Irit Davidson

Major histocompatibility complex class I is downregulated in Marek's disease virus infected chicken embryo fibroblasts and corrected by chicken interferon.

The major histocompatibility complex (MHC) is a part of the immune system which presents epitopes of intracellular antigens on the cell surface. MHC molecules have receptor-ligand binding affinities with T lymphocytes, permitting the latter to detect foreign intracellular infectious agents. Some pathogens, such as herpesviruses, have developed strategies of evading the host response by MHC. This pressure on the immune system brought, in turn, improvements in the antigen-presenting pathway, for example through the effect of interferon (IFN), which can upregulate MHC expression. The main objective of this work was on the one hand, to determine the abilities of three strains of Mareks disease virus (MDV), a chicken herpesvirus, in interfering with the expression of MHC class I molecules in chicken embryo fibroblasts. On the other hand, we analyzed the ability of IFN to reinstate this important immune capability to the infected cells. Our results show that only an oncogenic serotype 1 strain of MDV (RB1B) was able to markedly decrease MHC class I expression, and that addition of IFN reversed this MDV effect.

Genetic characterization of HA gene of low pathogenic H9N2 influenza viruses isolated in Israel during 2006-2012 periods

H9N2 influenza viruses are isolated in Israel since 2000 and became endemic. From November 2006 to the beginning of 2012, many H9N2 viruses were identified, all belonged to the Asian G1-like lineage represented by A/qu/Hong Kong/G1/97 (H9N2). In the present study, 66 isolates were selected for their hemagglutinin gene characterization. Most H9N2 isolates were distributed between two main groups, identified as the 4th and 5th introductions. The 5th introduction, was represented by a compact cluster containing viruses isolated in 2011–2012; the 4th introduction was subdivided into two subgroups, A and B, each containing at least two clusters, which can be identified as A-1, A-2, B-1, and B2, respectively. Genetic analysis of the deduced HA proteins of viruses, belonging to the 4th and 5th introductions, revealed amino acid variations in 79 out of 542 positions. All isolates had typical low pathogenicity motifs at the hemagglutinin (HA) cleavage site. Most viruses had leucine at position 216 in a receptor binding pocket that enables the virus to bind successfully with the cellular receptors intrinsic to mammals, including humans. It was shown that the differences between the HA proteins of viruses used for vaccine production and local field isolates increased in parallel with the duration and intensity of vaccine use, illustrating the genetic diversity of the H9N2 viruses in Israel.

Interferon-containing supernatants increase Marek's disease herpesvirus genomes and gene transcription levels, but not virion replication in vitro.

Viruses encounter the innate immune system immediately after infection of the host; specifically, soluble molecules that are both directly lethal and that initiate acquired immunity. Using the oncogenic Mareks disease alpha-herpesvirus (MDV) model, we quantified the effect of a interferon-containing supernatants (ICS), on MDV replication, gene transcription and antigen expression kinetics. We used an established cell culture system and a well-defined virulent MDV (RB-1B). RB-1B was cultured without ICS, or pretreated and then continuously treated with ICS. We compared (i) RB-1B infectivity; (ii) RB-1B growth by microscopy; (iii) numbers of cells expressing RB-1B antigens by flow cytometry; (iv) RB-1B-DNA load per cell by duplex real-time PCR, and (v) gene transcription kinetics for key MDV-life stages by duplex real-time reverse-transcriptase PCR (RT-PCR). ICS inhibited RB-1B infection, completion of productive life cycle and cell-to-cell infection. The numbers of cells expressing glycoprotein B (a kinetically late antigen) greatly decreased, but the numbers of cells expressing pp38 (a kinetically early antigen) decreased only slightly. The two greatest effects were increases in both RB-1B-DNA per infected cell and pp38 mRNA. We propose MDV has evolved to increase specific gene transcription and genome copies per cell to compensate for ICS. We speculate that the bi-directional shared pp38/origin of replication promoter, is central to this mechanism.

Quantitation of Marek's disease and chicken anemia viruses in organs of experimentally infected chickens and commercial chickens by multiplex real-time PCR.

