Guillermo Zavala

Relevance of Akt phosphorylation in cell transformation induced by Jaagsiekte sheep retrovirus

Expression of the JSRV envelope (Env) is sufficient to transform immortalized rodent fibroblasts. A putative docking site for the PI3-K kinase (Y(590)-X-X-M(593)) in the cytoplasmic tail of the transmembrane domain of the JSRV Env is a major determinant of viral-induced cell transformation. Akt is constitutively phosphorylated in rodent fibroblasts transformed by the JSRV Env. However, recent data suggest that Y590 and M593 are not necessary for JSRV Env-induced transformation of the immortalized chicken fibroblasts cell line DF-1. In this study we found that JSRV-induced transformation of DF-1 cells is Akt-independent. In addition, a replication-competent avian vector expressing the JSRV Env (RCASBP(A)+JE) was also able to induce transformation of primary chicken embryo fibroblasts (CEF). Vectors expressing JSRV Env Y590 mutants were still able to induce CEF cells transformation but not as efficiently as the vectors expressing the wild-type Env. In CEF cells, as in DF-1 cells, only the expression of the wild-type Env induced constitutive phosphorylation of Akt. Thus, in chicken cells, the degree of transformation induced by the JSRV Env is maximum in the presence of Y590 and Akt phosphorylation. We addressed the significance of Akt phosphorylation in rat 208F cells transformed by the JSRV Env and showed that Akt is indeed activated and shows kinase activity. Inhibitors of the PI-3K/Akt pathway reproducibly decreased the transformation efficiency of the JSRV Env. In vivo, we found phosphorylated Akt only in nasal tumors induced by the enzootic nasal tumor virus (ENTV), a JSRV-related beta-retrovirus. No evidence of Akt phosphorylation was found in lung tumor sections of sheep affected by pulmonary adenocarcinoma. As a whole, these results suggest that the activation of the PI-3K/Akt pathway contributes to the process of JSRV-induced cell transformation but most likely is not the primary determinant both in vitro and in vivo.

Specific Detection and Typing of Mycoplasma synoviae Strains in Poultry with PCR and DNA Sequence Analysis Targeting the Hemagglutinin Encoding Gene vlhA

Abstract Mycoplasma synoviae is a major pathogen of chickens and turkeys, causing economic losses to the poultry industry worldwide. In this study, we validated and applied polymerase chain reaction (PCR) and DNA sequence analysis on the N-terminal end of the hemagglutinin encoding gene vlhA as an alternative for the detection and initial typing of field strains of M. synoviae in commercial poultry. PCR primers were tested against isolates of M. synoviae from various sources along with other avian mycoplasma and other bacterial species. The vlhA gene–targeted PCR assay was highly specific in the identification of M. synoviae, with a detection limit of 4.7 × 102 color changing units /ml. DNA sequence analysis of amplified products was also conducted to validate the potential for typing M. synoviae strains using the N-terminal region of the vlhA gene. To evaluate the test, we applied the PCR assay to tracheal swabs collected from chickens challenged with M. synoviae strain K1968 and compared the results to the serologic detection. The PCR assay was also evaluated directly on tracheal samples collected from commercial layers. Overall, this vlhA gene–targeted PCR is a useful tool for detection and initial typing of M. synoviae and can be applied in the preliminary identification of M. synoviae isolates directly from clinical samples.

Protection Against Infectious Laryngotracheitis by In Ovo Vaccination with Commercially Available Viral Vector Recombinant Vaccines

Abstract Infectious laryngotracheitis (ILT) is a highly contagious respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). The disease is mainly controlled through biosecurity and by vaccination with live-attenuated vaccines. The chicken embryo origin (CEO) vaccines, although proven to be effective in experimental settings, have limited efficacy in controlling the disease in dense broiler production sites due to unrestricted use and poor mass vaccination coverage. These factors allowed CEO vaccines to regain virulence, causing long lasting and, consequently, severe outbreaks of the disease. A new generation of viral vector fowl poxvirus (FPV) and herpesvirus of turkey (HVT) vaccines carrying ILTV genes has been developed and such vaccines are commercially available. These vaccines are characterized by their lack of transmission, lack of ILTV-associated latent infections, and no reversion to virulence. HVT-vectored ILTV recombinant vaccines were originally approved for subcutaneous HVT or transcutaneous (pox) delivery. The increased incidence of ILTV outbreaks in broiler production sites encouraged the broiler industry to deliver the FPV-LT and HVT-LT recombinant vaccines in ovo. The objective of this study was to evaluate the protection induced by ILTV viral vector recombinant vaccines after in ovo application in 18-day-old commercial broiler embryos. The protection induced by recombinant ILTV vaccines was assessed by their ability to prevent clinical signs and mortality; to reduce challenge virus replication in the trachea; to prevent an increase in body temperature; and to prevent a decrease in body weight gain after challenge. In this study, both recombinant-vectored ILTV vaccines provided partial protection, thereby mitigating the disease, but did not reduce challenge virus loads in the trachea.

