Virus Stocks Research Articles

Cas9-Mediated Poxvirus Recombinant Recovery (CASPRR) for Fast Recovery of Recombinant Vaccinia Viruses.

Generation of recombinant vaccinia viruses opens many avenues for poxvirus research; however current methods for virus production can be laborious. Traditional methods rely on recombination strategies that produce engineered viruses at a low frequency, which then need to be identified and isolated from a large background of parent virus. For this reason, marker and reporter genes are often included, but in many cases these require removal in subsequent steps and the entire process is relatively inefficient. Cas9-mediated selection is a technique that repurposes Cas9/guide RNA complexes to amplify chosen subsets of vaccinia viruses. We refer to this approach as Cas9-mediated poxvirus recombinant recovery (CASPRR). Transient introduction of appropriately guided Cas9 allows for recovery of marker-free recombinant viruses in just 5days, and requires no additional virus modification. Following three rounds of purification, pure virus stocks are obtained. A newer method, stable expression of Cas9 and guide RNAs in a permissive cell line, allows for additional process streamlining, removing cell type-specific concerns related to transient transfection of Cas9. Within this chapter, the protocol for CASPRR is described in both a transient and stable expression model. These methods can be utilized to accelerate recovery of recombinant vaccinia viruses and be applied to generation of vaccinia libraries or novel therapeutic agents.

Isolation and Propagation of Marburgviruses.

Marburgviruses (Marburg virus and Ravn virus) have caused ~20 outbreaks of human Marburg virus disease since their discovery in 1967. These zoonotic viruses are categorized as priority pathogens with no licensed vaccines or treatment options. The foundation for all experimental work including deciphering disease parameters and countermeasure development is a defined virus stock. This chapter provides a guide for the isolation and growth of marburgviruses in tissue culture.

Why is next-generation sequencing essential in modern virology?

A contaminated viral stock results in considerable loss of time, resources and financial means and is generally discovered only by chance after years of research. Thus, it is necessary to implement a technique that can detect contamination without prior knowledge or assumptions, such as next-generation sequencing (NGS). Here, we describe the discovery of a cross-contaminated viral stock from a biological repository of an African Zika virus isolate with Toscana virus after performing NGS on infected cells. In addition, we also describe the consequences that we faced using this contaminated stock. These included the economic and time loss to the lab that needed to repeat all previously performed experiments, the generation of biologically flawed results with a subsequent potential retraction and the severe risk of infection of lab members who manipulated the contaminated stock.

Efficacy of CP-COV03 (a niclosamide-based inorganic nanohybrid product) against severe fever with thrombocytopenia syndrome virus in an in vitro model.

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease caused by the SFTS virus (SFTSV). CP-COV03 is a novel antiviral candidate that significantly enhanced the bioavailability of niclosamide through inorganic-based drug delivery technology. The active pharmaceutical ingredient of CP-COV03, niclosamide, has been previously shown to possess broad-spectrum antiviral activity against over 30 different viruses in the in vitro tests. The aim of this study is to confirm the antiviral activity of CP-COV03 against the SFTSV in an in vitro model. Vero cells and SFTS viral stock NCCP43270, a 2015 Gangwon Province isolate, were used to obtain the 50% tissue culture infective dose of the virus. Vero cells seeded in 96-well plates were infected with SFTSV for 1 h. SFTSV-infected cells were treated with CP-COV03 at various concentrations of 0.1-100 μM and incubated for 7 days. On the seventh day of the culture, the cytopathic effect (CPE) of SFTSV was checked by microscopy and the cell viability was checked by using Cell Counting Kit-8 assay. The CPE reduced as the CP-COV03 concentration increased. The 50% inhibitory concentration (IC50) range of CP-COV03 was below 0.125 µM, as determined from the viral titers of culture supernatants collected on the third day posttreatment of CP-COV03. The plaque reduction assay showed that the IC50 of CP-COV03 was 1.893 µM, as determined from the percentage reduction of plaque counts for each drug concentration on the second day posttreatment with CP-COV03. This study suggests that CP-COV03 could be used as a potential antiviral agent for SFTS.IMPORTANCEWe demonstrated a concentration-dependent response and identified low a IC50 of CP-COV03. This result is comparable to other antiviral drugs used against viruses like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We believe that our study makes a significant contribution to the literature as our findings suggest that CP-COV03 may serve as a potential treatment for SFTS, highlighting its importance in the field of antiviral research.

