaries during development in a cell type-specific distribution, suggesting that these differences may be regarded as an important cause in the process of asymmetric ovarian development in the chicken.