Pathogenicity and growth characteristics of selected infectious laryngotracheitis virus strains from the United States

In a recent study, several US infectious laryngotracheitis virus (ILTV) strains and field isolates were genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) into nine different genotypes. All of the commercial poultry isolates were identified within genotypes IV, V, and VI. Based on the PCR-RFLP, Group IV isolates were characterized as genetically identical to the chicken embryo origin (CEO) vaccines, Group V as genetically closely related to the CEO vaccines, and Group VI as genetically different to the vaccine strains. The objective of this study was to determine the pathogenicity and growth characteristics of six ILTV commercial poultry isolates as compared with the CEO vaccine. Two isolates representative of PCR-RFLP Groups IV, V, and VI were selected. Differences in disease severity, viral tissue distribution in chickens, and plaque formation ability in cell culture were observed among viral genotypes IV, V, and VI, and between V-A and V-B isolates. Mild respiratory clinical signs were produced by IV-A, IV-B and the CEO vaccine, while VI-A and VI-B isolates produced severe respiratory signs and severe depression, and during the peak of clinical signs both isolates were re-isolated from the conjunctiva, sinus, trachea and thymus. Similarly to Group VI isolates, V-A and V-B produced severe respiratory signs, depression, and were re-isolated from conjunctiva, sinus, and trachea; on cell culture, both isolates produced significant larger plaques than any of the other isolates analysed. Overall, differences in pathogenicity and growth characteristics were observed among genetically closely related US ILTV isolates; however, complete genomes will be necessary to identify molecular determinants linked to the pathogenic viral phenotypes.

Detection and Characterization of Avian Leukosis Virus in Marek's Disease Vaccines

Abstract Avian leukosis virus (ALV) infection in chickens is known to induce increased mortality, tumors, delayed growth, and suboptimal egg production. Countries importing specified pathogen-free eggs, vaccines, and poultry breeding stock require freedom of infection or contamination with ALV in such products among other avian pathogens. Recently, ALV was found as a contaminant in a limited number of commercial poultry vaccines, even after routine quality assurance procedures cleared the vaccines for commercialization. The contaminated vaccines were promptly withdrawn from the market, and no direct detrimental effects were reported in poultry vaccinated with such vaccines. We describe herein the characterization in vitro of the contaminant viruses. All exogenous viruses detected in four vaccine lots belong to subgroup A of ALV based on cell receptor interaction, subgroup-specific polymerase chain reaction (PCR), envelope gene sequencing, and virus neutralization. A combination of thermal treatment and serial dilutions of the contaminated vaccines facilitated detection of contaminating ALVs in cell culture coupled with antigen-capture enzyme-linked immunosorbent assay. Subgroup-specific PCR readily detected ALV-A directly in the contaminated vaccines but not in naive vaccines or cell controls. Our methods are proposed as complementary procedures to the currently required complement fixation for avian leukosis test for detection of ALV in commercial poultry vaccines.