Shelf Life of Freeze-dried Foot and Mouth Disease Virus Stock Seeds

Shelf Life of Freeze-dried Foot and Mouth Disease Virus Stock Seeds

Propagating and banking genetically diverse human sapovirus strains using a human duodenal cell line: investigating antigenic differences between strains.

The control of human sapovirus, which causes acute gastroenteritis in individuals of all ages, is challenging because of its association with outbreaks similar to those caused by human norovirus. The establishment of conditions for efficient viral propagation of various viral strains is essential for understanding the infection mechanism and identifying potential control methods. In this study, two critical factors for human sapovirus propagation in a conventional human duodenal cell line were identified, and 15 strains of different genotypes that differed at the genetic and antigenic levels were isolated and used to prepare virus stocks. The preparation of virus stocks has not been successful for noroviruses, which belong to the same family as sapoviruses. Securing virus stocks of multiple human sapovirus strains represents a significant advance toward establishing a reliable experimental system that does not depend on limited virus-positive fecal material.

Unraveling dynamics of paramyxovirus-receptor interactions using nanoparticles displaying hemagglutinin-neuraminidase.

Sialoglycan-binding enveloped viruses often possess receptor-destroying activity to avoid being immobilized by non-functional decoy receptors. Sialic acid (Sia)-binding paramyxoviruses contain a hemagglutinin-neuraminidase (HN) protein that possesses both Sia-binding and -cleavage activities. The multivalent, dynamic receptor interactions of paramyxovirus particles provide virion motility and are a key determinant of host tropism. However, such multivalent interactions have not been exhaustively analyzed, because such studies are complicated by the low affinity of the individual interactions and the requirement of high titer virus stocks. Moreover, the dynamics of multivalent particle-receptor interactions are difficult to predict from Michaelis-Menten enzyme kinetics. Therefore, we here developed Ni-NTA nanoparticles that multivalently display recombinant soluble HN tetramers via their His tags (HN-NPs). Applying this HN-NP platform to Newcastle disease virus (NDV), we investigated using biolayer interferometry (BLI) the role of important HN residues in receptor-interactions and analyzed long-range effects between the catalytic site and the second Sia binding site (2SBS). The HN-NP system was also applicable to other paramyxoviruses. Comparative analysis of HN-NPs revealed and confirmed differences in dynamic receptor-interactions between type 1 human and murine parainfluenza viruses as well as of lab-adapted and clinical isolates of human parainfluenza virus type 3, which are likely to contribute to differences in tropism of these viruses. We propose this novel platform to be applicable to elucidate the dynamics of multivalent-receptor interactions important for host tropism and pathogenesis, particularly for difficult to grow sialoglycan-binding (paramyxo)viruses.

Genetic diversity accelerates canine distemper virus adaptation to ferrets.

RNA viruses adapt rapidly to new host environments by generating highly diverse genome sets, so-called "quasispecies." Minor genetic variants promote their rapid adaptation, allowing for the emergence of drug-resistance or immune-escape mutants. Understanding these adaptation processes is highly relevant to assessing the risk of cross-species transmission and the safety and efficacy of vaccines and antivirals. We hypothesized that genetic memory within a viral genome population facilitates rapid adaptation. To test this, we investigated the adaptation of the Morbillivirus canine distemper virus to ferrets and compared an attenuated, Vero cell-adapted virus isolate with its recombinant derivative over consecutive ferret passages. Although both viruses adapted to the new host, the reduced initial genetic diversity of the recombinant virus resulted in delayed disease onset. The non-recombinant virus gradually increased the frequencies of beneficial mutations already present at very low frequencies in the input virus. In contrast, the recombinant virus first evolved de novo mutations to compensate for the initial fitness impairments. Importantly, while both viruses evolved different sets of mutations, most mutations found in the adapted non-recombinant virus were identical to those found in a previous ferret adaptation experiment with the same isolate, indicating that mutations present at low frequency in the original virus stock serve as genetic memory. An arginine residue at position 519 in the carboxy terminus of the nucleoprotein shared by all adapted viruses was found to contribute to pathogenesis in ferrets. Our work illustrates the importance of genetic diversity for adaptation to new environments and identifies regions with functional relevance.IMPORTANCEWhen viruses encounter a new host, they can rapidly adapt to this host and cause disease. How these adaptation processes occur remains understudied. Morbilliviruses have high clinical and veterinary relevance and are attractive model systems to study these adaptation processes. The canine distemper virus is of particular interest, as it exhibits a broader host range than other morbilliviruses and frequently crosses species barriers. Here, we compared the adaptation of an attenuated virus and its recombinant derivative to that of ferrets. Pre-existing mutations present at low frequency allowed faster adaptation of the non-recombinant virus compared to the recombinant virus. We identified a common point mutation in the nucleoprotein that affected the pathogenesis of both viruses. Our study shows that genetic memory facilitates environmental adaptation and that erasing this genetic memory by genetic engineering results in delayed and different adaptation to new environments, providing an important safety aspect for the generation of live-attenuated vaccines.

