Serial Passage In Chicken Embryos Research Articles

Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos

Previously, we have demonstrated that the recombinant Newcastle disease virus (NDV) expressing the infectious laryngotracheitis virus (ILTV) glycoprotein D (gD) conferred protection against both virulent NDV and ILTV challenges in chickens. In this study, we evaluated the genetic stability of the recombinant vaccine after eight serial passages in embryonated chicken eggs (ECE). The vaccine master seed virus at the original egg-passage level 3 (EP3) was diluted and passaged in three separate repetitions (A, B and C) in ECE eight times (EP4 to EP11). RT-PCR analysis of the vaccine seed and egg-passaged virus stocks showed that there was no detectable insertion/deletion in the ILTV gD insert region. Next-generation sequencing analysis of the EP3 and EP11 virus stocks confirmed their genome integrity and revealed a total of thirteen single-nucleotide polymorphisms (SNPs). However, none of these SNPs were located in the ILTV gD insert or any of the known critical biological determinant positions. Virological and immunofluorescent assays provided additional evidence that the EP11 virus stocks retained their growth kinetics, low pathogenicity, and robust level of gD expression comparable to that of the vaccine master seed virus. This indicated that the SNPs were non-detrimental sporadic mutations. These results demonstrated that the insertion of ILTV gD gene into the NDV LaSota backbone did not significantly affect the genetic stability of the recombinant virus and that the rLS/ILTV-gD virus is a safe and genetically stable vaccine candidate after at least eight serial passages in ECE.

DHAV-1 Inhibits Type I Interferon Signaling to Assist Viral Adaption by Increasing the Expression of SOCS3.

Duck hepatitis A virus type 1 (DHAV-1) is one of the most lethal pathogens in the duck industry. The attenuated vaccine (the CH60 strain) is cultivated through serial passage in chicken embryos and is widely used for the prevention and control of the disease. However, the specific mechanism underlying its adaptation in chicken embryos has not been fully elucidated. In this study, we first infected chicken embryo fibroblasts (CEFs) with the DHAV-1 CH60 strain. The peak of viral proliferation occurred within 36–48 h post-infection. The different DHAV-1 strains significantly induced the expression of IFNα, IFNγ, and Suppressor of cytokine signaling 3 (SOCS3) in CEFs, and we found that SOCS3 overexpression significantly promoted viral replication. Furthermore, SOCS3 overexpression significantly inhibited the expression of IFNα but promoted the expression of IFNγ. In addition, SOCS3 overexpression clearly decreased the mRNA levels of STAT1 and STAT3 in the Janus kinase (JAK)-STAT signaling pathway and inhibited the expression of the antiviral proteins MX1 and OASL. Immune-precipitation assays indicated that SOCS3 and IFNα do not physically interact. Subcellular localization of SOCS3 and IFNα revealed that SOCS3 was mainly located in the nucleus and cytoplasm, while IFNα was located only in the cytoplasm. Co-localization of these two proteins was not observed in the cytoplasm. In conclusion, the DHAV-1 CH60 strain may inhibit the expression of IFNα by increasing the SOCS3 protein and SOCS3 can in turn decrease STAT1 and STAT3 mRNA levels, thereby inhibiting the antiviral protein MX1 and ultimately promoting viral proliferation, indirectly assisting in viral adaptation in chicken embryos.

Genomic Characteristics and Changes of Avian Infectious Bronchitis Virus Strain CK/CH/LDL/97I after Serial Passages in Chicken Embryos

Background: We previously attenuated the infectious bronchitis virus (IBV) strain CK/CH/LDL/97I and found that it can convey protection against the homologous pathogenic virus. Objective: To compare the full-length genome sequences of the Chinese IBV strain CK/CH/LDL/97I and its embryo-passaged, attenuated level to identify sequence substitutions responsible for the attenuation and define markers of attenuation. Methods: The full-length genomes of CK/CH/LDL/97I P5 and P115 were amplified and sequenced. The sequences were assembled and compared using the MEGALIGN program (DNAStar) and a phylogenetic tree was constructed using MEGA4 software. Results: The CK/CH/LDL/97I virus population contained subpopulations with a mixture of genetic mutants. Changes were observed in nsp4, nsp9, nsp11/12, nsp14, nsp15, nsp16, and ORF3a, but these did not result in amino acid substitutions or did not show functional variations. Amino acid substitutions occurred in the remaining genes between P5 and P115; most were found in the S region, and some of the nucleotide mutations resulted in amino acid substitutions. Among the 9 nsps in the ORF1 region, nsp3 contained the most nucleotide substitutions. Conclusions: Sequence variations in different genes, especially the S gene and nsp3, in the genomes of CK/CH/LDL/97I viruses might contribute to differences in viral replication, pathogenicity, antigenicity, immunogenicity, and tissue tropism.

