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Emerging Oseltamivir-resistant influenza strains pose a critical public health threat due to antigenic shifts and drifts. We report an innovative strategy for controlling influenza A infections by use of a novel minibody of the 3D8 single chain variable fragment (scFv) showing intrinsic viral RNA hydrolyzing activity, cell penetration activity, and epidermal cell penetration ability. In this study, we examined 3D8 scFv’s antiviral activity in vitro on three different H1N1 influenza strains, one Oseltamivir-resistant (A/Korea/2785/2009pdm) strain, and two Oseltamivir-sensitive (A/PuertoRico/8/1934 and A/X-31) strains. Interestingly, the 3D8 scFv directly digested viral RNAs in the ribonucleoprotein complex. scFv’s reduction of influenza viral RNA including viral genomic RNA, complementary RNA, and messenger RNA during influenza A infection cycles indicated that this minibody targets all types of viral RNAs during the early, intermediate, and late stages of the virus’s life cycle. Moreover, we further addressed the antiviral effects of 3D8 scFv to investigate in vivo clinical outcomes of influenza-infected mice. Using both prophylactic and therapeutic treatments of intranasal administered 3D8 scFv, we found that Oseltamivir-resistant H1N1-infected mice showed 90% (prophylactic effects) and 40% (therapeutic effects) increased survival rates, respectively, compared to the control group. The pathological signs of influenza A in the lung tissues, and quantitative analyses of the virus proliferations supported the antiviral activity of the 3D8 single chain variable fragment. Taken together, these results demonstrate that 3D8 scFv has antiviral therapeutic potentials against a wide range of influenza A viruses via the direct viral RNA hydrolyzing activity.

The virus behind the current pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the etiology of novel coronavirus disease (COVID-19) and poses a critical public health threat worldwide. Effective therapeutics and vaccines against multiple coronaviruses remain unavailable. Single-chain variable fragment (scFv), a recombinant antibody, exhibits broad-spectrum antiviral activity against DNA and RNA viruses owing to its nucleic acid-hydrolyzing property. The antiviral activity of 3D8 scFv against SARS-CoV-2 and other coronaviruses was evaluated in Vero E6 cell cultures. Viral growth was quantified with quantitative RT-qPCR and plaque assay. The nucleic acid-hydrolyzing activity of 3D8 was assessed through abzyme assays of in vitro viral transcripts and cell viability was determined by MTT assay. We found that 3D8 inhibited the replication of SARS-CoV-2, human coronavirus OC43 (HCoV-OC43), and porcine epidemic diarrhea virus (PEDV). Our results revealed the prophylactic and therapeutic effects of 3D8 scFv against SARS-CoV-2 in Vero E6 cells. Immunoblot and plaque assays showed the reduction of coronavirus nucleoproteins and infectious particles, respectively, in 3D8 scFv-treated cells. These data demonstrate the broad-spectrum antiviral activity of 3D8 against SARS-CoV-2 and other coronaviruses. Thus, it could be considered a potential antiviral countermeasure against SARS-CoV-2 and zoonotic coronaviruses.

Probiotics are well known for their beneficial effects for animals, including humans and livestock. Here, we tested the probiotic activity of Lactobacillus paracasei expressing 3D8 scFv, a nucleic acid-hydrolyzing mini-antibody, in mice intestine. A total of 18 fecal samples derived from three different conditions at two different time points were subjected to high-throughput 16S ribosomal RNA (rRNA) metagenomic analyses. Bioinformatic analyses identified an average of 290 operational taxonomic units. After administration of L. paracasei, populations of the probiotics L. paracasei, Lactobacillus reuteri, and Pediococcus acidilactici increased, whereas the population of harmful bacteria such as Helicobacter species decreased. Furthermore, continuous administration of L. paracasei resulted in L. paracasei emerging as the dominant probiotic after competition with other existing probiotics. Expression of 3D8 scFv protein specifically increased the population of P. acidilactici, which is another probiotic. In summary, our results showed that L. paracasei expressing 3D8 scFv protein enhanced probiotic activity in mice intestine with no observable side effects. Thus, the system developed in this study may be a good tool for the expression of recombinant protein using probiotics.