Comparative analysis of multiple consensus genomes of the same strain of Marek's disease virus reveals intrastrain variation.

Current strategies to understand the molecular basis of Marek's disease virus (MDV) virulence primarily consist of cataloging divergent nucleotides between strains with different phenotypes. However, most comparative genomic studies of MDV rely on previously published consensus genomes despite the confirmed existence of MDV strains as mixed viral populations. To assess the reliability of interstrain genomic comparisons relying on published consensus genomes of MDV, we obtained two additional consensus genomes of vaccine strain CVI988 (Rispens) and two additional consensus genomes of the very virulent strain Md5 by sequencing viral stocks and cultured field isolates. In conjunction with the published genomes of CVI988 and Md5, this allowed us to perform three-way comparisons between multiple consensus genomes of the same strain. We found that consensus genomes of CVI988 can vary in as many as 236 positions involving 13 open reading frames (ORFs). By contrast, we found that Md5 genomes varied only in 11 positions involving a single ORF. Notably, we were able to identify 3 single-nucleotide polymorphisms (SNPs) in the unique long region and 16 SNPs in the unique short (US) region of CVI988GenBank.BAC that were not present in either CVI988Pirbright.lab or CVI988USDA.PA.field. Recombination analyses of field strains previously described as natural recombinants of CVI988 yielded no evidence of crossover events in the US region when either CVI988Pirbright.lab or CVI988USDA.PA.field were used to represent CVI988 instead of CVI988GenBank.BAC. We were also able to confirm that both CVI988 and Md5 populations were mixed, exhibiting a total of 29 and 27 high-confidence minor variant positions, respectively. However, we did not find any evidence of minor variants in the positions corresponding to the 19 SNPs in the unique regions of CVI988GenBank.BAC. Taken together, our findings suggest that continued reliance on the same published consensus genome of CVI988 may have led to an overestimation of genomic divergence between CVI988 and virulent strains and that multiple consensus genomes per strain may be necessary to ensure the accuracy of interstrain genomic comparisons.

Pathogenic differences of cynomolgus macaques after Taï Forest virus infection depend on the viral stock propagation.

Taï Forest virus (TAFV) is a negative-sense RNA virus in the Filoviridae family. TAFV has caused only a single human infection, but several disease outbreaks in chimpanzees have been linked to this virus. Limited research has been done on this human-pathogenic virus. We sought to establish an animal model to assess TAFV disease progression and pathogenicity at our facility. We had access to two different viral stock preparations from different institutions, both originating from the single human case. Type I interferon receptor knockout mice were inoculated with TAFV stock 1 or stock 2 by the intraperitoneal route. Inoculation resulted in 100% survival with no disease regardless of viral stock preparation or infectious dose. Next, cynomolgus macaques were inoculated with TAFV stock 1 or stock 2. Inoculation with TAFV stock 1 resulted in 100% survival and robust TAFV glycoprotein-specific IgG responses including neutralizing antibodies. In contrast, macaques infected with TAFV stock 2 developed disease and were euthanized 8-11 days after infection exhibiting viremia, thrombocytopenia, and increased inflammatory mediators identified by transcriptional analysis. Histopathologic analysis of tissue samples collected at necropsy confirmed classic filovirus disease in numerous organs. Genomic differences in both stock preparations were mapped to several viral genes which may have contributed to disease severity. Taken together, we demonstrate that infection with the two TAFV stocks resulted in no disease in mice and opposing disease phenotypes in cynomolgus macaques, highlighting the impact of viral stock propagation on pathogenicity in animal models.

