The widespread devastation from Huanglongbing, also known as citrus greening, and several damaging insect pests continues to hamper Florida citrus growers and requires environmentally harmful insecticides for adequate control. Repeated applications of these insecticides for sufficient control spurred research interest in insect-specific viruses (ISVs). ISVs used as a biocontrol may offer growers increased specificity of insect targets and the genomic potential for lethal modifications. To discover prospective ISVs of these citrus pests, we employed high-throughput sequencing, which has revolutionized virome studies of these biological organisms. With the intent to increase our source of potential viruses for future bio-insecticidal purposes, we screened for novel ISVs within seven economically destructive Florida citrus pests, specifically the black citrus aphid (Aphis aurantii), green citrus (or spirea) aphid (Aphis spiraecola), Asian citrus psyllid (Diaphorina citri), citrus leafminer (Phyllocnistis citrella), brown soft scale (Coccus hesperidum), Florida wax scale (Ceroplastes floridensis), and sharp-nosed leafhopper (Scaphytopius spp.). Most insects in this study originated from citrus grove populations, whereas some were collected from rutaceous greenhouse plants. For Asian citrus psyllid, the insect vector transmitting the bacterial agent of Huanglongbing, 'Candidatus Liberibacter asiaticus', we investigated differences in presumptive ISV profiles between laboratory and field-collected populations, as well as nymph versus adult populations. Ten putative, novel viral sequences were discovered and characterized collectively from these pests. Overall, this study contributes to the cumulative knowledge of ISVs associated with diverse insect populations, uncovers resources for future downstream bio-insecticidal modifications, and illuminates the ongoing viral diversity still in need of investigation.

Growers have depended on the specificity and efficacy of streptomycin and oxytetracycline as a part of their plant disease arsenal since the middle of the 20th century. With climate change intensifying plant bacterial epidemics, the established success of these antibiotics remains threatened. Our strong reliance on certain antibiotics for devastating diseases eventually gave way to resistance development. Although antibiotics in plant agriculture equal to less than 0.5% of overall antibiotic use in the United States, it is still imperative for humans to continue to monitor usage, environmental residues, and resistance in bacterial populations. This review provides an overview of the history and use, resistance and mitigation, regulation, environmental impact, and economics of antibiotics in plant agriculture. Bacterial issues, such as the ongoing Huanglongbing (citrus greening) epidemic in Florida citrus production, may need antibiotics for adequate control. Therefore, preserving the efficacy of our current antibiotics by utilizing more targeted application methods, such as trunk injection, should be a major focus. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

The citrus industry of Florida faces insurmountable challenges against the destructive diseases citrus tristeza and Huanglongbing (HLB, or citrus greening). Though the tristeza causal agent, citrus tristeza virus (CTV), has been in Florida decades longer than HLB, growers have concentrated most of their efforts on combating the more detrimental HLB. The Asian citrus psyllid (Diaphorina citri; ACP) is the insect vector of the bacterial pathogen Candidatus Liberibacter asiaticus and transmits the incurable HLB to all commercial citrus. During our searches for biological and viral controls against the ACP, we consistently detected sequences of CTV in Florida field populations of ACP. This unexpected finding led us to investigate whether ACPs collected from young shoots could be used as a tool to survey CTV in Florida citrus groves. We first surveyed for the most common CTV strains in Florida (T30, T36, and VT/T68) in citrus trees on mostly sour orange (Citrus aurantium) rootstock, the rootstock susceptible to CTV decline. Out of 968 trees sampled across five years (2018-2022), approximately 8.2% were positive for CTV, with more than half of the CTV-positive trees infected with strain T30. Simultaneously, we looked at CTV strains in ACPs during this time and found that approximately 88% of pooled adult and nymph ACPs also had CTV, with over half the positive samples having the T36 strain. As a result of the much higher CTV incidences in the ACPs, we conducted a second investigation into whether we could more easily detect the same CTV strains in ACP nymphs as in CTV-infected citrus tissue. After individually sampling 43 trees and pooling the nymphs from each tree, we detected CTV at about the same incidence in the citrus tissue and the nymphs, but with much less ACP tissue, time, and resources required for detection compared to citrus tissue. Results from this study illustrate the sustained threat of CTV to Florida citrus and demonstrate the ACP as a potential bioindicator for CTV.

