Henipaviruses and lyssaviruses target nucleolar treacle protein and regulate ribosomal RNA synthesis.

The nucleolus is a common target of viruses and viral proteins, but for many viruses the functional outcomes and significance of this targeting remains unresolved. Recently, the first intranucleolar function of a protein of a cytoplasmically-replicating negative-sense RNA virus (NSV) was identified, with the finding that the matrix (M) protein of Hendra virus (HeV) (genus Henipavirus, family Paramyxoviridae) interacts with Treacle protein within nucleolar subcompartments and mimics a cellular mechanism of the nucleolar DNA-damage response (DDR) to suppress ribosomal RNA (rRNA) synthesis. Whether other viruses utilise this mechanism has not been examined. We report that sub-nucleolar Treacle targeting and modulation is conserved between M proteins of multiple Henipaviruses, including Nipah virus and other potentially zoonotic viruses. Furthermore, this function is also evident for P3 protein of rabies virus, the prototype virus of a different RNA virus family (Rhabdoviridae), with Treacle depletion in cells also found to impact virus production. These data indicate that unrelated proteins of viruses from different families have independently developed nucleolar/Treacle targeting function, but that modulation of Treacle has distinct effects on infection. Thus, subversion of Treacle may be an important process in infection by diverse NSVs, and so could provide novel targets for antiviral approaches with broad specificity.

Reagents for detection of Rift Valley fever virus infection in sheep.

Rift Valley fever virus (RVFV) causes Rift Valley fever (RVF), resulting in morbidity and mortality in humans and ruminants. Evidence of transboundary outbreaks means that RVFV remains a threat to human health and livestock industries in countries that are free from the disease. To enhance surveillance capability, methods for detection of RVFV are required. The generation of reagents suitable for the detection of RVFV antigen in formalin-fixed, paraffin-embedded tissues from infected animals have been developed and are described herein. Recombinant nucleoprotein (rNP) was expressed in Escherichia coli and purified using immobilized metal ion affinity chromatography. Purified rNP was used as an immunogen to produce anti-NP polyclonal antisera in rabbits for use in detection of RVFV NP in experimentally infected animals by immunohistochemistry. Antisera raised in rabbits against rNP were able to recognize viral NP antigen in fixed infected Vero cell pellets and sheep liver. Therefore, the methods and reagents described herein are useful in assays for detection of RVFV infections in animals, for research and surveillance purposes.

Whole Transcriptome Analysis of Aedes albopictus Mosquito Head and Thorax Post-Chikungunya Virus Infection.

Chikungunya virus (CHIKV) is transmitted by Aedes mosquitoes and causes prolonged arthralgia in patients. After crossing the mosquito midgut barrier, the virus disseminates to tissues including the head and salivary glands. To better understand the interaction between Aedes albopictus and CHIKV, we performed RNASeq analysis on pools of mosquito heads and parts of the thorax 8 days post infection, which identified 159 differentially expressed transcripts in infected mosquitos compared to uninfected controls. After validation using RT-qPCR (reverse transcriptase-quantitative polymerase chain reaction), inhibitor of Bruton's tyrosine kinase (BTKi), which has previously been shown to be anti-inflammatory in mammals after viral infection, was further evaluated for its functional significance. Knockdown of BTKi using double-stranded RNA in a mosquito cell line showed no significant difference in viral RNA or infectivity titer. However, BTKi gene knocked-down cells showed increased apoptosis 24 hours post-infection compared with control cells, suggesting involvement of BTKi in the mosquito response to viral infection. Since BTK in mammals promotes an inflammatory response and has been shown to be involved in osteoclastogenesis, a hallmark of CHIKV pathogenesis, our results suggest a possible conserved mechanism at play between mosquitoes and mammals. Taken together, these results will add to our understanding of Aedes Albopictus interactions with CHIKV.

Evolution of high pathogenicity of H5 avian influenza virus: haemagglutinin cleavage site selection of reverse-genetics mutants during passage in chickens.

Low pathogenicity avian influenza viruses (LPAIVs) are generally asymptomatic in their natural avian hosts. LPAIVs can evolve into highly pathogenic forms, which can affect avian and human populations with devastating consequences. The switch to highly pathogenic avian influenza virus (HPAIV) from LPAIV precursors requires the acquisition of multiple basic amino acids in the haemagglutinin cleavage site (HACS) motif. Through reverse genetics of an H5N1 HPAIV, and experimental infection of chickens, we determined that viruses containing five or more basic amino acids in the HACS motif were preferentially selected over those with three to four basic amino acids, leading to rapid replacement with virus types containing extended HACS motifs. Conversely, viruses harbouring low pathogenicity motifs containing two basic amino acids did not readily evolve to extended forms, suggesting that a single insertion of a basic amino acid into the cleavage site motif of low-pathogenic viruses may lead to escalating selection for extended motifs. Our results may explain why mid-length forms are rarely detected in nature. The stability of the short motif suggests that pathogenicity switching may require specific conditions of intense selection pressure (such as with high host density) to boost selection of the initial mid-length HACS forms.