Replication kinetics and infectivity of SARS-CoV-2 Omicron variant sublineages recovered in the Republic of Korea.

We analyzed the correlation between the infectivity and transmissibility of the severe acute respiratory syndrome coronavirus 2 Omicron sublineages BA.1, BA. 2, BA.4, and BA.5. We assessed viral replication kinetics and infectivity at the cellular level. Nasopharyngeal and oropharyngeal specimens were obtained from patients with coronavirus disease 2019, confirmed using whole-genome sequencing to be caused by the Omicron sublineages BA.1, BA.2, BA.4, or BA.5. These specimens were used to infect Vero E6 cells, derived from monkey kidneys, for the purpose of viral isolation. Viral stocks were then passaged in Vero E6 cells at a multiplicity of infection of 0.01, and culture supernatants were harvested at 12-hour intervals for 72 hours. To evaluate viral replication kinetics, we determined the cycle threshold values of the supernatants using real-time reverse transcription polymerase chain reaction and converted these values to genome copy numbers. The viral load was comparable between BA.2, BA.4, and BA.5, whereas BA.1 exhibited a lower value. The peak infectious load of BA.4 was approximately 3 times lower than that of BA.2 and BA.5, while the peak load of BA.2 and BA.5 was about 7 times higher than that of BA.1. Notably, BA.1 demonstrated the lowest infectivity over the entire study period. Our results suggest that the global BA.5 wave may have been amplified by the higher viral replication and infectivity of BA.5 compared to other Omicron sublineages. These analyses could support the rapid assessment of the impact of novel variants on case incidence.

Spark Discharge Aerosol-Generated Copper-Based Nanoparticles: Structural & Optical Properties; Application on the Antiviral (SARS-CoV-2) and Antibacterial Improvement of Face Masks.

Nanoparticle formation by Spark Discharge Aerosol Generation offers low-cost fabrication of nanoparticles, without the use of chemicals or vacuum. It produces aerosol particles of a few nanometers in size with high purity. In this work, copper-based -CuO (tenorite) and Cu- nanoparticles are produced, characterized and used to modify face mask air filters, achieving the introduction of antibacterial and antiviral properties. A range of characterization techniques have been employed, down to the atomic level. The majority of the particles are CuO (of a few nanometers in size that agglomerate to form aggregates), the remainder being a small number of larger Cu particles. The particles were deposited on various substrates, mainly fiber filters in order to study them and use them as biocidal agents. On face masks, their antibacterial activity against Escherichia coli (E.coli) results in a 100 % decrease in bacteria cell viability. Their antiviral activity on face masks results in a 90 % reduction of the Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) viability, 15 minutes post the application of the virus stock solution. This highlights the effectiveness of this approach, its simplicity, its low cost and its excellent environmental credentials.

Proof of stability of an RSV Controlled Human Infection Model challenge agent

In 2018, SGS Belgium NV developed RSV-NICA (Respiratory Syncytial Virus-Nasobronchial Infective Challenge Agent), an RSV type A challenge agent for use in RSV Controlled Human Infection Model (CHIM) studies.It is widely recognized that the stability of RSV can be influenced by a variety of environmental parameters, such as temperature and pH. Consequently, our objective was to evaluate the stability of the viral titer of RSV-NICA following five years of controlled storage and to determine the uniformity of the viral titers across different vials of a GMP-qualified batch of RSV-NICA. In addition, we examined the capacity of RSV-NICA to infect human primary airway epithelial cells (MucilAir™), the principal target cells of RSV, and evaluated the influence of single and recurrent freeze–thaw cycles on the infectious viral titer of the challenge agent.The aliquoted RSV-NICA virus stock was subjected to standard virological and molecular methods to gather data on the titer and consistency of the viral titer contained within 24 representative vials of the stock. Our findings illustrate that over a span of five years of cryo-storage, the infectious viral titer in 75% of the tested vials exhibited a comparable average infectious viral titer (4.75 ± 0.06 vs 4.99 ± 0.11; p-value = 0.14). A considerable reduction down to an undetectable level of infectious virus was observed in the remaining vials. RSV-NICA demonstrated its capacity to effectively infect differentiated human airway epithelial cells, with active virus replication detected in these cells through increasing RSV genome copy number over time. Virus tropism for ciliated cells was suggested by the inhibition of cilia beating coupled with an increase in viral RNA titers. No discernable impact on membrane barrier function of the epithelial lung tissues nor cytotoxicity was detected. Pooling of vials with infectious titers > 4.0 log10 TCID50/ml and freeze-thawing of these combined vials showed no deterioration of the infectious titer. Furthermore, pooling and re-aliquoting of vials spanning the entire range of viral titers (including vials with undetectable infectious virus) along with subjecting the vials to three repeated freeze–thaw cycles did not result in a decrease of the infectious titers in the tested vials.Taken together, our findings indicate that long-term cryo-storage of vials containing RSV-NICA challenge agent may influence the infectious viral titer of the virus, leading to a decrease in the homogeneity of this titer throughout the challenge stock. However, our study also demonstrates that when heterogeneity of the infectious titer of an RSV stock is observed, rounds of pooling, re-aliquoting and subsequent re-titration serve as an effective method not only to restore the homogeneity of the infectious titer of an RSV-A stock, but also to optimize patient-safety, scientific and operational aspects of viral inoculation of study participants during at least the period of one RSV CHIM trial. RSV-NICA is a stable, suitable CHIM challenge agent that can be utilized in efficacy trials for RSV vaccines and antiviral entities.