The destructive citrus disease, Huanglongbing (HLB) or citrus greening, continues to devastate Florida's citrus industry. A hemipteran insect, the Asian citrus psyllid (ACP), disperses Candidatus Liberibacter asiaticus, one of the putative bacterial pathogens of HLB. This study builds upon ongoing research utilizing high-throughput sequencing to analyze the virome of ACP populations collected from citrus groves throughout Florida. Following the widespread detection of sequences aligning to the genome of citrus tristeza virus (CTV) across consecutive years in the Florida ACP virome, we continued to detect a pervasive amount of CTV in Florida ACPs during subsequent years. Simultaneously, we also detected mixed infections of CTV strains in pooled ACPs from different Florida regions. Predating the HLB epidemic, CTV has been present in Florida for many years and our results confirm its widespread and diverse persistence in Florida citrus groves through a unique lens, the ACP. CTV presence in the ACP likely results from feeding on CTV-infected citrus trees in Florida citrus groves, which may help to understand an overlapping presence of CTV and HLB, both endemic citrus pathosystems in the state, and their role in future integrated pest management strategies.

This report describes the partial (nearly complete) genome sequence of a novel reo-like virus tentatively named Diaphorina citri Cimodo-like virus. This putative virus has 10 double-stranded RNA segments and was detected in Asian citrus psyllid ( Diaphorina citri ) populations collected from Florida commercial citrus groves.

Huanglongbing is caused by Candidatus Liberibacter asiaticus (CLas) and transmitted by Diaphorina citri. D. citri harbors various insect-specific viruses, including the Diaphorina citri flavi-like virus (DcFLV). The distribution and biological role of DcFLV in its host and the relationship with CLas are unknown. DcFLV was found in various organs of D. citri, including the midgut and salivary glands, where it co-localized with CLas. CLas-infected nymphs had the highest DcFLV titers compared to the infected adults and CLas-free adults and nymphs. DcFLV was vertically transmitted to offspring from female D. citri and was temporarily detected in Citrus macrophylla and grapefruit leaves from greenhouse and field. The incidences of DcFLV and CLas were positively correlated in field-collected D. citri samples, suggesting that DcFLV might be associated with CLas in the vector. These results provide new insights on the interactions between DcFLV, the D. citri, and CLas.

The plant pathogenic bacterium Candidatus Liberibacter asiaticus (CLas), the causal agent of the citrus disease Huanglongbing (HLB), and its insect vector, the Asian citrus psyllid (ACP; Diaphorina citri), have been devastating the Florida citrus industry. To restore the competitive production presence of Florida in the worldwide citrus market, effective and sustainable control of HLB and the ACP needs to be identified. As alternatives for resistance-inducing insecticides, viruses are currently being considered for biological control of the ACP. To identify possible biological control candidates, we conducted one of the most comprehensive surveys of natural ACP populations in major citrus production regions spanning 21 counties in Florida. By optimizing PCRs and RT-PCRs, we were able to successfully detect and monitor the prevalence of five previously identified ACP-associated RNA and DNA viruses throughout Florida citrus groves, which include: Diaphorina citri-associated C virus (DcACV), Diaphorina citri flavi-like virus (DcFLV), Diaphorina citri densovirus (DcDNV), Diaphorina citri reovirus (DcRV), and Diaphorina citri picorna-like virus (DcPLV). Adult and nymph ACP populations from 21 of Florida's major citrus-producing counties were collected each month during approximately 18 consecutive months. RNA extracts used for these viral screens were also regionally combined and subjected to High Throughput Sequencing (HTS) to reveal a more comprehensive picture of known and unknown viruses in Florida ACP populations. We discovered that DcACV was the most prevalent ACP-associated virus throughout nymph and adult ACP populations in Florida, detected in more than 60% of all samples tested, followed by DcPLV and DcFLV. HTS allowed us to identify a novel ACP-associated reo-like virus and a picorna-like virus. The putative reo-like virus, tentatively named Diaphorina citri cimodo-like virus, was later surveyed and detected back in seasonal adult and nymph ACP samples collected in Florida during this study. HTS generated data also revealed that the most abundant virus in Florida ACP populations was Citrus tristeza virus (CTV), which is not an ACP-associated virus, suggesting persistent presence of CTV infection in citrus throughout Florida groves. Collectively, information obtained from our study may be able to help guide the direction of biotechnological pest control efforts involving a number of viruses that were detected for the first time in Florida ACP populations, including two newly identified ACP-associated viruses.