The identification of the first isolate of influenza B virus using a duplex RT-PCR DNA sequencing in Saudi Arabia (B/Riyadh/01/2007)

Although Influenza virus infections threaten thousands of lives each year worldwide and bear the risk of been an epidemic, little is known about the circulating strains in Saudi Arabia. The availability of a rapid and accurate diagnostic approach is essential for effective treatment and disease control. In the current study, we optimized a duplex RT-PCR assay for the simultaneous detection of influenza A and B viruses in clinical samples. The developed assay was utilized for testing 100 nasopharyngeal aspirates collected from young children hospitalized with acute respiratory tract infection in Riyadh. Influenza B not A viruses, were identified as possible causes of the disease syndrome. Recovery of influenza B virus from clinical samples was achieved by serial passage in chicken embryos and the virus isolate was designated as Influenza B/Riyadh/01/2007. The identity of B/Riyadh/01/2007 was confirmed by sequencing the RT-PCR product. The sequenced data was submitted to the Gene Bank under the accession number GU135839. An almost complete homology was recognized with all Influenza B virus strains available on Gene Bank. Although several studies suggested the presence of influenza B virus in respiratory tract infection of Saudi children, this is the first report that describes the isolation and partial identification of the circulating virus. Further studies that fully characterize the isolated strain are ongoing.

S1 gene sequence heterogeneity of a pathogenic infectious bronchitis virus strain and its embryo-passaged, attenuated derivatives

Infectious bronchitis virus CK/CH/LDL/97I was attenuated by serial passage in chicken embryos. Virus of passage 115 was attenuated as determined by clinical response to inoculation to 15-day-old specific pathogen free chickens. The vaccination-challenge test showed that the attenuated passage 115 virus could afford protection against the homologous pathogenic virus, passage 5, by the clinical response. Based on the sequence analysis and comparison of the S1 gene, both the pathogenic and attenuated CK/CH/LDL/97I viruses were populations that each included at least two subpopulations. Also, from analysis of the amino acid and nucleotide sequences of S1 gene, we speculate that recombination between the minor and dominant subpopulations plus accumulation of mutations in the S1 region of pathogenic passage 5 might lead to the formation of the embryo-passaged, attenuated passage 115.

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.

Avian Paramyxovirus Type 1 from Pigeons: Isolate Characterization and Pathogenicity after Chicken or Embryo Passage of Selected Isolates

Nine pigeon paramyxovirus type 1 isolates from the United States and Canada were characterized and three of the isolates were pathotyped before and after passage in chickens and serial passage in chicken embryos. One isolate previously passaged in Madin Darby bovine kidney cells was also pathotyped after chicken and embryo passage. Hemagglutination (HA) titers of all isolates were low when tested by microtiter procedures and all were negative by rapid-plate HA. The HA titers were increased by a factor of 8 to 32 by Tween-ether treatment, and treated antigen had the same reactivity as untreated antigen in hemagglutination-inhibition (HI) tests. All isolates had a slow elution rate and an HA thermostability equal to or greater than 60 minutes. Mean death times in embryos were 99 hours or greater, except for one isolate with a mean death time of 81 hours, and intracerebral pathogenicity indices of all isolates were greater than 1. Antigenic differences among the pigeon isolates were identified by three different binding patterns in HI tests against a battery of five Newcastle disease virus (NDV) monoclonal antibodies. Pathogenicity enhancement by bird, embryo, or cell passage was limited to an intravenous pathogenicity index increase for one of three viruses passaged in embryonated eggs. Cloacal samples collected during chicken passage contained higher virus titers than did oral samples. The pigeon isolates reported here, like those of earlier reports, have properties that prevent characterization within a single NDV pathotype. Finally, there was no evidence that any of these isolates was highly virulent for chickens.