Characterization of SARS-CoV-2 replication in human H1299/ACE2 cells: A versatile and practical infection model for antiviral research and beyond

A range of cell culture infection models have been used to study SARS-CoV-2 and perform antiviral drug research. Commonly used African green monkey Vero, human lung-derived Calu-3 and ACE2+TMPRSS2-expressing A549 cells, each have their limitations. Here, we describe human ACE2-expressing H1299 lung cells as a more efficient and robust model for SARS-CoV-2 research. These cells are as easy to handle as Vero cells, support SARS-CoV-2 replication to high titers, display a functional innate immune response and are suitable for plaque assays, microscopy, the production of (genetically stable) virus stocks and antiviral assays. H1299/ACE2-based (CPE reduction) assays can be performed without adding a P-gP drug efflux pump inhibitor, which is often required in Vero-based assays. Moreover, H1299/ACE2 cells allowed us to perform CPE reduction assays with omicron variants that did not work in Vero-based assays. In summary, H1299/ACE2 cells are a versatile infection model to study SARS-CoV-2 replication in the context of antiviral drug development and virus-host interaction studies.

Isolation and genetic characterization of waterfowl parvovirus in ducks in Northern Vietnam.

Short beak and dwarfism syndrome (SBDS), a highly contagious disease, has been reported in duck farms in Vietnam since 2019. In this study, we evaluated the virulence and characterized the virus obtained from SBDS cases in North Vietnam. Polymerase chain reaction was used to detect waterfowl parvovirus in ducks, and the virus from positive samples was inoculated into 10-day-old duck-embryonated eggs to reproduce the disease in young ducklings to determine the virulence and subjected to phylogenetic analysis of non-structural (NS) and VP1 gene sequences. Goose parvovirus (GPV) was isolated from ducks associated with SDBS in Vietnam. The virus Han-GPV2001 is highly virulent when inoculated into 10-day-old duck embryos and 3-day-old ducklings. The mortality rate of duck embryos was 94.35% within 6 days of virus inoculation. Inoculating 3-day-old ducks with the virus stock with 104.03 EID50 through intramuscular and neck intravenous administration resulted in 80% and 66.67% of clinical signs of SDBS, respectively, were shown. Phylogenetic analysis based on the partial NS and VP1 gene sequences revealed that the viral isolate obtained in this study belonged to novel GPV (NGPV) and was closely related to previous Vietnamese and Chinese strains. A GPV strain, Han-GPV2001, has been successfully isolated and has virulence in duck-embryonated eggs as well as caused clinical signs of SBDS in ducks. Phylogenetic analyses of partial genes encoding NS and capsid proteins indicated that the obtained GPV isolate belongs to the NGPV group.

