A. N. J. Scott

Discrimination of Mycobacterium tuberculosis complex bacteria using novel VNTR-PCR targets

The lack of a convenient high-resolution strain-typing method has hampered the application of molecular epidemiology to the surveillance of bacteria of the Mycobacterium tuberculosis complex, particularly the monitoring of strains of Mycobacterium bovis. With the recent availability of genome sequences for strains of the M. tuberculosis complex, novel PCR-based M. tuberculosis-typing methods have been developed, which target the variable-number tandem repeats (VNTRs) of minisatellite-like mycobacterial interspersed repetitive units (MIRUs), or exact tandem repeats (ETRs). This paper describes the identification of seven VNTR loci in M. tuberculosis H37Rv, the copy number of which varies in other strains of the M. tuberculosis complex. Six of these VNTRs were applied to a panel of 100 different M. bovis isolates, and their discrimination and correlation with spoligotyping and an established set of ETRs were assessed. The number of alleles varied from three to seven at the novel VNTR loci, which differed markedly in their discrimination index. There was positive correlation between spoligotyping, ETR- and VNTR-typing. VNTR-PCR discriminates well between M. bovis strains. Thirty-three allele profiles were identified by the novel VNTRs, 22 for the ETRs and 29 for spoligotyping. When VNTR- and ETR-typing results were combined, a total of 51 different profiles were identified. Digital nomenclature and databasing were intuitive. VNTRs were located both in intergenic regions and annotated ORFs, including PPE (novel glycine-asparigine-rich) proteins, a proposed source of antigenic variation, where VNTRs potentially code repeating amino acid motifs. VNTR-PCR is a valuable tool for strain typing and for the study of the global molecular epidemiology of the M. tuberculosis complex. The novel VNTR targets identified in this study should additionally increase the power of this approach.

Molecular Basis of the Attenuation Exhibited by Molecularly Cloned Highly Passaged Chicken Anemia Virus Isolates

ABSTRACT Chimeric virus experiments indicated that the pathogenicity and monoclonal antibody reactivity differences between two molecularly cloned, highly passaged chicken anemia virus isolates could be attributed to the VP1 amino acid change at residue 89. The introduction of this change into a pathogenic cloned low-passage isolate was not sufficient to cause attenuation.

Antigenicity and pathogenicity characteristics of molecularly cloned chicken anaemia virus isolates obtained after multiple cell culture passages

Summary. The Cux-1 isolate of chicken anaemia virus (CAV), which had received 310 (P310) cell culture passages, was substantially less pathogenic than virus that had been passaged 13 times (P13). Molecularly cloned virus isolates, selected from the P310 and P13 virus populations using recombinant DNA cloning and transfection procedures, reacted differently with 4 CAV-specific monoclonal antibodies (MAbs), which had been raised to low-passage Cux-1 virus. In contrast to the strong immunofluorescence (IF) reactivities exhibited by all P13 cloned isolates tested, 80% and 57% of the P310 cloned isolates reacted weakly with MAbs 2A9 and 4H4, which are directed against conformational epitopes on the capsid protein, VP1. Sequence analysis of the VP1 coding regions possessed by ten P310 and two P13 cloned isolates showed that 6 amino acid changes within VP1 had been selected by multiple-cell culture passage. One of these at position 89 in VP1 appeared to be crucial for determining reactivity with MAb 2A9. Of nine P310 cloned isolates evaluated, 8 were substantially attenuated compared to the low-passage Cux-1 virus pool. It is concluded that the individual virus variants comprising the P310 virus pool differ with regards to their antigenicity and pathogenicity.

Development of a blocking enzyme-linked immunosorbent assay for the serological diagnosis of chicken anaemia virus.

The development and evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of serum antibody to chicken anaemia virus (CAV) are described. This test depends on the abilities of CAV-specific antibodies present in convalescent chicken serum to block the reaction between virus antigen, adsorbed to the ELISA plate. and a CAV-specific mouse monoclonal antibody (MAb), 2A9, that has been conjugated to horseradish peroxidase. The 2A9 MAb has been shown to react with 10 geographically different field isolates of CAV, a finding which indicates that the test will find worldwide application. In comparative experiments involving 525 serum samples from specific pathogen free and commercial breeder flocks, there was 98.5% agreement between the results obtained with the blocking ELISA and those obtained with an indirect ELISA developed previously in this laboratory. The blocking ELISA was found to have advantages in terms of speed and cost compared with the indirect ELISA format.

Characterisation of a chicken anaemia virus variant population that resists neutralisation with a group-specific monoclonal antibody.