Protection induced by commercially available live-attenuated and recombinant viral vector vaccines against infectious laryngotracheitis virus in broiler chickens

Viral vector vaccines using fowl poxvirus (FPV) and herpesvirus of turkey (HVT) as vectors and carrying infectious laryngotracheitis virus (ILTV) genes are commercially available to the poultry industry in the USA. Different sectors of the broiler industry have used these vaccines in ovo or subcutaneously, achieving variable results. The objective of the present study was to determine the efficacy of protection induced by viral vector vaccines as compared with live-attenuated ILTV vaccines. The HVT-LT vaccine was more effective than the FPV-LT vaccine in mitigating the disease and reducing levels of challenge virus when applied in ovo or subcutaneously, particularly when the challenge was performed at 57 days rather than 35 days of age. While the FPV-LT vaccine mitigated clinical signs more effectively when administered subcutaneously than in ovo, it did not reduce the concentration of challenge virus in the trachea by either application route. Detection of antibodies against ILTV glycoproteins expressed by the viral vectors was a useful criterion to assess the immunogenicity of the vectors. The presence of glycoprotein I antibodies detected pre-challenge and post challenge in chickens vaccinated with HVT-LT indicated that the vaccine induced a robust antibody response, which was paralleled by significant reduction of clinical signs. The chicken embryo origin vaccine provided optimal protection by significantly mitigating the disease and reducing the challenge virus in chickens vaccinated via eye drop. The viral vector vaccines, applied in ovo and subcutaneously, provided partial protection, reducing to some degree clinical signs, and challenge VIRUS replication in the trachea.

Molecular Characterization of Three Recombinant Isolates of Avian Leukosis Virus Obtained from Contaminated Marek's Disease Vaccines

Abstract Three natural recombinant avian leukosis viruses (ALV; PDRC-1039, PDRC-3246, and PDRC-3249) expressing a subgroup A gp85 envelope protein and containing long terminal repeats (LTR) of endogenous ALV-E viruses were isolated from contaminated commercial Mareks disease vaccines, cloned, and completely sequenced. Their full genomes were analyzed and compared with representative strains of ALV. The proviral DNA of all three isolates displayed 99.3% identity to each other, suggesting a possible common ancestor, even though the contaminating viruses were obtained from three separate vaccine serials produced by two different vaccine manufacturing companies. The contaminating viruses have a genetic organization typical of replication-competent alpharetroviruses. The proviral genomes of PDRC-1039 and PDRC-3246 are 7497 bp long, and the PDRC-3249 is three base pairs shorter because of a deletion of a threonine residue within the gp85 coding region. The LTR, gag, pol, and the transmembrane (TM) region (gp37) of the env gene of all three viruses displayed high identity to endogenous counterpart sequences (>98%). Only the surface (SU) region (gp85) of the env gene displayed high identity with exogenous ALV-A (98.7%). Locus-specific polymerase chain reaction (PCR) analysis for ALV endogenous sequences (ev loci) in the chicken embryo fibroblasts used to produce the original vaccine vials identified the presence of ev-1, ev-2, ev-3, ev-4, and ev-6 in all three vaccines. Homologous recombination most likely took place to involve the SU region of the env gene because the recombinant viruses only differ in this particular region from the consensus ALV-E. These results suggest that the contaminating ALV isolates probably emerged by recombination of ALV-A with endogenous virus sequences before vaccine preparation.

Enzootic Reticuloendotheliosis in the Endangered Attwater's and Greater Prairie Chickens

Abstract Reticuloendotheliosis (RE) in captive greater prairie chickens (GPC, Tympanuchus cupido pinnatus) and Attwaters prairie chickens (APC, Tympanuchus cupido attwateri) was first reported in 1998. RE is caused by avian reticuloendotheliosis virus (REV), an oncogenic and immunosuppressive retrovirus infecting multiple species of wild and domestic birds. During August 2004 through May 2006 a captive population of prairie chickens was affected simultaneously with a neoplastic condition and also avian pox, the latter being detected in 7.4% (2 of 27) of all birds submitted for histopathology. A survey for REV was conducted in order to examine its possible role in mortality observed primarily in juvenile and adult specimens of prairie chickens. The investigative procedures included postmortem examinations, histopathology, molecular detection, and virus isolation. In total, 57 Attwaters prairie chickens and two greater prairie chickens were included in the study. REV infection was diagnosed using virus isolation or polymerase chain reaction (PCR) or both in 59.5% (28 of 47) of blood samples and/or tumors from suspect birds. Lymphosarcomas were detected in the tissues of 37% (10 of 27) of the birds submitted for histopathology. Such lymphosarcomas suggestive of RE represented the most frequent morphologic diagnosis on histopathology among 27 separate submissions of naturally dead prairie chickens. Overall, REV was detected or RE diagnosed in 34 of 59 prairie chickens (57.62%). The average death age of all birds diagnosed with lymphosarcomas on histopathology was 2.2 yr, ranging from <1 to 4 yr. Although deaths associated with neoplasia occurred in males and females in equal proportions based on submissions, overall more males were diagnosed as REV infected or RE affected (16 males vs. 7 females, and 11 birds of undetermined gender). Reticuloendotheliosis virus was confirmed as a significant cause of mortality in captive prairie chickens.