DengueSeq: a pan-serotype whole genome amplicon sequencing protocol for dengue virus

BackgroundThe increasing burden of dengue virus on public health due to more explosive and frequent outbreaks highlights the need for improved surveillance and control. Genomic surveillance of dengue virus not only provides important insights into the emergence and spread of genetically diverse serotypes and genotypes, but it is also critical to monitor the effectiveness of newly implemented control strategies. Here, we present DengueSeq, an amplicon sequencing protocol, which enables whole-genome sequencing of all four dengue virus serotypes.ResultsWe developed primer schemes for the four dengue virus serotypes, which can be combined into a pan-serotype approach. We validated both approaches using genetically diverse virus stocks and clinical specimens that contained a range of virus copies. High genome coverage (>95%) was achieved for all genotypes, except DENV2 (genotype VI) and DENV 4 (genotype IV) sylvatics, with similar performance of the serotype-specific and pan-serotype approaches. The limit of detection to reach 70% coverage was 10-100 RNA copies/μL for all four serotypes, which is similar to other commonly used primer schemes. DengueSeq facilitates the sequencing of samples without known serotypes, allows the detection of multiple serotypes in the same sample, and can be used with a variety of library prep kits and sequencing instruments.ConclusionsDengueSeq was systematically evaluated with virus stocks and clinical specimens spanning the genetic diversity within each of the four dengue virus serotypes. The primer schemes can be plugged into existing amplicon sequencing workflows to facilitate the global need for expanded dengue virus genomic surveillance.

Genomic profile of eGFP-tagged senecavirus A subjected to serial plaque-to-plaque transfers

Senecavirus A (SVA) belongs to the genus Senecavirus in the family Picornaviridae. This virus possesses a positive-sense, single-stranded RNA genome, approximately 7200 nt in length, composed of a single 5′ untranslated region, encoding region and 3′ untranslated region. In this study, a recombinant SVA tagged with enhanced green fluorescent protein (eGFP) sequence, rSVA-eGFP, was rescued from its cDNA clone using reverse genetics. The passage-5 (P5) rSVA-eGFP was totally subjected to 55 rounds of consecutive fluorescent plaque-to-fluorescent plaque (FP-FP) transfers, and one extra common passaging in vitro. The P61 viral stock was analyzed by next-generation sequencing. The result showed ten single-nucleotide mutations (SNMs) in the rSVA-eGFP genome, including nine transitions and only one transversion. The P61 progeny still showed a complete eGFP sequence, indicating no occurrence of copy-choice recombination within the eGFP region during serial FP-FP transfers. In other words, this progeny was genetically deficient in the recombination of eGFP sequence (RES), namely, an RES-deficient strain. Out of ten SNMs, three were missense mutations, leading to single-amino acid mutations (SAAMs): F15V in L protein, A74T in VP2, and E53R in 3D protein. The E53R was predicted to be spatially adjacent to the RNA channel of 3D protein, perhaps involved in the emergence of RES-deficient strain. In conclusion, this study uncovered a global landscape of rSVA-eGFP genome after serial FP-FP transfers, and moreover shed light on a putative SAAM possibly related to the RES-deficient mechanism.

Recombinant Vaccine Strain ASFV-G-Δ9GL/ΔUK Produced in the IPKM Cell Line Is Genetically Stable and Efficacious in Inducing Protection in Pigs Challenged with the Virulent African Swine Fever Virus Field Isolate Georgia 2010.

We have previously reported that the recombinant African Swine Fever (ASF) vaccine candidate ASFV-G-Δ9GL/ΔUK efficiently induces protection in domestic pigs challenged with the virulent strain Georgia 2010 (ASFV-G). As reported, ASFV-G-Δ9GL/ΔUK induces protection, while intramuscularly (IM), administered at doses of 104 HAD50 or higher, prevents ASF clinical disease in animals infected with the homologous ASFV g strain. Like other recombinant vaccine candidates obtained from ASFV field isolates, ASFV-G-Δ9GL/ΔUK stocks need to be produced in primary cultures of swine macrophages, which constitutes an important limitation in the production of large virus stocks at the industrial level. Here, we describe the development of ASFV-G-Δ9GL/ΔUK stocks using IPKM (Immortalized Porcine Kidney Macrophage) cells, which are derived from swine macrophages. We show that ten successive passages of ASFV-G-Δ9GL/ΔUK in IPKM cells induced small changes in the virus genome. The produced virus, ASFV-G-Δ9GL/ΔUKp10, presented a similar level of replication in swine macrophages cultures to that of the original ASFV-G-Δ9GL/ΔUK (ASFV-G-Δ9GL/ΔUKp0). The protective efficacy of ASFV-G-Δ9GL/ΔUKp10 was evaluated in pigs that were IM-inoculated with either 104 or 106 HAD50 of ASFV-G-Δ9GL/ΔUKp10. While animals inoculated with 104 HAD50 present a partial protection against the experimental infection with the virulent parental virus ASFV-G, those inoculated with 106 HAD50 were completely protected. Therefore, as was just recently reported for another ASF vaccine candidate, ASFV-G-ΔI177L, IPKM cells are an effective alternative to produce stocks for vaccine strains which only grow in swine macrophages.