Proteomics informed by transcriptomics reveals Hendra virus sensitizes bat cells to TRAIL-mediated apoptosis.

BACKGROUND: Bats are a major reservoir of emerging infectious viruses. Many of these viruses are highly pathogenic to humans however bats remain asymptomatic. The mechanism by which bats control viral replication is unknown. Here we utilize an integrated approach of proteomics informed by transcriptomics to compare the response of immortalized bat and human cells following infection with the highly pathogenic bat-borne Hendra virus (HeV). RESULTS: The host response between the cell lines was significantly different at both the mRNA and protein levels. Human cells demonstrated minimal response eight hours post infection, followed by a global suppression of mRNA and protein abundance. Bat cells demonstrated a robust immune response eight hours post infection, which led to the up-regulation of apoptosis pathways, mediated through the tumor necrosis factor-related apoptosis inducing ligand (TRAIL). HeV sensitized bat cells to TRAIL-mediated apoptosis, by up-regulating death receptor transcripts. At 48 and 72 hours post infection, bat cells demonstrated a significant increase in apoptotic cell death. CONCLUSIONS: This is the first study to comprehensively compare the response of bat and human cells to a highly pathogenic zoonotic virus. An early induction of innate immune processes followed by apoptosis of virally infected bat cells highlights the possible involvement of programmed cell death in the host response. Our study shows for the first time a side-by-side high-throughput analysis of a dangerous zoonotic virus in cell lines derived from humans and the natural bat host. This enables a way to search for divergent mechanisms at a molecular level that may influence host pathogenesis.

A nucleosomal region important for ensuring proper interactions between the transcription elongation factor Spt16 and transcribed genes in Saccharomyces cerevisiae.

The highly conserved FACT (FAcilitates Chromatin Transactions) histone chaperone assists in the transcription elongation process first by facilitating the removal of histones in front of transcribing RNA polymerase II (Pol II) and then by contributing to nucleosome reassembly in the wake of Pol II passage. Whereas it is well established that FACT localizes across actively transcribed genes, the mechanisms that regulate FACT recruitment to and disengagement from chromatin during transcription still remain to be elucidated. Using the Saccharomyces cerevisiae model system, we previously showed that a histone H3 mutant--H3-L61W--greatly perturbs interactions between the yeast FACT (yFACT) complex and chromatin during transcription, resulting in a pronounced shift in yFACT occupancy toward the 3' ends of transcribed genes. In the present study we report that two histone H4 mutants-H4-R36A and H4-K31E-alter the association pattern of the yFACT subunit Spt16 across transcribed genes in a fashion similar to that seen for H3-L61W. Interestingly, H4-R36, H4-K31, and H3-L61 are in close proximity to each other on the side of the nucleosome. We also provide evidence that the H4-R36A and H3-L61W mutants impair proper Spt16-chromatin interactions by perturbing a common process. Collectively, our results suggest that a nucleosomal region encompassing the H4-R36, H4-K31, and H3-L61 residues plays an important role in ensuring proper association of yFACT across transcribed genes.

Transcriptome analysis of pigeon milk production - role of cornification and triglyceride synthesis genes.

BACKGROUND: The pigeon crop is specially adapted to produce milk that is fed to newly hatched young. The process of pigeon milk production begins when the germinal cell layer of the crop rapidly proliferates in response to prolactin, which results in a mass of epithelial cells that are sloughed from the crop and regurgitated to the young. We proposed that the evolution of pigeon milk built upon the ability of avian keratinocytes to accumulate intracellular neutral lipids during the cornification of the epidermis. However, this cornification process in the pigeon crop has not been characterised. RESULTS: We identified the epidermal differentiation complex in the draft pigeon genome scaffold and found that, like the chicken, it contained beta-keratin genes. These beta-keratin genes can be classified, based on sequence similarity, into several clusters including feather, scale and claw keratins. The cornified cells of the pigeon crop express several cornification-associated genes including cornulin, S100-A9 and A16-like, transglutaminase 6-like and the pigeon 'lactating' crop-specific annexin cp35. Beta-keratins play an important role in 'lactating' crop, with several claw and scale keratins up-regulated. Additionally, transglutaminase 5 and differential splice variants of transglutaminase 4 are up-regulated along with S100-A10. CONCLUSIONS: This study of global gene expression in the crop has expanded our knowledge of pigeon milk production, in particular, the mechanism of cornification and lipid production. It is a highly specialised process that utilises the normal keratinocyte cellular processes to produce a targeted nutrient solution for the young at a very high turnover.