Molecular epidemiology of avian leukosis virus subgroup J and evolutionary history of its 3' untranslated region.

Abstract Avian leukosis subgroup J (ALV-J) causes a variety of tumors and mortality in meat-type chickens. Since its discovery in the late 1980s, ALV-J has spread to breeding stock produced by most primary breeding companies of North America, the European Union, and Asia. ALV-J seems to have been eradicated from elite breeding stock produced by most primary breeders, albeit ALV-J still circulates in some commercial poultry. This study was undertaken to examine the molecular epidemiology and evolution of ALV-J detected in breeding stock and broiler chickens representing eight primary breeding companies over a period of approximately 20 yr (1988–2007). The redundant transmembrane region of the envelope gene has been deleted in some isolates, suggesting that this region is dispensable for viral fitness. Within the 3′ untranslated region (3′ UTR), the direct repeat 1 was present in 100% of the ALV-J isolates studied. In contrast, the E element has undergone substantial deletions in >50% of the ALV-J proviruses studied. Overall, the unique region 3 was the least conserved within the 3′ long terminal repeat (LTR), albeit the transcriptional regulatory elements typical of avian retroviruses (CAAT, CArG, PRE, TATA, and Y boxes) were highly conserved. The direct repeat region of the LTR was identical in all of the proviruses, and the 3′ unique region 5 was relatively well conserved. Thus, the 3′ UTR of ALV-J has evolved rapidly, reflecting significant instability of this region. Some of the mutations in the 3′ UTR have resulted in the emergence of moderately distinct genetic lineages representing each primary breeding company from which ALV-J was isolated.

Protection Induced by Infectious Laryngotracheitis Virus Vaccines Alone and Combined with Newcastle Disease Virus and/or Infectious Bronchitis Virus Vaccines

Abstract Two types of live attenuated vaccines have been used worldwide for the control of infectious laryngotracheitis virus (ILTV): 1) chicken embryo origin (CEO) vaccines; and 2) tissue culture origin vaccines (TCO). However, the disease persists in spite of extensive use of vaccination, particularly in areas of intense broiler production. Among the factors that may influence the efficiency of ILTV live attenuated vaccines is a possible interference of Newcastle Disease virus (NDV) and infectious bronchitis virus (IBV) vaccines with the protection induced by ILTV vaccines. The protection induced by CEO and TCO vaccines was evaluated when administered at 14 days of age alone or in combination with the B1 type strain of NDV (B1) and/or the Arkansas (ARK) and Massachusetts (MASS) serotypes of IBV vaccines. Two weeks after vaccination (28 days of age), the chickens were challenged with a virulent ILTV field strain (63140 isolate, group V genotype). Protection was evaluated at 5 and 7 days postchallenge by scoring clinical signs and quantifying the challenge virus load in the trachea using real-time PCR (qPCR). In addition, the viral load of the vaccine viruses (ILTV, NDV, and IBV) was quantified 3 and 5 days postvaccination also using qPCR. The results of this study indicate that the NDV (B1) and IBV (ARK) vaccines and a multivalent vaccine constituted by NDV (B1) and IBV (ARK and MASS) did not interfere with the protection induced by the CEO ILTV vaccine. However, the NDV (B1) and the multivalent (B1/MASS/ARK) vaccines interfered with the protection induced by the TCO vaccine (P < 0.05). Either in combination or by themselves, the NDV and IBV vaccines decreased the tracheal replication of the TCO vaccine and the protection induced by this vaccine, since the ILTV-vaccinated and -challenged chickens displayed significantly more severe clinical signs and ILTV load (P < 0.05) than chickens vaccinated with the TCO vaccine alone. Although NDV and IBV challenges were not performed, the antibody responses elicited by NDV and/or the IBV vaccinations were significantly reduced (P < 0.05) when applied in combination with the CEO vaccine.