Experimental bluetongue virus infection of Culicoides austropalpalis, collected from a farm environment in Victoria, Australia.

Following the emerging bluetongue virus transmission in European temperate regions, we question the vector competence of the abundant Culicoides austropalpalis Lee and Reye in South-East temperate Australia. Field collected Culicoides midges were membrane fed with a bluetongue virus serotype 1 (BTV-1). The average feeding rate was 50%. After 13 days, survival rate was 25% and virus RNA presence was checked by quantitative PCR targeting viral genome segment 10. Virus RNA was found in 7.4% of individually tested females with relative viral RNA load values lower than freshly fed females, indicating that viral replication was low or null. A second qPCR targeting viral genome segment 1 confirmed the presence of virus RNA in only four out of 29 previously positive specimens. After 10 days culture on Culicoides cells, none of these four confimed positive samples did show subsequent cytopathogenic effect on Vero cells or BTV antigen detection by ELISA. As control for this virus activity detection, 12 days after microinjection of BTV-1, Culex annulirostris mosquitoes showed, after culture on Kc cells, cytopathogenic effect on Vero cells, with ELISA-confirmed infection. Despite its abundance in farm environment of the temperate Australian regions, the results of this study make C. austropalpalis of unlikely epidemiological importance in the transmission of BTV in Australia.

Attenuation of Bluetongue Virus (BTV) in an in ovo Model Is Related to the Changes of Viral Genetic Diversity of Cell-Culture Passaged BTV.

The embryonated chicken egg (ECE) is routinely used for the laboratory isolation and adaptation of Bluetongue virus (BTV) in vitro. However, its utility as an alternate animal model has not been fully explored. In this paper, we evaluated the pathogenesis of BTV in ovo using a pathogenic isolate of South African BTV serotype 3 (BTV-3) derived from the blood of an infected sheep. Endothelio- and neurotropism of BTV-3 were observed by immunohistochemistry of non-structural protein 1 (NS1), NS3, NS3/3a, and viral protein 7 (VP7) antigens. In comparing the pathogenicity of BTV from infectious sheep blood with cell-culture-passaged BTV, including virus propagated through a Culicoides-derived cell line (KC) or ECE, we found virus attenuation in ECE following cell-culture passage. Genomic analysis of the consensus sequences of segments (Seg)-2, -5, -6, -7, -8, -9, and -10 identified several nucleotide and amino-acid mutations among the cell-culture-propagated BTV-3. Deep sequencing analysis revealed changes in BTV-3 genetic diversity in various genome segments, notably a reduction of Seg-7 diversity following passage in cell culture. Using this novel approach to investigate BTV pathogenicity in ovo, our findings support the notion that pathogenic BTV becomes attenuated in cell culture and that this change is associated with virus quasispecies evolution.

Identification and genomic characterization of the first isolate of bluetongue virus serotype 5 detected in Australia.

Bluetongue virus (BTV), transmitted by midges (Culicoides sp), is distributed worldwide and causes disease in ruminants. In particular, BT can be a debilitating disease in sheep causing serious trade and socio-economic consequences at both local and global levels. Across Australia, a sentinel cattle herd surveillance program monitors the BTV activity. Prior to 2014, BTV-1, -2, -3, -7, -9, -15, -16, -20, -21 and -23 had been isolated in Australia, but no bluetongue disease has occurred in a commercial Australian flock. We routinely use a combination of serology, virus isolation, RT-PCR and next generation and conventional nucleotide sequencing technologies to detect and phylogenetically characterize incursions of novel BTV strains into Australia. Screening of Northern Territory virus isolates in 2015 revealed BTV-5, a serotype new to Australia. We derived the complete genome of this isolate and determined its phylogenetic relationship with exotic BTV-5 isolates. Gene segments 2, 6, 7 and 10 exhibited a close relationship with the South African prototype isolate RSArrrr/5. This was the first Australian isolation of a Western topotype of segment 10. Serological surveillance data highlighted the antigenic cross-reactivity between BTV-5 and BTV-9. Phylogenetic investigation of segments 2 and 6 of these serotypes confirmed their unconventional relationships within the BTV serogroup. Our results further highlighted a need for a revision of the current serologically based system for BTV strain differentiation and importantly, implied a potential for genome segments of pathogenic Western BTV strains to rapidly enter Southeast Asia. This emphasized a need for continued high-level surveillance of vectors and viruses at strategic locations in the north of Australia The expansion of routine characterization and classification of BTV to a whole genome approach is recommended, to better monitor the presence and level of establishment of novel Western topotype segments within the Australian episystem.

Purification and characterisation of immunoglobulins from the Australian black flying fox (Pteropus alecto) using anti-fab affinity chromatography reveals the low abundance of IgA.

