Replication of Bunyamwera Virus in Aedes albopictus C6/36 Cells: Establishment and Maintenance of a Persistent Infection

Bunyamwera virus is a member of the Bunyaviridae, a large family of mainly arthropod-borne viruses which possess a tri-segmented RNA genome comprising three segments of single-stranded RNA, designated L (large), M (medium) and S (small). There are five recognised genera within the Bunyaviridae-Bunyavirus, Hantavirus, Nairovirus, Phlebovirus and Tospovirus. Bunyamwera virus is the prototype virus of the Bunyavirus genus and each of its genome segments are of negative polarity. In common with other arboviruses Bunyamwera virus replicates in its vector species (the mosquito) without causing overt detrimental effects and establishes a persistent infection which lasts for the life time of the mosquito. Upon transmission to a susceptible vertebrate host, Bunyamwera virus causes an acute infection. These disparate host-dependent outcomes of infection can be reproduced in tissue culture. Bunyamwera virus undergoes a lytic replication cycle in mammalian cells which results in cell death, but establishes a long-term, non-cytocidal, persistent infection in cultures of mosquito cells. In this thesis, the molecular mechanisms leading to the establishment of persistent Bunyamwera virus infection of a cloned mosquito cell line, Aedes albopictus C6/36 (C6/36) cells, and the factors responsible for the maintenance of the persistent infection, were investigated. Viral protein and RNA synthesis were compared in BHK and C6/36 cells infected with Bunyamwera virus. In BHK cells, host protein synthesis was inhibited and viral protein synthesis was detected until the cells died. In C6/36 cells, host protein synthesis continued throughout the infection, but viral protein synthesis declined from maximum levels at 24 h pi to barely detectable levels at 36 h pi, despite the presence of translatable S mRNA. The levels of encapsidated L and M RNAs in the C6/36 cells declined after 24 h pi, but encapsidated S RNA species continued to accumulate. In the BHK cell infection, encapsidated L, M and S RNA species continued to accumulate up to 48 h pi. Overall, relatively more S segment RNA than L or M segment RNA accumulated in infected C6/36 cells compared to BHK cells. The decline in viral protein syrithesis and genome replication observed in the C6/36 cells after 24 h pi, was paralleled by an accumulation of encapsidated S mRNA. Encapsidation of S mRNA may have a role to play in preventing the cytopathic effects of Bunyamwera virus infection in C6/36 cells by limiting translation of this viral mRNA. A persistent infection of C6/36 cells was established with a biologically cloned stock of Bunyamwera virus (wtL9BUN virus) and the persistently infected cell line (C6/36/BUN) was passaged at weekly intervals for over a year without cytopathic effects. The titre of virus released from the cells, the susceptibility of the cells to superinfection with homologous virus, and the level of viral RNA in the cells at different passages fluctuated markedly, but there was no simple relationship between virus titre, superinfectibility and levels of viral RNA. There was an alteration in the plaque-phenotype of virus released from the persistently infected culture with increasing passage level. By passage 15 the virus released from the C6/36/BUN cell line produced small, cloudy plaques on BHK cell monolayers, in contrast to the clear lytic plaques produced by the wtL9BUN virus. Direct RNA sequence analysis was carried out on the S RNA segments of viruses plaque-purified from the supernatant culture fluid of the C6/36-PI LO cell line established by Elliott and Wilkie. Only one nucleotide substitution, in a non-conserved region of the viral genome, was detected in viral isolate BUN18.10 (which was isolated from the persistently infected culture after it had been passaged for four months) suggesting that genetic drift during persistent Bunyamwera virus infection of C6/36 cells was minimal. However, Northern blot analysis of viral RNA extracted from different passage levels of the C6/36/BUN cell line revealed that polymerase errors which generated subgenomic L RNAs, and S RNA species larger than the standard S RNA segment, did occur in the persistently infected cells. The defective viral RNA species were not efficiently encapsidated by N protein. There was a strong selection for encapsidation of full-length S RNAs in the persistently infected cells and the majority of intracellular nucleocapsids contained standard-sized S RNA species. Stable cell lines were established with cells cloned from the persistently infected C6/36/BUN cell line. The cloned cells were heterogeneous in their ability to produce virus, and their susceptibility to superinfection with wtL9BUN virus, and in the levels of intracellular viral RNA they contained. Individual clones contained a single prominent defective L RNA species and resistance of the cells to superinfection appeared to correlate more with the levels of full-length viral genomic RNA resident in the cells than with the amount or occurence of defective viral RNAs