SUMMARY.  The worldwide distribution of chicken anemia virus (CAV) and Mareks disease virus (MDV) is well documented. In addition to their economic significance in single- or dual-virus infections, the two viruses can often accompany various other pathogens and affect poultry health either directly, by causing tumors, anemia, and delayed growth, or indirectly, by aggravating other diseases, as a result of their immunosuppressive effects. After a decade of employing the molecular diagnosis of those viruses, which replaced conventional virus isolation, we present the development of a real-time multiplex PCR for the simultaneous detection of both viruses. The real-time PCRs for MDV and for CAV alone are more sensitive than the respective end-point PCRs. In addition, the multiplex real-time shows a similar sensitivity when compared to the single real-time PCR for each virus. The newly developed real-time multiplex PCR is of importance in terms of the diagnosis and detection of low copies of each virus, MDV and CAV in single- and in multiple-virus infections, and its applicability will be further evaluated. RESUMEN.  Cuantificación del virus de la enfermedad de Marek y del virus de la anemia infecciosa en los órganos de pollos infectados experimentalmente y de pollos comerciales mediante una prueba de PCR múltiple en tiempo real. La distribución mundial del virus de la anemia infecciosa del pollo (CAV) y del virus de la enfermedad de Marek (MDV) está bien documentada. Además de su importancia económica en infecciones simples o por los dos virus. Estos dos agentes a menudo pueden acompañar otros patógenos diversos y afectan a la salud de las aves de corral, ya sea directamente, por los tumores que causan, por la anemia o por retraso en el crecimiento, o indirectamente, al aumentar la severidad de otras enfermedades, como consecuencia de sus efectos inmunosupresores. Después de una década de emplear el diagnóstico molecular, que sustituyó el aislamiento viral convencional, se presenta el desarrollo de un método de PCR múltiple en tiempo real para la detección simultánea de ambos virus. Los métodos de PCR en tiempo real para el virus de Marek y para el virus de la anemia por separado son más sensibles que los respectivos métodos de PCR de punto final. Además, el procedimiento de PCR múltiple en tiempo real muestra una sensibilidad similar en comparación con los métodos de PCR en tiempo real por separado para cada virus. El método de PCR múltiple en tiempo real recién desarrollado es de importancia en términos de diagnóstico y para la detección de bajos niveles de copias para cada virus, para diagnosticar infecciones simples o múltiples y su aplicabilidad se seguirá evaluando.

Development of a reliable dual-gene amplification RT-PCR assay for the detection of Turkey Meningoencephalitis virus in Turkey brain tissues.

The Turkey Meningoencephalitis virus (TMEV) causes neuroparalytic signs, paresis, in-coordination, morbidity and mortality in turkeys. In parallel to the increased worldwide scientific interest in veterinary avian flaviviruses, including the Bagaza, Tembusu and Tembusu-related BYD virus, TMEV-caused disease also reemergence in commercial turkeys during late summer of 2010. While initially TMEV was detected by NS5-gene RT-PCR, subsequently, the env-gene RT-PCR was employed. As lately several inconsistencies were observed between the clinical, serological and molecular detection of the TMEV env gene, this study evaluated whether genetic changes occurred in the recently isolated viruses, and sought to optimize and improve the direct TMEV amplification from brain tissues of affected turkeys. The main findings indicated that no changes occurred during the years in the TMEV genome, but the PCR detection sensitivities of the env and NS5 genes differed. The RT-PCR and RNA purification were optimized for direct amplification from brain tissues without pre-replication of clinical samples in tissue cultures or in embryonated eggs. The amplification sensitivity of the NS5-gene was 10-100 times more than the env-gene when separate. The new dual-gene amplification RT-PCR was similar to that of the NS5 gene, therefore the assay can be considered as a reliable diagnostic assay. Cases where one of the two amplicons would be RT-PCR negative would alert and warn on the virus identity, and possible genetic changes. In addition, the biochemical environment of the dual-gene amplification reaction seemed to contribute in deleting non-specific byproducts that occasionally appeared in the singular RT-PCR assays on RNA purified from brain tissues.

Infectious laryngotracheitis virus (ILTV) vaccine intake evaluation by detection of virus amplification in feather pulps of vaccinated chickens.

Infectious laryngotracheitis (ILT) is a respiratory disease of poultry caused by an alphaherpesvirus, ILTV. The live vaccine is applied worldwide by drinking water or by the respiratory route, and by the vent application in Israel. No system of direct evaluation of the efficacy of vaccination exists today, except of antibody elicitation, which is an indirect indication of vaccination intake and might happen due to environment exposure. We suggest for the first time an assay for evaluating the accuracy of the vaccination process by spotting the spread of the live vaccine systemically, namely by virus detection in the feather shafts of the vaccinated birds. The feathers are particularly beneficial as they are easy to collect, non-lethal for the bird, therefore advantageous for monitoring purposes. Moreover, the continuous survey of the vaccine virus unveiled the different kinetics of viremia by the different vaccination routes; while after the vent vaccination the systemic viremia peaks during the first week afterwards, after two consecutive vaccine administration by drinking water with 6 day interval, the vireamia peaks only after the second administration. A robust amplification was needed because the vaccine ILTV was present in the bird in minute quantities compared to the wild-type virus. For the vaccine virus identification in feather shafts a nested real-time PCR for the TK ILTV gene was developed. The sensitivity of detection of the nested rtPCR was greater by 1000 compared to conventional nested PCR and 10 times that real-time PCR.

