Aly M. Fadly

Pathogenicity of variant Marek's disease virus isolants in vaccinated and unvaccinated chickens.

Mareks disease (MD) virus isolants Md/5 and Md/11, obtained from commercial broiler flocks vaccinated with turkey herpesvirus (HVT) but having excessive condemnation losses from MD, seemed similar to prototype MD viruses in oncogenicity for susceptible chickens, immunodepressive ability, and antigenicity. Compared with prototype MD viral strains JM/102W and GA/22, however, both field isolants were classed as biological variants on the basis of: 1) higher induction of acute cytolytic infection, characterized by atrophy of bursa and thymus and early death in the absence of lymphomas; 2) higher oncogenicity in genetically resistant chickens; and 3) higher oncogenicity in chickens immunized with HVT. A standard field dose (l103 plaque-forming units) of HVT vaccine protected 92-95% of chickens challenged with JM/ 102W but only 57-72% of chickens challenged with Md/5; the calculated dose of HVT required to protect 90% of chickens was at least 1000-fold as great for challenge with Md/5 as for challenge with JM/102W. The extent to which such variant MD viruses are responsible for MD vaccine failures in commercial flocks was not determined. These studies suggest that susceptibilities of chickens to acute cytolytic infection and MD lymphomas are mediated independently, and confirm that the severity of acute cytolytic infection in MD is influenced by virus strain.

An Enzyme-Linked Immunosorbent Assay For Detecting Avian Leukosis-Sarcoma Viruses

Immunoglobulins from antiserum raised against chromatographically purified avian myeloblastosis virus (AMV) group-specific (gs) antigens were used in enzyme-linked immunosorbent assay (ELISA). Readily discernible color was produced with 2--3 ng of AMV protein in microplate wells coated with 4 micrograms of salt-precipitated immunoglobulins. When a biological assay, i.e., phenotypic mixing (PM), was the criterion for the infectious status of specimens, the ELISA consistently identified a greater percentage of virus-positive specimens than direct complement-fixation (DCF) tests. Over 95% concordance was obtained between the ELISA and PM bioassays when meconia and whole-blood samples were tested. Moreover, three DCF(-) egg albumens from one virus shedder hen were positive by the direct ELISA. Complete agreement was found between a biological assay for endogenous virus and the ELISA when blood and albumens from inbred chickens were tested. The ELISA is a rapid and convenient alternative to the DCF test for identifying infected chickens in eradication programs, because virus-rich sources such as meconia and blood that are unsuitable for DCF can be tested directly.

Isolation and some characteristics of a subgroup J-like avian leukosis virus associated with myeloid leukosis in meat-type chickens in the United States.

Several subgroup J-like avian leukosis viruses (ALV-Js) were isolated from broiler breeder (BB) and commercial broiler flocks experiencing myeloid leukosis (ML) at 4 wk of age or older. In all cases, diagnosis of ML was based on the presence of typical gross and microscopic lesions in affected tissues. The isolates were classified as ALV-J by 1) their ability to propagate in chicken embryo fibroblasts (CEF) that are resistant to avian leukosis virus (ALV) subgroups A and E (C/AE) and 2) positive reaction in a polymerase chain reaction with primers specific for ALV-J. The prototype strain of these isolates, an isolate termed ADOL-Hc1, was obtained from an adult BB flock that had a history of ML. The ADOL-Hc1 was isolated and propagated on C/AE CEF and was distinct antigenically from ALV of subgroups A, B, C, D, and E, as determined by virus neutralization tests. Antibody to ADOL-Hc1 neutralized strain HPRS-103, the prototype of ALV-J isolated from meat-type chickens in the United Kingdom, but antibody to HPRS-103 did not neutralize strain ADOL-Hc1. On the basis of both viremia and antibody, prevalence of ALV-J infection in affected flocks was as high as 87%. Viremia in day-old chicks of three different hatches from a BB flock naturally infected with ALV-J varied from 4% to 25%; in two of the three hatches, 100% of chicks that tested negative for virus at hatch had evidence of viremia by 8 wk of age. The data document the isolation of ALV-J from meat-type chickens experiencing ML as young as 4 wk of age. The data also suggest that strain ADOL-Hc1 is antigenically related, but not identical, to strain HPRS-103 and that contact transmission of ALV-J is efficient and can lead to tolerant infection.

Development and characterization of monoclonal antibodies to subgroup J avian leukosis virus.