The protein 3D8 single-chain variable fragment (3D8 scFv) has potential anti-viral activity due to its ability to penetrate into cells and hydrolyze nucleic acids. Probiotic Lactobacillus paracasei engineered to secrete 3D8 scFv for oral administration was used to test the anti-viral effects of 3D8 scFv against gastrointestinal virus infections. We found that injection of 3D8 scFv into the intestinal lumen resulted in the penetration of 3D8 scFv into the intestinal villi and lamina propria. 3D8 scFv secreted from engineered L. paracasei retained its cell-penetrating and nucleic acid-hydrolyzing activities, which were previously shown with 3D8 scFv expressed in Escherichia coli. Pretreatment of RAW264.7 cells with 3D8 scFv purified from L. paracasei prevented apoptosis induction by murine norovirus infection and decreased messenger RNA (mRNA) expression of the viral capsid protein VP1. In a mouse model, oral administration of the engineered L. paracasei prior to murine norovirus infection reduced the expression level of mRNA encoding viral polymerase. Taken together, these results suggest that L. paracasei secreting 3D8 scFv provides a basis for the development of ingestible anti-viral probiotics active against gastrointestinal viral infection.

Outbreaks of viral diseases, which cause morbidity and mortality in animals and humans, are increasing annually worldwide. Vaccines, antiviral drugs, and antibody therapeutics are the most effective tools for combating viral infection. The ongoing coronavirus disease 2019 pandemic, in particular, raises an urgent need for the development of rapid and broad-spectrum therapeutics. Current antiviral drugs and antiviral antibodies, which are mostly specific at protein levels, have encountered difficulties because the rapid evolution of mutant viral strains resulted in drug resistance. Therefore, degrading viral genomes is considered a novel approach for developing antiviral drugs. The current article highlights all potent candidates that exhibit antiviral activity by digesting viral genomes such as RNases, RNA interference, interferon-stimulated genes 20, and CRISPR/Cas systems. Besides that, we introduce a potential single-chain variable fragment (scFv) that presents antiviral activity against various DNA and RNA viruses due to its unique nucleic acid hydrolyzing characteristic, promoting it as a promising candidate for broad-spectrum antiviral therapeutics.

. Many natural anti-DNA antibodies (Abs) have the ability to translocate across the plasma membrane and localize in the nucleus of mammalian cells, frequently leading to cytotoxicity to cells. Herein, we report detailed intracellular trafficking routes and cytotoxicity in HeLa cells for a single chain variable fragment (scFv) Ab, 3D8, which is an anti-DNA Ab capable of hydrolyzing both DNA and RNA. The intracellular penetration of 3D8 scFv occurred by caveolae/lipid raft endocytosis. The time-course chasing experiments revealed that 3D8 scFv escaped directly from the caveosome into the cytosol and remained in the cytosol without further trafficking into endosomes, lysosomes, endoplasmic reticulum, Golgi, or nucleus. The cytosolically localized 3D8 scFv maintained its nuclease activity to hydrolyze cellular RNAs, mainly mRNAs, eventually triggering apoptotic cell death. Our results demonstrate that 3D8 scFv has a unique intracellular trafficking route of localizing in the cytosol, thereby exhibiting cytotoxicity due to its nuclease activity.