Biochemical characterization of an aphthovirus type C 3 strain resende attenuated for cattle by serial passages in chicken embryos

We have compared several aspects of an aphthovirus strain attenuated for cattle (C 3R-O/E) with the original strain (C 3Res) from which it was derived after serial passages in chicken embryos. Biochemical differences detected by protein analysis in regular polyacrylamide gels (SDS-PAGE) and on electrofocusing gels (NEPHGE) suggest the presence of mutations throughout the genome. Changes were located in coat proteins VP1 and VP3 and in the polymerase precursor P100 (P3/ABCD). No other differences were found at the protein level by means of the techniques used. Polypeptide P100 of the attenuated strain showed a faster electrophoretic mobility in SDS-PAGE with respect to that of the wild-type strain, and the change seems to be located on its amino terminus half. Several functional differences were also found between the two viruses. Both strains grew equally well in BHK cells reaching roughly similar titers in plaque assays. However, the wild-type strain maintained its titer in cells of bovine origin (BK), whereas the titer of C 3R-O/E strain decreased approximately one log in this cell system; moreover, plaques elicited by the attenuated strain were much smaller than the ones produced by C 3Res. A diminution in the rate of RNA synthesis induced by C 3R-O/E in BK cells compared with that of the wild-type strain was also detected; this trait was not observed in BHK cells. A delay in the kinetics of RNA synthesis was also detected in this strain. The virus yield of attenuated strain in BK cells was four times lower than in BHK cells.

Attenuation of Eimeria tenella by serial passage in chicken embryos.

A strain of Eimeria tenella of Japan origin was serially passaged 51 times in chicken embryos. Any obvious changes of reproductivity and lethality to chicken embryos of the passage strain were not observed. Chickens infected with oocysts passaged 41 times in chicken embryos showed less severe cecal lesions and hemorrhage, and higher values of body weight gain, feed intake and lower value of feed requirment, compared with chickens infected with oocysts passaged once in chicken embryos. Less severe lesions were also observed in chickens infected with oocysts passaged 51 times in chicken embryos than those in chickens with oocysts passaged once. It was indicated that pathogenicity of the strain was attenuated after serial passage in chicken embryos.

Biochemical characterization of an aphthovirus type 0 1 strain campos attenuated for cattle by serial passages in chicken embryos

The biochemical properties of a virulent and an attenuated strain of foot-and-mouth disease virus (FMDV) Type 0 1 Campos (0 1C) were compared in order to establish differences that could account for their altered biological functions. The avirulent strain (0 1C-O/E) was derived from the virulent strain 0 1C by serial passages in chicken embryos. Analysis of the RNase T 1-generated oligonucleotides of the viral RNA through one- and two-dimensional (2D) gel electrophoresis (fingerprints) revealed a few changes in the genome structure of the 0 1C-O/E strain compared to the wild type strain. In addition there was a significant decrease in the length of the poly(C) rich tract of the 0 1C-0/E RNA. All virion structural proteins, except VP4, their precursors, and the viral RNA polymerase (p56a) show charge differences. In addition a significant decrease in the apparent molecular weight of polypeptide p100 (primary translational product from the 3′ end region of the genome) of the attenuated strain was observed.

Effect of Serial Embryo Passage of an Arkansas-Type Avian Infectious Bronchitis Virus Isolate on Clinical Response, Virus Recovery, and Immunity

Infectious bronchitis virus (IBV) Arkansas-type DPI strain (Ark DPI) was attenuated by serial passage in chicken embryos. Virus of passage 50 was less pathogenic for day-old maternally immune broiler-type chickens than virus of passage 10 or 25 as determined by clinical response to vaccination, virus isolation in respiratory (trachea) and nonrespiratory (kidney and cloaca) tissues, and weight-gain studies. Chickens vaccinated with virus of passage 10, 25, or 50 were at least 80% resistant to homologous virus challenge of the upper respiratory tract 4 and 6 weeks postvaccination. Serum antibody production by passage-50-vaccinated chickens was comparatively low at 4 weeks but at 6 weeks was similar to that in chickens vaccinated with virus of passage 10 or 25.

