The Effects of Pulsed Electric Field On The Interaction Between SARS-Cov-2 And Human ACE2: Y505 Is A Key Target

A novel coronavirus has rapidly spread to almost every country in the world, causing over 233 million confirmed cases of coronavirus disease 2019 (COVID-19) and over 209,761,242 deaths by late September 2021. Binding the receptor binding domain (RBD) to the host cell surface receptor protein, angiotensin converter enzyme (ACE2), is a key step in virus infection. In this study, we applied a pulsed electric field to the RBD/ACE2 complex based on molecular dynamics simulation and demonstrated that the electric field affects the structure and binding affinity of the complex. Additionally, residue Y505 is the crucial medium for the effects of electric field on the complex. Overall, these results may help apply an external electric field to virus suppression.

Estimating the transmissibility of SARS-CoV-2 during periods of high, low and zero case incidence

Against a backdrop of widespread global transmission, a number of countries have successfully brought large outbreaks of COVID-19 under control and maintained near-elimination status. A key element of epidemic response is the tracking of disease transmissibility in near real-time. During major outbreaks, the reproduction rate can be estimated from a time-series of case, hospitalisation or death counts. In low or zero incidence settings, knowing the potential for the virus to spread is a response priority. Absence of case data means that this potential cannot be estimated directly. We present a semi-mechanistic modelling framework that draws on time-series of both behavioural data and case data (when disease activity is present) to estimate the transmissibility of SARS-CoV-2 from periods of high to low -- or zero -- case incidence, with a coherent transition in interpretation across the changing epidemiological situations. Of note, during periods of epidemic activity, our analysis recovers the effective reproduction number, while during periods of low -- or zero -- case incidence, it provides an estimate of transmission risk. This enables tracking and planning of progress towards the control of large outbreaks, maintenance of virus suppression, and monitoring the risk posed by re-introduction of the virus. We demonstrate the value of our methods by reporting on their use throughout 2020 in Australia, where they have become a central component of the national COVID-19 response.

MULTIPLEXED SIV-SPECIFIC PAIRED RNA-GUIDED CAS9 NICKASES INACTIVATE PROVIRAL DNA

Human and simian immunodeficiency virus infections establish lifelong reservoir of cells harboring an integrated proviral genome. Genome editing CRISPR-associated Cas9 nucleases, combined with SIV-specific guiding RNA (gRNA) molecules, inactive integrated provirus DNA in vitro and in animal models. We generated RNA-guided Cas9 nucleases (RGNu) and nickases (RGNi) targeting conserved SIV regions with no homology in the human or rhesus macaque genome. Assays in cells co-transfected with SIV provirus and plasmids coding for RGNus identified SIV LTR, TAR, and RSS regions as the most effective at virus suppression; RGNi targeting these same regions inhibited virus production significantly. Multiplex plasmids that co-expressed these three RGNu (Nu3), or six (three pairs) RGNi (Ni6), were more efficient at virus suppression than any combination of individual RGNu and RGNi plasmids. Both Nu3 and Ni6 plasmids were tested in lymphoid cells chronically infected with SIVmac239, and whole genome sequencing was used to determine on- and off-target mutations. Treatment with these all-in-one plasmids resulted in similar levels of mutations of viral sequences from the cellular genome; Nu3 induced indels at the 3 SIV-specific sites, whereas for Ni6 indels were present at the LTR and TAR sites. Levels of off-target effects detected by two different algorithms were indistinguishable from background mutations. In summary, we demonstrate that Cas9 nickase in association with gRNA pairs can specifically eliminate parts of the integrated provirus DNA; also, we show that careful design of an all-in-one plasmid coding for 3 gRNAs and Cas9 nuclease inhibits SIV production with undetectable off-target mutations making these tools a desirable prospect for moving into animal studies. Importance: Our approach to HIV cure, utilizing the translatable SIV/rhesus macaque model system, aims at provirus inactivation and its removal with the least possible off-target side effects. We developed single molecules that delivered either three truncated SIV-specific gRNAs along with Cas9 nuclease, or three pairs of SIV-specific gRNAs (six individual gRNAs) along with Cas9 nickase to enhance efficacy of on-target mutagenesis. Whole genome sequencing demonstrated effective SIV sequence mutation and inactivation, and absence of demonstrable off-target mutations. These results open the possibility to employ Cas9 variants that introduce single-strand DNA breaks to eliminate integrated proviral DNA.

Whitefly Control Strategies against Tomato Leaf Curl New Delhi Virus in Greenhouse Zucchini

(1) Background: Tomato leaf curl New Delhi virus (ToLCNDV), transmitted by tobacco whitefly (Bemisia tabaci Gennadius) (Hemiptera: Aleyrodidae), is of major concern in the cultivation of zucchini. The threat of this virus motivates reliance on chemical vector control but European consumers’ demands for vegetables grown free of pesticides provides an important incentive for alternative pest management; (2) Methods: Different whitefly management strategies and ToLCNDV incidences were surveyed in commercial zucchini greenhouses in south-east Spain. In an experimental greenhouse, three different whitefly control strategies, biological, chemical, and integrated (IPM), were evaluated in a replicated trial to determine the most effective strategy for virus suppression (3) Results: Whitefly was present in all commercial zucchini crops surveyed, whereas fewer crops had Amblyseius swirskii or other natural enemies. During three consecutive years, pest management was increasingly based on chemical treatments. Yet, ToLCNDV was widespread in zucchini greenhouses. Experimental results showed that the order of best strategy for virus suppressing was integrated management (73%) > biological control (58%) > chemical control (44%); and (4) Conclusions: IPM was the best strategy for virus suppression. The results can assist in the design of appropriate control strategies for chemical pesticide reduction and decision-making in pest management.

Combinatorial CRISPR-Cas9 and RNA Interference Attack on HIV-1 DNA and RNA Can Lead to Cross-Resistance

ABSTRACT Many potent antiviral drugs have been developed against HIV-1, and their combined action is usually successful in achieving durable virus suppression in infected individuals. This success is based on two effects: additive or even synergistic virus inhibition and an increase in the genetic threshold for development of drug resistance. More recently, several genetic approaches have been developed to attack the HIV-1 genome in a gene therapy setting. We set out to test the combinatorial possibilities for a therapy based on the CRISPR-Cas9 and RNA interference (RNAi) mechanisms that attack the viral DNA and RNA, respectively. When two different sites in the HIV-1 genome were targeted, either with dual CRISPR-Cas9 antivirals or with a combination of CRISPR-Cas9 and RNAi antivirals, we observed additive inhibition, much like what was reported for antiviral drugs. However, when the same or overlapping viral sequence was attacked by the antivirals, rapid escape from a CRISPR-Cas9 antiviral, assisted by the error-prone nonhomologous end joining (NHEJ) DNA repair machinery, accelerated the development of cross-resistance to the other CRISPR-Cas9 or RNAi antiviral. Thus, genetic antiviral approaches can be combined, but overlap should be avoided.