Summary. A variant population of chicken anaemia virus (CAV), termed P310 2A9-resist, that resists neutralisation by the monoclonal antibody (MAb) 2A9, was selected from Cux-1 virus that had been passaged 310 times (P310) in MDCC-MSB1 cells. Substantially higher concentrations of MAb 2A9 were required to neutralise the selected virus compared to those required to neutralise a low-passage (P13) Cux-1 isolate. Virus neutralisation tests showed that serum from chickens infected with the P310 2A9-resist virus neutralised P13 virus and that serum from chickens infected with P13 virus conversely neutralised the P310 2A9-resist virus. MDCC-MSB1 cells infected with the P310 2A9-resist virus produced no staining with low dilutions (1:100) of Mab 2A9 in an indirect immunofluorescence (IF) test, whereas cells infected with P13 virus reacted positively at high MAb dilutions (1:80,000). Experimental infections of 1-day-old SPF chicks showed that the P310 2A9-resist virus was substantially attenuated. Chimaeric viruses constructed using PCR-amplified regions from the P310 2A9-resist and a pathogenic low-passage cloned Cux-1 isolate showed that the reduced MAb reactivity and attenuation exhibited by the P310 2A9-resist virus were mainly associated with a region encoding the N-terminal half of the 50 kDa capsid protein VP1 and C-terminal regions of VP2 and VP3. The nucleotide sequence of the protein-coding region of the P310 2A9-resist virus is reported and the amino acid sequences of the 3 encoded proteins compared with those of other Cux-1 isolates.

Investigation of the unstable attenuation exhibited by a chicken anaemia virus isolate

An attenuated chicken anaemia virus (CAV) isolate, cloned isolate 10, which was molecularly cloned from the Cuxhaven-1 CAV after 173 cell-culture passages, was shown previously to recover pathogenicity following 10 passages in young chicks. The consensus nucleotide sequence of the ‘revertant’ (Rev) virus, present as a tissue homogenate, differed from cloned isolate 10 at a single nucleotide residue (nucleotide 1739) that changed amino acid 287 of the capsid protein from alanine to aspartic acid. Subjecting Rev virus to 10 cell-culture passages re-selected viruses with an alanine at this amino acid position. Experimental infections using a molecularly cloned Rev virus isolate demonstrated that the mutation at nucleotide 1739 was not in itself responsible for the recovery of pathogenicity exhibited by the Rev virus. Additional sequence analyses of cloned amplicons provided evidence that the Rev virus population comprised minor, genetically different subpopulations, and provided an indication of CAVs potential for genetic change.

Transcriptomic Profiling of Virus-Host Cell Interactions following Chicken Anaemia Virus (CAV) Infection in an In Vivo Model

Chicken Anaemia Virus (CAV) is an economically important virus that targets lymphoid and erythroblastoid progenitor cells leading to immunosuppression. This study aimed to investigate the interplay between viral infection and the host’s immune response to better understand the pathways that lead to CAV-induced immunosuppression. To mimic vertical transmission of CAV in the absence of maternally-derived antibody, day-old chicks were infected and their responses measured at various time-points post-infection by qRT-PCR and gene expression microarrays. The kinetics of mRNA expression levels of signature cytokines of innate and adaptive immune responses were determined by qRT-PCR. The global gene expression profiles of mock-infected (control) and CAV-infected chickens at 14 dpi were also compared using a chicken immune-related 5K microarray. Although in the thymus there was evidence of induction of an innate immune response following CAV infection, this was limited in magnitude. There was little evidence of a Th1 adaptive immune response in any lymphoid tissue, as would normally be expected in response to viral infection. Most cytokines associated with Th1, Th2 or Treg subsets were down-regulated, except IL-2, IL-13, IL-10 and IFNγ, which were all up-regulated in thymus and bone marrow. From the microarray studies, genes that exhibited significant (greater than 1.5-fold, false discovery rate <0.05) changes in expression in thymus and bone marrow on CAV infection were mainly associated with T-cell receptor signalling, immune response, transcriptional regulation, intracellular signalling and regulation of apoptosis. Expression levels of a number of adaptor proteins, such as src-like adaptor protein (SLA), a negative regulator of T-cell receptor signalling and the transcription factor Special AT-rich Binding Protein 1 (SATB1), were significantly down-regulated by CAV infection, suggesting potential roles for these genes as regulators of viral infection or cell defence. These results extend our understanding of CAV-induced immunosuppression and suggest a global immune dysregulation following CAV infection.

Chicken anaemia virus evades host immune responses in transformed lymphocytes

Chicken anaemia virus (CAV) is a lymphotropic virus that causes anaemia and immunosuppression in chickens. Previously, we proposed that CAV evades host antiviral responses in vivo by disrupting T-cell signalling, but the precise cellular targets and modes of action remain elusive. In this study, we examined gene expression in Mareks disease virus-transformed chicken T-cell line MSB-1 after infection with CAV using both a custom 5K immune-focused microarray and quantitative real-time PCR at 24, 48 and 72 h post-infection. The data demonstrate an intricate equilibrium between CAV and the host gene expression, displaying subtle but significant modulation of transcripts involved in the T-cell, inflammation and NF-κB signalling cascades. CAV efficiently blocked the induction of type-I interferons and interferon-stimulated genes at 72 h. The cell expression pattern implies that CAV subverts host antiviral responses and that the transformed environment of MSB-1 cells offers an opportunistic advantage for virus growth.