Pathogenicity of Chlamydia psittaci after serial passage in chicken embryos

The virulence in pregnant ewes of a single strain of Chlamydia psittaci was tested after the following treatments: (a) 110 serial passages in chicken embryos (CE) incubated at 37 C, and (b) 7 CE passages at 37 C followed by 10 CE passages at 40 C. Neither treatment caused significant reduction in abortions (P > 0.3) nor incidence of ewes shedding chlamydia in placentas or vaginal discharges (P > 0.5) compared to ewes inoculated with the original virulent strain.

Tetratrichomonas gallinarum in the blood of chickens infected with spirochetes.

SYNOPSIS. Tetratrichomonas gallinarum was found in the blood of 3 chickens experimentally infected with Borrelia anserina. The blood infection was successfully reproduced in healthy chickens and was accompanied by evidence that the trichomonads multiplied in the blood, ingested red blood cells, and were themselves phagocytized by leucocytes. T. gallinarum was maintained in serial passage in chicken embryos as well as in the usual trichomonad culture media.

Experimental Infections of Wild Birds with the Viruses of Eastern Equine Encephalitis, Newcastle Disease and Vesicular Stomatitis

Received for publication April 7, 1959. * On leave from the Central Institute of Hygiene, Zagreb, Yugoslavia. Paper NS 270 from the Department of Veterinary Science, University of Wisconsin, Madison, published with the approval of the director of the Wisconsin Agricultural Experiment Station_ sin. The crows, hawks and owl were captured in southern Wisconsin by personnel of the Wisconsin Conservation Department. All birds appeared healthy at the beginning of the experiments. The eastern equine encephalitis virus (EEEV) used was originally isolated in 1956 from the brain of a horse which died during an encephalitis epizootic in southeastern Georgia. Allantoic fluids from the thirteenth and twenty-third serial passages in chicken embryos were used in these experiments. The vesicular stomatitis virus (VSV) used was the Wisconsin strain (New Jersey serotype) isolated from a cow in 1949 and transmitted serially in chicken embryos. The Newcastle disease virus (NDV) was Texas GB strain isolated from an outbreak of severe nervous-respiratory disease in chickens in 1948 and maintained by serial passage in chicken embryos. The routes of viral exposure used in the experiments were oral, subcutaneous, intracardial and intracerebral. Dosage varied according to the route of exposure employed and the estimated susceptibility of the individual bird. Birds were visually examined at least once daily after viral exposure to record clinical signs of illness. Body temperatures were not measured, since it was felt that the excitement produced by handling would greatly influence thermometer readings. Blood was drawn on alternate days after infection and examined for evidence of viremia. Fecal samples obtained by cloacal washing were collected daily from crows exposed to the viruses by ingestion of infected chicken embryos. Immune response was judged on the basis of virus neutralization (VN) tests on paired pre-exposure and convalescent serums. Convalescent serums were usually collected on the fourteenth day post exposure. Neutralization indices of 50 or higher in convalescent serums were considered indicative of infection and in birds which possessed no antibody before experimental exposure, this was recorded as a positive immune response. Most of the birds which did not die as a result of the infections were killed by bleeding on the fourteenth day post exposure and tissues, usually brain, lung, liver and spleen, were examined for histopathology and viral content. Tissues were prepared for histopathological examination by

Enzymatic variants of influenza virus. I. Isolation and characterization of slowly reacting enzymatic variants of influenza B virus.

Slowly reacting enzymatic variants of the Lee strain of influenza B virus were isolated by the serial application of a technique which concentrated the fraction of the virus population obtained by slow elution followed by passage of this fraction at high dilution in chicken embryos. The variants were stable upon serial passage in chicken embryos and did not differ significantly from the parent virus in any of the characteristics tested other than those relating to the virus enzyme. Enzymatically, the variants had a much slower rate of activity on chicken mucoproteins than the parent virus. Their activity was greater at 25 degrees C. than at 37 degrees C. and was much enhanced by calcium ions and depressed by a calcium chelating agent. The enzyme of the variant virus was more labile to heat than that of the parent virus although their hemagglutinins were equally stable. It was estimated that the variants comprised less than 3 per cent of the original population of the Lee strain of influenza B virus particles.