In an attempt to develop a specific diagnostic test for avian leukosis virus (ALV) subgroup J (ALV-J) strain Hc1, four monoclonal antibodies (MAbs), JE9, G2, 145, and J47, were generated that are specific for ALV-J envelope glycoprotein, gp85. Polymerase chain reaction (PCR) was used to amplify genomic pro-viral DNA of Avian Disease and Oncology Laboratory (ADOL)-Hc1 and ADOL-4817 envelope genes. Both open reading frames encoding glycoproteins gp85 and gp37 were cloned into baculoviruses. Abundant expression of gp85 and gp37 was detected in the recombinant viruses with specific antibody to Hc1 strain of the ALV-J. The expressed proteins were used for immunization of mice to produce hybridoma cell lines secreting MAbs specific to ALV-J envelope protein. A panel of MAbs was generated by fusing NS1 myeloma cells and spleen cells from mice immunized with the recombinant baculoviruses. With the use of an immunofluorescence assay, three MAbs (JE9, G2, 145) reacted with ALV-J but not with subgroups A, B, C, D, or E of ALV. MAb J47 reacted with all exogenous subgroups of ALV including A, B, C, D, and J but not with endogenous subgroup E viruses. Western blot analysis was performed with all four MAbs against recombinant baculovirus and Hc1-infected chicken embryo fibroblast (CEF) lysates. A major band with a molecular weight about 90 kD corresponding to the size of ALV-J envelope was consistently obtained. With these MAbs, we detected the Hc1 antigen in CEFs infected with several ALV-J viruses isolated in the United States and also in tissue sections from chickens infected with Hc1 strain of ALV-J. These MAbs will be useful reagents for the diagnosis of ALV-J infection because they recognize a common antigenic epitope in six isolates tested thus far.

Characteristics of CVI988/Rispens and R2/23, Two Prototype Vaccine Strains of Serotype 1 Marek's Disease Virus

Studies were focused on two attenuated serotype 1 Mareks disease (MD) vaccine viruses, CVI988/Rispens (passage 42) and R2/23 (passage 105). Both serotype 1 vaccine viruses provided much higher levels of protection than the prototype MD vaccine, turkey herpesvirus (HVT); the best protection was generally provided by CVI988/Rispens when compared with other vaccines. The efficacy of neither serotype 1 vaccine was improved by mixture with viruses of other serotypes (synergism). No differences between the two serotype 1 vaccines were revealed by cross-neutralization tests, thus excluding preferential in vivo neutralization by maternal antibodies as an explanation for differences in protective efficacy. Neither vaccine strain induced MD lesions or reduced growth rates in 8- or 18-week trials. Neither virus depressed humoral or cellular immune responses to antigenic challenge at 3 or 15 days after vaccination. Both virus strains exhibited altered characteristics during serial backpassage; R2/23 acquired increased oncogenic potential, and CVI988/Rispens acquired the potential for increased viremia titers, accompanied by an increased frequency of both histologic nerve lesions and gross thymic atrophy. During backpassage trials, contact spread was not observed for R2/23 and, surprisingly, seemed relatively limited for CVI988/Rispens. Studies on these two serotype 1 strains generally support the safety and efficacy of the serotype 1 class of MD vaccines.

Influence of Endogenous Viral (ev) Gene Expression and Strain of Exogenous Avian Leukosis Virus (ALV) on Mortality and ALV Infection and Shedding in Chickens

Two experiments were conducted to determine the influence of endogenous viral (ev) gene expression on the response of chicken to day-old inoculation with two laboratory and two field strains of avian leukosis virus (ALV). The ev2- and ev3-negative semi-congenic chickens had much higher mortality from a unique non-neoplastic syndrome (NNS) than ev2- and ev3-positive semi-congenic chickens after inoculation with ALV strain RAV-1. The three other ALV strains induced little NNS. All four strains of ALV induced higher incidences of neutralizing antibody and lower incidences of viremia in ev2- and ev3-negative chickens than ev2- and ev3-positive chickens. The semi-congenic ev2- and ev3-negative chickens were only slightly less likely to shed ALV in the cloaca. Chickens of line 0, an unrelated line lacking ev genes, had a much higher rate of antibody production and lower rate of viremia and shedding than the semi-congenic chickens. Surprisingly, line 0 failed to get NNS after RAV-1 inoculation. There were major differences among strains of ALV in induction of antibody response, viremia, and shedding in the cloaca. The two field strains of ALV tended to be less immunogenic than the laboratory strains. We conclude that both the genetics of the host, including variation in ev gene expression, and the strain of ALV can influence the probability of ALV shedding and congenital transmission after horizontal infection.

Development of a Polymerase Chain Reaction to Differentiate Avian Leukosis Virus (ALV) Subgroups: Detection of an ALV Contaminant in Commercial Marek's Disease Vaccines

Abstract Avian leukosis viruses (ALVs) are common in many poultry flocks and can be detected using an enzyme-linked immunosorbent assay or any other test designed to identify p27, the group-specific antigen located in gag. However, endogenous retroviruses expressing p27 are often present and can be confused with exogenous ALVs. A more specific and informative assay involves targeting the variable envelope glycoprotein gene (gp85) that is the basis for dividing ALVs into their different subgroups. We designed polymerase chain reaction (PCR) primers that would specifically detect and amplify viruses from each of the six ALV subgroups: A, B, C, D, E, and J. Subgroup B and D envelopes are related, and our B-specific primers also amplified subgroup D viruses. We also designed a set of common primers to amplify any ALV subgroup virus. To demonstrate the usefulness of these primers, we obtained from the Center for Veterinary Biologics in Iowa culture supernatant from chicken embryo fibroblasts infected with an ALV that was found to be a contaminant in two commercial Mareks disease vaccines. Using our PCR primers, we demonstrate that the contaminant was a subgroup A ALV. We cloned and sequenced a portion of the envelope gene and confirmed that the ALV was a subgroup A virus. Unlike typical subgroup A viruses, the contaminant ALV grew very slowly in cell culture. We also cloned and sequenced a portion of the long terminal repeat (LTR) from the contaminant virus. The LTR was found to be similar to those LTRs found in endogenous ALVs (subgroup E) and very dissimilar to LTRs normally found in subgroup A viruses. The E-like LTR probably explains why the contaminant grew so poorly in cell culture.

