Deletion of the EP402R Gene from the Genome of African Swine Fever Vaccine Strain ASFV-G-∆I177L Provides the Potential Capability of Differentiating between Infected and Vaccinated Animals.

The African swine fever virus (ASFV) mutant ASFV-G-∆I177L is a safe and efficacious vaccine which induces protection against the challenge of its parental virus, the Georgia 2010 isolate. Although a genetic DIVA (differentiation between infected and vaccinated animals) assay has been developed for this vaccine, still there is not a serological DIVA test for differentiating between animals vaccinated with ASFV-G-∆I177L and those infected with wild-type viruses. In this report, we describe the development of the ASFV-G-∆I177L mutant having deleted the EP402R gene, which encodes for the viral protein responsible for mediating the hemadsorption of swine erythrocytes. The resulting virus, ASFV-G-∆I177L/∆EP402R, does not have a decreased ability to replicates in swine macrophages when compared with the parental ASFV-G-∆I177L. Domestic pigs intramuscularly (IM) inoculated with either 102 or 106 HAD50 of ASFV-G-∆I177L/∆EP402R remained clinically normal, when compared with a group of mock-vaccinated animals, indicating the absence of residual virulence. Interestingly, an infectious virus could not be detected in the blood samples of the ASFV-G-∆I177L/∆EP402R-inoculated animals in either group at any of the time points tested. Furthermore, while all of the mock-inoculated animals presented a quick and lethal clinical form of ASF after the intramuscular inoculation challenge with 102 HAD50 of highly virulent parental field isolate Georgia 2010 (ASFV-G), all of the ASFV-G-∆I177L/∆EP402R-inoculated animals were protected, remaining clinically normal until the end of the observational period. Most of the ASFV-G-∆I177L/∆EP402R-inoculated pigs developed strong virus-specific antibody responses against viral antigens, reaching maximum levels at 28 days post inoculation. Importantly, all of the sera collected at that time point in the ASFV-G-∆I177L/∆EP402R-inoculated pigs did not react in a direct ELISA coated with the recombinant EP402R protein. Conversely, the EP402R protein was readily recognized by the pool of sera from the animals immunized with recombinant live attenuated vaccine candidates ASFV-G-∆I177L, ASFV-G-∆MGF, or ASFV-G-∆9GL/∆UK. Therefore, ASFV-G-∆I177L/∆EP402R is a novel, safe and efficacious candidate with potential to be used as an antigenically DIVA vaccine.

Restoration of virulence in the attenuated Candid#1 vaccine virus requires reversion at both positions 168 and 427 in the envelope glycoprotein GPC.

Live-attenuated virus vaccines provide long-lived protection against viral disease but carry inherent risks of residual pathogenicity and genetic reversion. The live-attenuated Candid#1 vaccine was developed to protect Argentines against lethal infection by the Argentine hemorrhagic fever arenavirus, Junín virus. Despite its safety and efficacy in Phase III clinical study, the vaccine is not licensed in the US, in part due to concerns regarding the genetic stability of attenuation. Previous studies had identified a single F427I mutation in the transmembrane domain of the Candid#1 envelope glycoprotein GPC as the key determinant of attenuation, as well as the propensity of this mutation to revert upon passage in cell culture and neonatal mice. To ascertain the consequences of this reversion event, we introduced the I427F mutation into recombinant Candid#1 (I427F rCan) and investigated the effects in two validated small-animal models: in mice expressing the essential virus receptor (human transferrin receptor 1; huTfR1) and in the conventional guinea pig model. We report that I427F rCan displays only modest virulence in huTfR1 mice and appears attenuated in guinea pigs. Reversion at another attenuating locus in Candid#1 GPC (T168A) was also examined, and a similar pattern was observed. By contrast, virus bearing both revertant mutations (A168T+I427F rCan) approached the lethal virulence of the pathogenic Romero strain in huTfR1 mice. Virulence was less extreme in guinea pigs. Our findings suggest that genetic stabilization at both positions is required to minimize the likelihood of reversion to virulence in a second-generation Candid#1 vaccine.IMPORTANCELive-attenuated virus vaccines, such as measles/mumps/rubella and oral poliovirus, provide robust protection against disease but carry with them the risk of genetic reversion to the virulent form. Here, we analyze the genetics of reversion in the live-attenuated Candid#1 vaccine that is used to protect against Argentine hemorrhagic fever, an often-lethal disease caused by the Junín arenavirus. In two validated small-animal models, we find that restoration of virulence in recombinant Candid#1 viruses requires back-mutation at two positions specific to the Candid#1 envelope glycoprotein GPC, at positions 168 and 427. Viruses bearing only a single change showed only modest virulence. We discuss strategies to genetically harden Candid#1 GPC against these two reversion events in order to develop a safer second-generation Candid#1 vaccine virus.

A replication competent Plasmodium falciparum parasite completely attenuated by dual gene deletion.