There is now an overwhelming body of evidence that implicates bats in the dissemination of a long list of emerging and re-emerging viral agents, often causing illnesses or death in both animals and humans. Despite this, there is a paucity of information regarding the immunological mechanisms by which bats coexist with highly pathogenic viruses. Immunoglobulins are major components of the adaptive immune system. Early studies found bats may have quantitatively lower antibody responses to model antigens compared to conventional laboratory animals. To further understand the antibody response of bats, the present study purified and characterised the major immunoglobulin classes from healthy black flying foxes, Pteropus alecto. We employed a novel strategy, where IgG was initially purified and used to generate anti-Fab specific antibodies. Immobilised anti-Fab specific antibodies were then used to capture other immunoglobulins from IgG depleted serum. While high quantities of IgM were successfully isolated from serum, IgA was not. Only trace quantities of IgA were detected in the serum by mass spectrometry. Immobilised ligands specific to IgA (Jacalin, Peptide M and staphylococcal superantigen-like protein) also failed to capture P. alecto IgA from serum. IgM was the second most abundant serum antibody after IgG. A survey of mucosal secretions found IgG was the dominant antibody class rather than IgA. Our study demonstrates healthy P. alecto bats have markedly less serum IgA than expected. Higher quantities of IgG in mucosal secretions may be compensation for this low abundance or lack of IgA. Knowledge and reagents developed within this study can be used in the future to examine class-specific antibody response within this important viral host.

Arabidopsis thaliana Y-family DNA polymerase eta catalyses translesion synthesis and interacts functionally with PCNA2.

SUMMARY: Upon blockage of chromosomal replication by DNA lesions, Y-family polymerases interact with monoubiquitylated proliferating cell nuclear antigen (PCNA) to catalyse translesion synthesis (TLS) and restore replication fork progression. Here, we assessed the roles of Arabidopsis thaliana POLH, which encodes a homologue of Y-family polymerase eta (Poleta), PCNA1 and PCNA2 in TLS-mediated UV resistance. A T-DNA insertion in POLH sensitized the growth of roots and whole plants to UV radiation, indicating that AtPoleta contributes to UV resistance. POLH alone did not complement the UV sensitivity conferred by deletion of yeast RAD30, which encodes Poleta, although AtPoleta exhibited cyclobutane dimer bypass activity in vitro, and interacted with yeast PCNA, as well as with Arabidopsis PCNA1 and PCNA2. Co-expression of POLH and PCNA2, but not PCNA1, restored normal UV resistance and mutation kinetics in the rad30 mutant. A single residue difference at site 201, which lies adjacent to the residue (lysine 164) ubiquitylated in PCNA, appeared responsible for the inability of PCNA1 to function with AtPoleta in UV-treated yeast. PCNA-interacting protein boxes and an ubiquitin-binding motif in AtPoleta were found to be required for the restoration of UV resistance in the rad30 mutant by POLH and PCNA2. These observations indicate that AtPoleta can catalyse TLS past UV-induced DNA damage, and links the biological activity of AtPoleta in UV-irradiated cells to PCNA2 and PCNA- and ubiquitin-binding motifs in AtPoleta.

NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens.

For over 30 years a phospholipase C enzyme called alpha-toxin was thought to be the key virulence factor in necrotic enteritis caused by Clostridium perfringens. However, using a gene knockout mutant we have recently shown that alpha-toxin is not essential for pathogenesis. We have now discovered a key virulence determinant. A novel toxin (NetB) was identified in a C. perfringens strain isolated from a chicken suffering from necrotic enteritis (NE). The toxin displayed limited amino acid sequence similarity to several pore forming toxins including beta-toxin from C. perfringens (38% identity) and alpha-toxin from Staphylococcus aureus (31% identity). NetB was only identified in C. perfringens type A strains isolated from chickens suffering NE. Both purified native NetB and recombinant NetB displayed cytotoxic activity against the chicken leghorn male hepatoma cell line LMH; inducing cell rounding and lysis. To determine the role of NetB in NE a netB mutant of a virulent C. perfringens chicken isolate was constructed by homologous recombination, and its virulence assessed in a chicken disease model. The netB mutant was unable to cause disease whereas the wild-type parent strain and the netB mutant complemented with a wild-type netB gene caused significant levels of NE. These data show unequivocally that in this isolate a functional NetB toxin is critical for the ability of C. perfringens to cause NE in chickens. This novel toxin is the first definitive virulence factor to be identified in avian C. perfringens strains capable of causing NE. Furthermore, the netB mutant is the first rationally attenuated strain obtained in an NE-causing isolate of C. perfringens; as such it has considerable vaccine potential.