Small datasets were generated for each sample and used in bioinformatic analysis through de novo assemblies and read-mapping. Assembled contigs were identified and classified according to the sequence they aligned to with the highest bit score in BLAST searches against the NCBI non-redundant DNA and protein databases. The Capillovirus, ASGV was identified in the known sample together with several CTV genotypes. However, the atypical “psorosis” sample had a more complex virome that include three viroids , as well as several CTV genotypes. The presence of multiple CTV genotypes was confirmed for both samples by read-mapping to full length reference genomes. The results of this proof of principle experiment indicate that the metagenomic sequencing approach of dsRNA can be successfully implemented to establish the virome of citrus trees with an unknown virus etiology.Research subsequently focused on other aspects of the disease epidemiology: impact of ambient temperatures and graft transmission and multiplication of the pathogens in citrus and orange jasmine Jack as incorrectly referred to in other publications. The research on the impact of ambient temperatures focused on exposing Lam+ve and Las+ve trees to distinct daily temperature regimes . The research was motivated by the already known contrasting responses to ambient temperatures of plants affected by Las or Ca. L. africanus and by the complete lack of information on this subject for Lam. After a series of growth chamber experiments it was demonstrated that Lam is more heat sensitive than Las. Fully symptomatic orange trees affected by Lam exposed to daily regimes of 27 to 32°C, 24 to 32°, or 35 to 38ºC for 60 days were totally cleared of symptoms and of the pathogen,cultivo arandanos en maceta while fully symptomatic trees affected by Las were only partially cleared of symptoms and the pathogen only when exposed to 24 to 38ºC for the same duration.
More recently it was shown that this same temperature regime leads to a decline in Las titers in new flushes on symptomatic branches, an impact which would lead to a significant reduction in pathogen acquisition rates by the insect vectors feeding on them . Although field work will add important information on this aspect of the HLB pathosystem, data so far accumulated indicate that high summer temperatures may restrict rates of spread of the disease and help to explain the irregular dissemination patterns of HLB in SPS. Field and greenhouse experiments involving even higher temperatures for different durations were also conducted, with the aim of curing Las+ve trees, but with limited success . The reasons for the limited success were apparently related to the sensitivity of the citrus tree to high temperatures and to the ability of the pathogen to survive in roots. The temperature-time combinations necessary to kill the bacterium were apparently close to those that would kill a citrus tree, and in the roots, the bacterium remains protected from heat. Studies on graft transmission of Las and Lam were conducted with the objective of comparing graft transmission efficiencies and the ability of both bacteria to multiply, individually or simultaneously, in potted Valencia, Hamlin, Pera, and Natal , under conditions favorable for disease development .The percentage of plants that became infected varied from 10.0 to 23.3% for Lam and 66.7 to 73.3% for Las, and the cycle threshold values varied from 24.14 to 24.97 for Lam and 19.42 to 20.92 for Las. These Cts corresponded to average 106 and 107 cells per gram of tissue for Lam and Las, respectively. Similar values were obtained also when field samples, collected from three distinct regions of SPS, were analyzed . No apparent effect of one species over the other was observed in plants inoculated simultaneously with both pathogens. Lower titers of Lam appear to be the main factor explaining its conspicuous decline over the years in SPS. Lower titer would reduce the chances of pathogen acquisition by the insect vector and its consequent transmission to healthy trees, in a pattern similar to the one observed for Las in new flushes exposed to heat .
This work also showed that, contrary to Lam+ve plants, Las+ve plants harbored the bacterium attiters close to maximal values, three months before symptom expression, an indication that asymptomatic trees may be serving as a source of inoculum, contributing to dissemination of HLB in the field. The research on orange jasmine aimed at determination of the distribution, based on sampling at 76 urban locations over two time intervals, of orange jasmine trees infected by Lam or Las, and determination of levels of genetic and pathogenic similarities among the orange jasmine and citrus liberibacters, based on sequences of the rplJ gene and on cross inoculation experiments . The work was motivated by the detection of Lam in a single mature orange jasmine tree growing in front of the manager’s house in the citrus farm most affected by Lam in 2004, by the detection of Las in 2005 in orange jasmine trees growing in urban areas and, more importantly, by suspicion that infected M. exotica trees may play an important role in the HLB epidemics. In the years 2005/2006 Lam was detected in 56 and Las in 2 of the 477 orange jasmine trees from 10 locations and, in 2009, Lam was detected in an additional 5 and Las in 28 of the 309 orange jasmine trees from seven locations. Lam titers were higher in Lam+ve than in Las+ve trees . As happens with infected citrus under favorable conditions for disease development, symptom severity was stronger on the orange jasmine trees infected by Lam than on those infected by Las. The higher symptom severity in M. exotica may not be related only to the higher bacterium titers in this host since in citrus, Lam reaches lower titers than Las. In Las infected orange jasmine the infection seemed to be transient. This was observed in naturally infected field trees and in graft-inoculated plants. This work also showed that the infected orange jasmine trees were in locations relatively close to each other and, coincidently, in the area of highest incidence of HLB in citrus at that time, a clear indication of pathogen transmission from host to host by D. citri. Similarity among citrus and orange jasmine liberibacters,grow hydroponic in terms of pathogenicity, could not be fully determined due to the strong tissue incompatibility observed between citrus and orange jasmine during the cross inoculation experiments. Most budwood used as inoculum died in heterologous combinations. On those plants in which the budwood survived, only Lam was successfully transmitted and the plants remained infected.