In current studies we assessed the ability the of capsid-optimized adeno-associated virus (AAV) vectors expressing antigen to activate a specific T-cell response in vivo. We have showed that the efficacy of wild-type (WT) AAV vectors can be significantly enhanced by substituting critical serine (S) and threonine (T) residues on their capsids to valine (V). These residues were identified by analysis of the AAV capsid crystal structure and they can be recognized and phosphorylated by common serine/threonine cellular kinases such as JNK and p38 MAPK. Several different amino acids were tested and (V) was chosen because of the similarity of its structure with both (S) and (T), and lack of recognition by kinases. Thus, these modifications can prevent kinase-mediated phosphorylation of the AAV capsid, subsequent ubiquitination and proteasome-mediated degradation of the vectors. Next, the ovarian albumin (OVA), commonly used in mouse models as an antigen for immunization studies was used to evaluate ability of these vectors to initiate immune response. AAV6-WT-OVA and capsid-optimized AAV6-S662V+T492V-OVA vectors were injected intramuscularly in C57BL/6 mice with a dose of 5x10e11 vgs/mouse. Enchased green fluorescent protein (AAV6-EGFP) was used as negative control. In two weeks after injection, blood was collected and a number of OVA-specific T-cells were analyzed by stain with iTAg MHC Class I Murine Tetramer. The data suggest that the administration of AAV vectors expressing OVA led to a robust activation (approximately 9%) of specific T-cells compared to the mock and AAV-WT treated animals (less than 1%). Next, we evaluated the killing ability of these OVA specific T-cell isolated from splenocytes of C57BL/6 mice i.m. injected with AAV6-WT and AAV6-mutant vectors. Two-color fluorescence assay of cell-mediated cytotoxicity was used to estimate percentage of dead/alive target cells and generate a killing curve with different effectors to target cell ratio. Results of these experiments suggest that OVA-CD8 cells isolated from mice injected with AAV-S662V+T492V-OVA vectors have approximately 2-fold higher killing activity compared with OVA-CD8 cells from AAV-WT-OVA injected mice. Finally, we evaluated the ability of capsid-optimized AAV6 vectors to initiate a protective anti-cancer immune response. Prostatic acid phosphatase (PAP), a gene up regulated in both human and mouse prostate cancer, was used as a specific target. C57BL/6 mouse subcutaneously injected prostate cancer cell line, RM1, was used as an animal model. We genetically modified this cell line for a stable expression of firefly luciferase (FLuc) to monitor progression of tumor reduction or growth in live animals. First, animals were immunized with AAV6-S663V+T492-PAP or AAV6-WT-PAP vectors. Two weeks later mice were challenged with RM1-FLuc cells by subcutaneous injection. Results indicate suppression of tumor growth by AAV6-S663V+T492-PAP for approximately four weeks in comparison to one week and two weeks for negative control AAV6-GFP and AAV6-WT-PAP treated mice, respectively. In conclusion, successful inhibition of tumor growth in this artificial animal model would set the stage for potential clinical application. Citation Format: Munjal Pandy, Kellee Britt, George Aslanidi. Reprogramming immune response with capsid-optimized AAV vectors for immunotherapy of cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A072.

Cancer, a leading cause of death in the human population today, is generally being treated with chemotherapeutic methods, which have adverse toxic side effects. Immunotherapy represents an attractive alternative to common treatment since it is based on activation of host immune system. In current studies we assessed the ability the of AAV vectors expressing antigen to activate a specific T-cell response in vivo. The ovarian albumin (OVA), commonly used in mouse models as an antigen for immunization studies was used to evaluate immune response. AAV6-WT-OVA and capsid-optimized AAV6-S662V+T492V-OVA vectors were injected intramuscularly in C57BL/6 mice with a dose of 5×10e11 vgs/mouse. Enchased green fluorescent protein (AAV6-EGFP) was used as negative control. In two weeks after injection, blood was collected and a number of OVA-specific T-cells were analyzed by stain with iTAg MHC Class I Murine Tetramer. The data shown suggest that the administration of AAV vectors expressing OVA led to a robust activation (approximately 9%) of specific T-cells compared to the mock and AAV-WT treated animals (less than 1%). Next, we evaluated the killing ability of these OVA specific T-cell. Splenocytes from C57BL/6 mice i.m. injected with AAV6-WT and AAV6-mutant vectors were isolated in 14 days after injection. OVA-CD8 cells were expanded in vitro in the presence of a predominant for C57BL/6 mice, SIINFEKL peptide. Stimulated T-cells were used for killing assay against mouse prostate cell line RM1 stably expressing OVA. Two-color fluorescence assay of cell-mediated cytotoxicity was used as described above to estimate percentage of dead/alive target cells and generate a killing curve with different effectors to target cell ratio. Results of these experiments suggest that OVA-CD8 cells isolated from mice injected with AAV-S662V+T492V-OVA vectors have higher killing activity compared with OVA-CD8 cells from AAV-WT-OVA injected mice. Control T-cell do not show significant cytotoxicity which eliminates the possibility of autoreactive response. Finally, we evaluated the ability of capsid-optimized AAV6 vectors to initiate a protective anti-cancer immune response. Prostatic acid phosphatase (PAP), a gene up regulated in both human and mouse prostate cancer, was used as a specific target. C57BL/6 mouse subcutaneously injected prostate cancer cell line, RM1, was used as an animal model. We genetically modified this cell line for a stable expression of firefly luciferase (FLuc) to monitor progression of tumor growth in live animals. First, animals were immunized with AAV6-S663V+T492-PAP or AAV6-WT-PAP vectors. Two weeks later mice were challenged with RM1-FLuc cells by subcutaneous injection. The effect of treatment on tumor growth was evaluated by whole body life imaging. Results indicate suppression of tumor growth by AAV6-S663V+T492-PAP for approximately four weeks in comparison to one week and two weeks for negative control AAV6-GFP and AAV6-WT-PAP treated mice, respectively. In conclusion, successful inhibition of tumor growth in this artificial animal model would set the stage for potential clinical application.