Novel paramyxoviruses in free-ranging European bats.

The zoonotic potential of paramyxoviruses is particularly demonstrated by their broad host range like the highly pathogenic Hendra and Nipah viruses originating from bats. But while so far all bat-borne paramyxoviruses have been identified in fruit bats across Africa, Australia, South America, and Asia, we describe the detection and characterization of the first paramyxoviruses in free-ranging European bats. Moreover, we examined the possible impact of paramyxovirus infection on individual animals by comparing histo-pathological findings and virological results. Organs from deceased insectivorous bats of various species were sampled in Germany and tested for paramyxovirus RNA in parallel to a histo-pathological examination. Nucleic acids of three novel paramyxoviruses were detected, two viruses in phylogenetic relationship to the recently proposed genus Jeilongvirus and one closely related to the genus Rubulavirus. Two infected animals revealed subclinical pathological changes within their kidneys, suggestive of a similar pathogenesis as the one described in fruit bats experimentally infected with Hendra virus.Our findings indicate the presence of bat-born paramyxoviruses in geographic areas free of fruit bat species and therefore emphasize a possible virus-host co-evolution in European bats. Since these novel viruses are related to the very distinct genera Rubulavirus and Jeilongvirus, a similarly broad genetic diversity among paramyxoviruses in other Microchiroptera compared to Megachiroptera can be assumed. Given that the infected bats were either found in close proximity to heavily populated human habitation or areas of intensive agricultural use, a potential risk of the emergence of zoonotic paramyxoviruses in Europe needs to be considered.

Menangle virus, a pteropid bat paramyxovirus infectious for pigs and humans, exhibits tropism for secondary lymphoid organs and intestinal epithelium in weaned pigs.

This study is the first report of experimental infection and transmission of Menangle virus (MenPV) in pigs. Isolated in 1997 from piglets that were stillborn at a large commercial piggery in New South Wales, Australia, MenPV is a recently identified paramyxovirus of bat origin that causes severe reproductive disease in pigs and an influenza-like illness, with a rash, in humans. Although successfully eradicated from the infected piggery, the virus was only isolated from affected fetuses and stillborn piglets during the period of reproductive disease, and thus the mode of transmission between pigs was not established. To investigate the pathogenesis of MenPV, we undertook time-course studies in 6-week-old pigs following intranasal administration of a low-passage, non-plaque-purified isolate from the lung of an infected stillborn piglet. Viraemia was of short duration and low titre, as determined by real-time RT-PCR and virus isolation. Following an incubation period of 2-3 days, virus was shed in nasal and oral secretions, faeces and urine, typically for less than 1 week. Cessation of shedding correlated with the development of neutralizing antibodies in sera. Secondary lymphoid organs and intestine were identified, using quantitative real-time RT-PCR, as major sites of viral replication and dissemination, and this was confirmed by positive immunolabelling of viral antigen within various lymphoid tissues and intestinal epithelium. These data provide new insights into the pathogenesis of MenPV in weaned pigs, and will facilitate future control and eradication programmes should it ever re-emerge in the pig population.

Mutant versions of the S. cerevisiae transcription elongation factor Spt16 define regions of Spt16 that functionally interact with histone H3.

In eukaryotic cells, the highly conserved FACT (FAcilitates Chromatin Transcription) complex plays important roles in several chromatin-based processes including transcription initiation and elongation. During transcription elongation, the FACT complex interacts directly with nucleosomes to facilitate histone removal upon RNA polymerase II (Pol II) passage and assists in the reconstitution of nucleosomes following Pol II passage. Although the contribution of the FACT complex to the process of transcription elongation has been well established, the mechanisms that govern interactions between FACT and chromatin still remain to be fully elucidated. Using the budding yeast Saccharomyces cerevisiae as a model system, we provide evidence that the middle domain of the FACT subunit Spt16--the Spt16-M domain--is involved in functional interactions with histone H3. Our results show that the Spt16-M domain plays a role in the prevention of cryptic intragenic transcription during transcription elongation and also suggest that the Spt16-M domain has a function in regulating dissociation of Spt16 from chromatin at the end of the transcription process. We also provide evidence for a role for the extreme carboxy terminus of Spt16 in functional interactions with histone H3. Taken together, our studies point to previously undescribed roles for the Spt16 M-domain and extreme carboxy terminus in regulating interactions between Spt16 and chromatin during the process of transcription elongation.