Comparative analysis of the rplJ gene from the liberibacters found in orange jasmine with those found in citrus showed that Lam or Las from both hosts were identical. The importance of orange jasmine and citrus as source of Lam to citrus in SPS was investigated in further work involving the insect vector for bacterium inoculation . Higher Lam transmission rates occurred from orange jasmine than from citrus. As orange jasmine trees infected with liberibacter are not systematically eliminated in urban areas, and vector populations not suppressed, orange jasmine may represent a constant risk to neighboring citrus orchards. Also, since nursery production and sale of orange jasmine are not regulated , asymptomatic orange jasmine trees may be important for distributing liberibacters to distant citrus areas still free from the disease. An overview of the HLB epidemics in Brazil, particularly in SPS, and the main research findings on the HLB pathosystem were briefly presented here. Other field work and studies , and the daily experience of the citrus growers with the disease, have confirmed the necessity of eliminating symptomatic trees and controlling the insect vector on an area-wide basis in order to optimize opportunities for successfully minimizing the spread and impact of HLB. Although many research questions still require answers, research has provided a better understanding of the distinct patterns of spatio-temporal progress of the disease, and knowledge required for official responses and establishment of management practices. Among research outcomes, impacts of high temperatures on Las multiplication in new flushes may have some potential for the development of new, less costly and less insecticide dependent strategies to manage HLB. The impact of the COVID-19 pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 was foreshadowed by earlier epidemics of new or re-emerging diseases such as SARS , influenza , Middle East Respiratory Syndrome , Ebola , and Zika affecting localized regions . These events showed that novel and well-known viral diseases alike can pose a threat to global health. In 2014, an article published in Nature Medicine stated that the Ebola outbreak should have been “a wake-up call to the research and pharmaceutical communities, and to federal governments, of the continuing need to invest resources in the study and cure of emerging infectious diseases” . Recommendations and even new regulations have been implemented to reduce the risk of zoonotic viral infections , but the extent to which these recommendations are applied and enforced on a regional and, more importantly, local level remains unclear. Furthermore, most vaccine programs for SARS, MERS, and Zika are still awaiting the fulfillment of clinical trials, sometimes more than 5 years after their initiation, due to the lack of patients .
In light of this situation, and despite the call to action, the SARS-CoV-2 pandemic has resulted in nearly 20 million infections and more than 700,000 deaths at the time of writing based on the Johns Hopkins University Hospital global database.The economic impact of the pandemic is difficult to assess, but support programs are likely to cost more than €4 trillion in the United States and EU alone. Given the immense impact at both the personal and economic levels, this review considers how the plant-based production of recombinant proteins can contribute to a global response in such an emergency scenario. Several recent publications describe in broad terms how plant-made countermeasures against SARS CoV-2 can contribute to the global COVID-19 response . This review will focus primarily on process development, manufacturing considerations, and evolving regulations to identify gaps and research needs, as well as regulatory processes and/or infrastructure investments that can help to build a more resilient pandemic response system. We first highlight the technical capabilities of plants, such as the speed of transient expression, making them attractive as a first-line response to counter pandemics, and then we discuss the regulatory pathway for plant-made pharmaceuticals in more detail. Next, we briefly present the types of plant-derived proteins that are relevant for the prevention, treatment, or diagnosis of disease. This sets the stage for our assessment of the requirements in terms of production costs and capacity to mount a coherent response to a pandemic, given currently available infrastructure and the intellectual property landscape. We conclude by comparing plant-based expression with conventional cell culture and highlight where investments are needed to adequately respond to pandemic diseases in the future. Due to the quickly evolving information about the pandemic, our statements are supported in some instances by data obtained from web sites . Accordingly, the scientific reliability has to be treated with caution in these cases.The development of a protein-based vaccine, therapeutic, or diagnostic reagent for a novel disease requires the screening of numerous expression cassettes, for example, to identify suitable regulatory elements that achieve high levels of product accumulation, a sub-cellular compartment that ensures product integrity, as well as different product candidates to identify the most active and most amenable to manufacturing in plants . A major advantage of plants in this respect is the ability to test multiple product candidates and expression cassettes in parallel by the simple injection or infiltration of leaves or leaf sections with a panel of Agrobacterium tumefaciens clones carrying each variant cassette as part of the transferred DNA in a binary transformation vector . This procedure does not require sterile conditions, transfection reagents, or skilled staff, and can, therefore, be conducted in standard biosafety level 1 laboratories all over the world. The method can produce samples of even complex proteins such as glycosylated monoclonal antibodies for analysis ~14 days after the protein sequence is available.