In the current studies we generated novel capsid-optimized adeno-associated virus (AAV) serotype 6 (AAV6) vectors expressing a tumor-associated antigen, and assessed their ability to activate a protective T-cell response in an animal model. First, we showed that specific mutations in the AAV6 capsid increase the transduction efficiency of these vectors in mouse bone marrow-derived dendritic cells in vitro for approximately 5-fold compared with the wild-type (WT) AAV6 vectors. Next, we evaluated the ability of the mutant AAV6 vectors to initiate specific T-cell clone proliferation in vivo. Our data indicate that the intramuscular administration of AAV6-S663V+T492V vectors expressing ovalbumin (OVA) led to a strong activation (approximately 9%) of specific T cells in peripheral blood compared with AAV6-WT treated animals (<1%). These OVA-specific T cells have a superior killing ability against mouse prostate cancer cell line RM1 stably expressing the OVA antigen when propagated in vitro. Finally, we evaluated the ability of capsid-optimized AAV6-S663V+T492V vectors to initiate a protective anticancer immune response in vivo. Our results document the suppression of subcutaneous tumor growth in animals immunized with AAV6-S663V+T492V vectors expressing prostatic acid phosphatase (PAP) for approximately 4 weeks in comparison with 1 week and 2 weeks for the negative controls, AAV6-EGFP, and AAV6-WT-PAP treated mice, respectively. These studies suggest that successful inhibition of tumor growth in an animal model would set the stage for potential clinical application of the capsid-optimized AAV6-S663V+T492V vectors.

Abstract Dendritic cell (DC) based immunotherapy has recently demonstrated great potential for clinical application. However, additional progress in methods of tumor specific antigen delivery to DC is necessary for the further development of the anti-tumor vaccine. In our studies of the adeno-associated virus (AAV) crystal structure combined with data from various mutagenesis experiments on the capsid genes, we have identified specific residues which play a critical role in intracellular trafficking of these vectors. Genetic modifications of these regions increase transduction efficiency of AAV vectors most likely by preventing phosphorylation of serine (S) and threonine (T) residues by cellular kinases and subsequent degradation of vectors by host proteosomes. These studies led to development of several vectors such as AAV6-S663V, AAV6-T492V and AAV6-T492+S663V which can increase transduction efficiency of the monocytes-derived DCs (moDCs) by up to 5-fold compared to AAV6-WT. This significant improvement in infectivity of capsid-optimized over AAV6-WT vectors was associated with advanced nuclear translocation rather than facilitated viral entry into cells, which was confirmed by qPCR analysis on viral genome distribution in cytoplasmic and nuclear fractions. Our data indicate that approximately 85% of viral genomes of mutant AAV vectors accumulated in the nucleus 24 hrs post-infection compared with approximately 50% of AAV6-WT vectors at the same time-point. Considering the relatively short period of time between the initiation of maturation and cytopathic changes in DCs, rapid expression of antigen is critical to provide appropriate signals for T-cell activation. Another important advantage of the employment of such capsid-modified AAV6 vectors for vaccination is that efficient nuclear translocation in DCs will provide less material for potential presentation, and might prevent induction of immuno-competition in immune responses against vector-derived and antigen-derived epitopes. We next evaluated the possibility of whether high transduction efficiency of the new vectors will be associated with better antigen presentation and generating antigen-specific cytotoxic T-cells (CTLs). Our data show that DCs loaded with AAV6-T492+S663V carrying human prostate specific antigen (hPSA) resulted specific T-cell clone proliferation and generation of superior CTLs with higher killing capability of human prostate adenocarcinoma cells, LNCaP, compare CTLs stimulated by AAV6-WT vectors. In summary, our studies lead to the development of effective novel AAV vectors by manipulating viral capsid structure for the potential use in DC-based immunotherapy. Citation Format: Pandya Jheel, Kellee Britt, George Aslanidi. Bioengineering of the adeno-associated virus (AAV) vectors for dendritic cell (DC)-based immunotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 714. doi:10.1158/1538-7445.AM2014-714