AAV2-mediated in vivo immune gene therapy of solid tumours

Abstract Background Many strategies have been adopted to unleash the potential of gene therapy for cancer, involving a wide range of therapeutic genes delivered by various methods. Immune therapy has become one of the major strategies adopted for cancer gene therapy and seeks to stimulate the immune system to target tumour antigens. In this study, the feasibility of AAV2 mediated immunotherapy of growing tumours was examined, in isolation and combined with anti-angiogenic therapy. Methods Immune-competent Balb/C or C57 mice bearing subcutaneous JBS fibrosarcoma or Lewis Lung Carcinoma (LLC) tumour xenografts respectively were treated by intra-tumoural administration of AAV2 vector encoding the immune up-regulating cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) and the co-stimulatory molecule B7-1 to subcutaneous tumours, either alone or in combination with intra-muscular (IM) delivery of AAV2 vector encoding Nk4 14 days prior to tumour induction. Tumour growth and survival was monitored for all animals. Cured animals were re-challenged with tumourigenic doses of the original tumour type. In vivo cytotoxicity assays were used to investigate establishment of cell-mediated responses in treated animals. Results AAV2-mediated GM-CSF, B7-1 treatment resulted in a significant reduction in tumour growth and an increase in survival in both tumour models. Cured animals were resistant to re-challenge, and induction of T cell mediated anti-tumour responses were demonstrated. Adoptive transfer of splenocytes to naïve animals prevented tumour establishment. Systemic production of Nk4 induced by intra-muscular (IM) delivery of Nk4 significantly reduced subcutaneous tumour growth. However, combination of Nk4 treatment with GM-CSF, B7-1 therapy reduced the efficacy of the immune therapy. Conclusions Overall, this study demonstrates the potential for in vivo AAV2 mediated immune gene therapy, and provides data on the inter-relationship between tumour vasculature and immune cell recruitment

Validation of a Lysis Buffer Containing 4 M Guanidinium Thiocyanate (GITC)/ Triton X-100 for Extraction of SARS-CoV-2 RNA for COVID-19 Testing: Comparison of Formulated Lysis Buffers Containing 4 to 6 M GITC, Roche External Lysis Buffer and Qiagen RTL Lysis Buffer

The COVID-19 pandemic has resulted in increased need for diagnostic testing using reverse transcriptase real-time PCR (RT-PCR). An exponential increase in demand has resulted in a shortage of numerous reagents in particular those associated with the lysis buffer required to extract the viral RNA. Herein, we describe a rapid collective effort by hospital laboratory scientists, academic researchers and the biopharma industry to generate a validated lysis buffer. We have formulated a 4M Guanidinium thiocyanate (GITC)/ Triton X-100 Lysis buffer which provides comparable results with the recommended reagents. This buffer will ease the burden on hospital labs in their heroic efforts diagnose a large population of patients

Cas9/gRNA targeted excision of cystic fibrosis-causing deep-intronic splicing mutations restores normal splicing of CFTR mRNA.

Cystic Fibrosis is an autosomal recessive disorder caused by mutations in the CFTR gene. CRISPR mediated, template-dependent homology-directed gene editing has been used to correct the most common mutation, c.1521_1523delCTT / p.Phe508del (F508del) which affects ~70% of individuals, but the efficiency was relatively low. Here, we describe a high efficiency strategy for editing of three different rare CFTR mutations which together account for about 3% of individuals with Cystic Fibrosis. The mutations cause aberrant splicing of CFTR mRNA due to the creation of cryptic splice signals that result in the formation of pseudoexons containing premature stop codons c.1679+1634A>G (1811+1.6kbA>G) and c.3718-2477C>T (3849+10kbC>T), or an out-of-frame 5' extension to an existing exon c.3140-26A>G (3272-26A>G). We designed pairs of Cas9 guide RNAs to create targeted double-stranded breaks in CFTR either side of each mutation which resulted in high efficiency excision of the target genomic regions via non-homologous end-joining repair. When evaluated in a mini-gene splicing assay, we showed that targeted excision restored normal splicing for all three mutations. This approach could be used to correct aberrant splicing signals or remove disruptive transcription regulatory motifs caused by deep-intronic mutations in a range of other genetic disorders

Pax5 and E47 regulate AID and class switch recombination in young and aged BALB/c mice (84.6)

Abstract Our laboratory has shown that stimulated B cells from senescent mice (19 to 24 months) have an intrinsic defect in class switch recombination (CSR) that is coincident with reduced E47. In aging, the decrease in E47 correlates proportionally with levels of activation induced cytidine deaminase (Aicda/AID) transcripts; but, other transcription factors may be involved. Pax5 has been shown to play a role in CSR. The purpose of the present study was to determine whether 1) Pax5 is decreased with age, 2) the decrease in Pax5 correlates with decreased CSR in aged B cells, 3) increased E47 and/or Pax5 can rescue the decreased CSR observed in these cells and 4) depletion of Pax5 down-regulates E47 and/or AID expression. Here, we demonstrate that E47 and Pax5 are both down-regulated in mature splenic B cells of aged mice. Endogenous E47, Pax5 and IgG1 are increased in old as well as young primary stimulated B cells in response to exogenously expressed E47. We successfully rescued levels of both E47 and Pax5 mRNA and protein in transduced splenic B cells of aged mice when compared to young untransduced controls. Restoration of Pax5 expression increased E47, AID and CSR in B cells from aged mice. Conversely, silencing Pax5 by means of siRNA transfection decreased E47 and AID expression significantly. These results suggest a possible feedback mechanism between E47 and Pax5. Our results show that E47 and Pax5 cooperate to regulate AID and CSR in both young and old B lymphocytes