Differential diagnosis of fowlpox and infectious laryngotracheitis viruses in chicken diphtheritic manifestations by mono and duplex real-time polymerase chain reaction.

Infectious laryngotracheitis virus (ILTV) and fowlpox virus (FPV) cause diphtheritic lesions in chicken tracheas and can simultaneously infect the same bird. A differential molecular diagnostic test, the duplex real-time polymerase chain reaction, is now reported using ILTV and FPV vaccine viruses and clinical samples from chickens, either uninfected or naturally infected with ILTV or FPV, or with both viruses. The dual virus amplification by real-time polymerase chain reaction was demonstrated to behave similarly to monoplex amplification, in spite of the fact that the real-time exponential amplification plots of the vaccine viruses were more illustrative than those of the clinical samples.

Isolation and Identification of Highly Pathogenic Avian Influenza Virus Subtype H5N1 from Emus from the Ein Gedi Oasis by the Dead Sea

SUMMARY. An avian influenza virus (AIV), A/Emu/Israel/552/2010/ (H5N1), was isolated from a dead emu that was found in the Ein Gedi oasis near the Dead Sea. The virus molecular characterization was performed by reverse transcriptase–polymerase chain reaction (RT-PCR) and real-time RT-PCR using AIV subtype-specific primers. The virus was of high pathogenicity, according to its intravenous pathogenicity index of 2.85 and the nucleotide sequencing at the cleavage site of the hemagglutinin gene, GERRRKKR, which is typical for highly pathogenic chicken influenza A viruses.

Differential amplification of Marek’s disease CVI988 vaccine and of wild-type isolates from organs of commercial chickens using single or duplexed probes in real-time PCR

ABSTRACT The differentiation of Mareks disease virus (MDV)-infected and vaccinated animal (DIVA) test, based on the MDV pp38 gene was described by Baigent et al. [(2016). Real-time PCR for differential quantification of CVI988 vaccine and virulent MDV strains. Journal of Virological Methods, 233, 23-36], using similar primers and alternate probes for virulent MDV-1 and the vaccine CVI988 virus. We explored the assay’s applicability for commercial vaccines and commercial chickens, as the above-mentioned study employed tissue-cultured MDV strains and tissues from experimental trials. DNA of visceral organs and feathers of vaccinated or naturally infected chickens was used. Further, the applicability of the DIVA assay was evaluated using single or duplexed probes for the two viruses in the same amplification tube. Due to the high viral content in the commercial vaccines and in the clinical cases of MDV-1 infected commercial chickens, their examination by the MDV-1 DIVA real-time PCR was performed in one step. However, for the feather DNAs of commercially vaccinated birds, a step of pre-amplification was required. The MDV-1 DIVA real-time PCR performed as single probe in separate tubes using the Vir3 probe was very sensitive for virulent MDV-1 strains, but not very specific, as it also gave a clear signal with CVI988 vaccine virus. In contrast, the CVI vaccine probe was specific for CVI988, and did not recognize the MDV-1 strains. When both probes were present in one tube, the CVI probe showed a greater sensitivity for CV1988, while the Vir3 probe showed a much better specificity for virulent MDV-1.

Development of duplex dual-gene and DIVA real-time RT-PCR assays and use of feathers as a non-invasive sampling method for diagnosis of Turkey Meningoencephalitis Virus

ABSTRACT The avian flavivirus Turkey Meningoencephalitis Virus (TMEV) causes a neuroparalytic disease of commercial turkeys, expressed in paresis, incoordination, drooping wings and mortality that is controlled by vaccination. The molecular diagnosis using brain tissue RNA has now been upgraded by the development of a diagnostic dual-gene multiplex real-time PCR targeting the envelope and the non-structural NS5 gene, increasing the sensitivity by 10–100-fold compared to the previously existing assays. Based on the recent complete sequences of five TMEV isolates we have now developed a Differentiating Infected from Vaccinated Animals (DIVA) assay, to distinguish between wild-type TMEV strains and the vaccine virus. The DIVA assay was evaluated on commercial vaccines produced by two manufacturers, on RNA purified from brains of experimentally infected turkeys with TMEV strains, and on clinical samples collected between the years 2009 and 2015. We also investigated turkey feather pulps for their suitability to serve for TMEV detection, to avoid invasive sampling and bird killing. The parallel TMEV diagnosis in brain and feather-pulp RNA were similarly useful for diagnosis, at least in experimentally infected turkeys and in three cases of disease encountered in commercial flocks.