Background The 3D8 single chain variable fragment (scFv) is a mini-antibody sequence that exhibits independent nuclease activity against all types of nucleic acids. In this research, crossing a 3D8 scFv G1 transgenic rooster with wild-type hens produced 3D8 scFv G 2 transgenic chickens to evaluate suppression of viral transmission. Result The transgenic chickens were identified using genomic PCR and immunohistochemistry. To evaluate Newcastle disease virus (NDV) protection conferred by 3D8 scFv expression, transgenic, non-transgenic, and specific pathogen-free (SPF) chickens were challenged with virulent NDV by direct injection or aerosol exposure. The three groups of chickens showed no significant differences ( p  < 0.05) in mean death time after being directly challenged with NDV; however, in contrast to chickens in the non-transgenic and SPF groups, chickens in the transgenic group survived after aerosol exposure. Although the transgenic chickens did not survive after direct challenge, we found that the chickens expressing the 3D8 scFv survived aerosol exposure to NDV. Conclusions Our finding suggest that the 3D8 scFv could be a useful tool to prevent chickens from spreading NDV and control virus transmission.

Chrysanthemum stunt viroid (CSVd), the smallest plant pathogen known to infect chrysanthemums, is a single-stranded circular RNA viroid that induces stunting that results in an overall height reduction of 30–50 % in mature plants. A catalytic single-chain variable antibody, 3D8 scFv, which exhibits intrinsic DNase and RNase activities, was expressed in chrysanthemums to generate transgenic plant resistance to CSVd infection. Moreover, a codon-optimized version of the 3D8 scFv gene for chrysanthemums was also transformed into plants; these codon-optimized transgenic chrysanthemums expressed twice as much 3D8 scFv and displayed 60 % more resistance to CSVd infection, compared with transgenic chrysanthemums harboring the original 3D8 scFv gene. CSVd challenge experiments with codon-optimized and original 3D8 scFv-transgenic chrysanthemums showed that CSVd in newly produced leaves of both codon-optimized and original 3D8 scFv-transgenic plants was not detected by RT-PCR. This is the first report describing the development of a CSVd-resistant chrysanthemum harboring a catalytic single-chain antibody, 3D8 scFv, which has intrinsic RNase activity.

Plant viruses continue to cause diseases on economically important crops. Therefore, numerous attempts to produce virus resistant plants have been reported by using the mechanisms such as host mediated protection and virus mediated protection. Here, a novel strategy of targeting viral RNA itself, rather than viral gene products, is presented to generate virus-resistant transgenic plants. A catalytic single chain variable antibody, 3D8 scFv, which has RNase activities, was functionally expressed in the cytosol of Nicotiana tabacum. We found that progenies of the transgenic tobacco plant acquired complete resistances against four ss-RNA tobamoviruses and one cucumovirus tested without viral accumulation and delayed onset of disease symptoms. The results showed that the resistance observed in 3D8 scFv transgenic plants was caused by the RNase activity of 3D8 scFv itself, not by RNA-mediated gene silencing mechanism. Taken together, we suggested that newly gained resistance of the 3D8 scFv transgenic plants to five ss-RNA viruses most likely resulted from the RNase activity of 3D8 scFv.

The antiviral effect of a catalytic RNA-hydrolyzing antibody, 3D8 scFv, for intranasal administration against avian influenza virus (H1N1) was described. The recombinant 3D8 scFv protein prevented BALB/c mice against H1N1 influenza virus infection by degradation of the viral RNA genome through its intrinsic RNA-hydrolyzing activity. Intranasal administration of 3D8 scFv (50 μg/day) for five days prior to infection demonstrated an antiviral activity (70% survival) against H1N1 infection. The antiviral ability of 3D8 scFv to penetrate into epithelial cells from bronchial cavity via the respiratory mucosal layer was confirmed by immunohistochemistry, qRT-PCR, and histopathological examination. The antiviral activity of 3D8 scFv against H1N1 virus infection was not due to host immune cytokines or chemokines, but rather to direct antiviral RNA-hydrolyzing activity of 3D8 scFv against the viral RNA genome. Taken together, our results suggest that the RNase activity of 3D8 scFv, coupled with its ability to penetrate epithelial cells through the respiratory mucosal layer, directly prevents H1N1 virus infection in a mouse model system.