An outbreak of lymphomas in commercial broiler breeder chickens vaccinated with a fowlpox vaccine contaminated with reticuloendotheliosis virus.

Gross and microscopic examinations of affected tissues from chickens of two commercial broiler breeder flocks aged 27 and 31 weeks revealed lesions of visceral lymphomas with bursal involvement in some chickens. Reticuloendotheliosis virus (REV), but not avian leukosis virus (ALV), was isolated from blood of affected chickens. Furthermore, DNA extracted from tumours tested positive for REV, but not for ALV or Mareks disease virus by polymerase chain reaction (PCR) test. Attempts to determine the source of REV infection included testing a commercial fowlpox (FP) vaccine used to immunize flocks at 7 days of age. Chicken-embryo fibroblasts inoculated with the FP vaccine tested positive for REV by PCR and immunofluorescent tests. REV was also isolated from plasma of pathogen-free chickens experimentally inoculated with FP vaccine at hatch; two of eight (25%) inoculated chickens developed lymphomas by 34 weeks of age. Antigenic characterization of REV isolated from commercial broiler breeder chickens and from FP vaccine, using monoclonal antibodies, revealed that both isolates belong to subtype 3 of REV. The data represent the first report of an outbreak of REV-induced lymphomas in commercial chickens. The data also indicate that the source of REV infection is an REV-contaminated commercial FP vaccine.

Effect of Endogenous Leukosis Virus Genes on Response to Infection with Avian Leukosis and Reticuloendotheliosis Viruses

We examined the effect of the presence or absence of endogenous viral gene (ev) 3, which controls expression of group-specific viral and envelope antigens (gs+chf+ phenotype), and ev2, which controls the production of a complete subgroup E virus (V-E+ phenotype), on the response of chickens to RAV-1, an exogenous avian leukosis virus (ALV) with an antigenic relationship to endogenous virus. After inoculation at one day of age, the chickens lacking either ev gene expression had a lower frequency of virus isolations and higher frequency and titer of neutralizing antibodies than those expressing ev genes. This relationship was not seen in groups inoculated with chick syncytial virus (CSV), a reticuloendotheliosis-associated virus with no relationship to endogenous virus, but the ev2+ birds tended to yield more CSV isolations than the ev2- birds. We suggest that chickens expressing ev genes may be immunologically tolerant to antigens common to exogenous and endogenous viruses. In addition, ev3- birds inoculated with RAV-1 at one day of age or as embryos died at a high rate between 6 and 12 weeks of age with a non-neoplastic syndrome characterized by severe atrophy of lymphoid organs, an inflammatory reaction in the liver, and a lower immune response to particulate antigens.

Molecular Characterization of Reticuloendotheliosis Virus Insertions in the Genome of Field and Vaccine Strains of Fowl Poxvirus

Abstract Evidence of the widespread occurrence of reticuloendotheliosis virus (REV) sequence insertions in fowl poxvirus (FPV) genome of field isolates and vaccine strains has increased in recent years. However, only those strains carrying a near intact REV provirus are more likely to cause problems in the field. Detection of the intact provirus or REV protein expression from FPV stocks has proven to be technically difficult. The objective of the present study was to evaluate current and newly developed REV and FPV polymerase chain reaction (PCR) assays to detect the presence of REV provirus in FPV samples. The second objective was to characterize REV insertions among recent “variant” FPV field isolates and vaccine strains. With REV, FPV, and heterologous REV-FPV primers, five FPV field isolates and four commercial vaccines were analyzed by PCR and nucleotide sequence analysis. Intact and truncated REV 5′ long terminal repeat (LTR) sequences were detected in all FPV field isolates and vaccine strains, indicating heterogeneous REV genome populations. However only truncated 3′ LTR and envelope sequences were detected among field isolates and in one vaccine strain. Amplifications of the REV envelope and 3′ LTR provided strong evidence to indicate that these isolates carry a near intact REV genome. Three of the four FPV vaccine strains analyzed carried a solo complete or truncated 5′ LTR sequence, indicating that intact REV provirus was not present. Comparison of PCR assays indicated that assays amplifying REV envelope and REV 3′ LTR sequences provided a more accurate assessment of REV provirus than PCR assays that amplify the REV 5′ LTR region. Therefore, to differentiate FPV strains that carry intact REV provirus from those that carry solo 5′ LTR sequences, positive PCR results with primers that amplify the 5′ LTR should be confirmed with more specific PCR assays, such as the envelope, or the REV 3′ LTR PCR.