Abstract 6360: A novel CAR-NK cell therapy to target lung adenocarcinoma cells

Abstract Cell Adhesion Molecule (CADM1) is a Bonafede tumor suppressor with 40% of NSCLC tumors lose CADM1 expression either by loss of heterozygosity or promoter hypermethylation. The tumor suppressive ability of CADM1 is attributed to its cell adhesion function. However, we previously demonstrated that CADM1 also mediates its tumor suppression through regulation of NK-mediated immunosurveillance. In tumors that do not lose CADM1, it is expressed on cell surface and up regulated during TGF-β-induced EMT making it a biomarker for potential therapeutic targeting. Here we report the design and development of T and NK specific CAR that would specifically target lung cancer cells in CADM1 dependent manner. Anti-CADM1 single chain variable fragment (CADM1-scFv) was fused to NK or T cell specific transmembrane and co-stimulatory domains. The resulting CAR constructs were cloned into a retroviral vector, from which viral stocks were generated to transduce human and mouse cells. Next, we introduced anti-CADM1-CAR-NK construct into NK92 cell line and tested it in a cytotoxicity assay using A549-GFP-ffluc cell line as a target. We observed a significant increase in the killing of target cells with anti-CADM1-CAR-NK cells compared with non-transduced NK92 cells. This effect was significantly reduced when A549- GFP-ffluc-CADM1 KO cells were used as target. Next, we generated anti-CADM1-CAR-T cells by transducing human CD3+ T-cells purified from human PBMCs. Similar to anti -CADM1-CAR-NK, anti-CADM1-CAR-T cells showed increased T-cell specific cytotoxicity in a CADM1-dependent manner. Next, we evaluated the efficacy of anti-CADM1-CAR-T cells through adoptive transfer into NSG mice xenografted with human lung adenocarcinoma cells into mouse lungs. The administration of anti-CADM1-CAR-T cells markedly reduced tumor size and enhanced the survival rate of the mice. Importantly this effect was dependent on CADM1 expression, as mice xenografted with human CADM1 KO cells showed neither tumor size reduction nor increased survival. Finally, using IHC staining, we demonstrated that anti-CADM1-CAR-T cells directly target xenografted tumors in the mouse lungs. In conclusion, our findings indicate that CAR-NK and CAR-T cells targeting CADM1-expressing lung tumors have a significant therapeutic potential for the treatment of NSCLC. Citation Format: Sergey Zolov, Sergei Chuikov, Shiva Krishna Katkam, Delaney Shield, Venkateshwar G. Keshamouni. A novel CAR-NK cell therapy to target lung adenocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6360.

Optimizing heat inactivation for SARS-CoV-2 at 95 °C and its implications: A standardized approach

BackgroundStandardized and validated heat inactivation procedure for Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not available. For heat inactivation, various protocols were reported to prepare External Quality Assessment Programme (EQAP) samples without direct comparison between different durations. ObjectiveTo assess the heat inactivation procedures against SARS-CoV-2. The efficacy of the optimized condition was reflected by the results from laboratories testing the EQAP samples. Study designThe SARS-CoV-2 strain was exposed to 95 °C in a water bath for three different time intervals, 5 min, 10 min and 15 min, respectively. The efficacy of inactivation was confirmed by the absence of cytopathic effects and decreasing viral load in 3 successive cell line passages. The viral stock inactivated by the optimal time interval was dispatched to EQAP participants and the result returned were analyzed. ResultsAll of the three conditions were capable of inactivating the SARS-CoV-2 of viral load at around cycle threshold value of 10. When the 95 °C 10 min condition was chosen to prepare SARS-CoV-2 EQAP samples, they showed sufficient homogeneity and stability. High degree of consensus was observed among EQAP participants in all samples dispatched. ConclusionsThe conditions evaluated in the present study could be helpful for laboratories in preparing SARS-CoV-2 EQAP samples.