Vaccination with infectious Plasmodium falciparum (Pf) sporozoites (SPZ) administered with antimalarial drugs (PfSPZ-CVac), confers superior sterilizing protection against infection when compared to vaccination with replication-deficient, radiation-attenuated PfSPZ. However, the requirement for drug administration constitutes a major limitation for PfSPZ-CVac. To obviate this limitation, we generated late liver stage-arresting replication competent (LARC) parasites by deletion of the Mei2 and LINUP genes (mei2-/linup- or LARC2). We show that Plasmodium yoelii (Py) LARC2 sporozoites did not cause breakthrough blood stage infections and engendered durable sterilizing immunity against various infectious sporozoite challenges in diverse strains of mice. We next genetically engineered a PfLARC2 parasite strain that was devoid of extraneous DNA and produced cryopreserved PfSPZ-LARC2. PfSPZ-LARC2 liver stages replicated robustly in liver-humanized mice but displayed severe defects in late liver stage differentiation and did not form liver stage merozoites. This resulted in complete abrogation of parasite transition to viable blood stage infection. Therefore, PfSPZ-LARC2 is the next-generation vaccine strain expected to unite the safety profile of radiation-attenuated PfSPZ with the superior protective efficacy of PfSPZ-CVac.

CA-CAS-01-A: A Permissive Cell Line for Isolation and Live Attenuated Vaccine Development Against African Swine Fever Virus.

African swine fever virus (ASFV) is the causative agent of the highly lethal African swine fever disease that affects domestic pigs and wild boars. In spite of the rapid spread of the virus worldwide, there is no licensed vaccine available. The lack of a suitable cell line for ASFV propagation hinders the development of a safe and effective vaccine. For ASFV propagation, primary swine macrophages and monocytes have been widely studied. However, obtaining these cells can be time-consuming and expensive, making them unsuitable for mass vaccine production. The goal of this study was to validate the suitability of novel CA-CAS-01-A (CAS-01) cells, which was identified as a highly permissive cell clone for ASFV replication in the MA-104 parental cell line for live attenuated vaccine development. Through a screening experiment, maximum ASFV replication was observed in the CAS-01 cell compared to other sub-clones of MA-104 with 14.89 and log10 7.5 ± 0.15 Ct value and TCID50/ml value respectively. When CAS-01 cells are inoculated with ASFV, replication of ASFV was confirmed by Ct value for ASFV DNA, HAD50/ml assay, TCID50/ml assay, and cytopathic effects and hemadsoption were observed similar to those in primary porcine alveolar macrophages after 5th passage. Additionally, we demonstrated stable replication and adaptation of ASFV over the serial passage. These results suggest that CAS-01 cells will be a valuable and promising cell line for ASFV isolation, replication, and development of live attenuated vaccines.

A novel vaccine strategy using quick and easy conversion of bacterial pathogens to unnatural amino acid-auxotrophic suicide derivatives.

We propose a novel strategy for quick and easy preparation of suicide live vaccine candidates against bacterial pathogens. This method requires only the transformation of one or more plasmids carrying genes encoding for two types of biological devices, an unnatural amino acid (uAA) incorporation system and toxin-antitoxin systems in which translation of the antitoxins requires the uAA incorporation. Escherichia coli BL21-AI laboratory strains carrying the plasmids were viable in the presence of the uAA, whereas the free toxins killed these strains after the removal of the uAA. The survival time after uAA removal could be controlled by the choice of the uAA incorporation system and toxin-antitoxin systems. Multilayered toxin-antitoxin systems suppressed escape frequency to less than 1 escape per 109 generations in the best case. This conditional suicide system also worked in Salmonella enterica and E. coli clinical isolates. The S. enterica vaccine strains were attenuated with a >105 fold lethal dose. Serum IgG response and protection against the parental pathogenic strain were confirmed. In addition, the live E. coli vaccine strain was significantly more immunogenic and provided greater protection than a formalin-inactivated vaccine. The live E. coli vaccine was not detected after inoculation, presumably because the uAA is not present in the host animals or the natural environment. These results suggest that this strategy provides a novel way to rapidly produce safe and highly immunogenic live bacterial vaccine candidates. IMPORTANCE: Live vaccines are the oldest vaccines with a history of more than 200 years. Due to their strong immunogenicity, live vaccines are still an important category of vaccines today. However, the development of live vaccines has been challenging due to the difficulties in achieving a balance between safety and immunogenicity. In recent decades, the frequent emergence of various new and old pathogens at risk of causing pandemics has highlighted the need for rapid vaccine development processes. We have pioneered the use of uAAs to control gene expression and to conditionally kill host bacteria as a biological containment system. This report proposes a quick and easy conversion of bacterial pathogens into live vaccine candidates using this containment system. The balance between safety and immunogenicity can be modulated by the selection of the genetic devices used. Moreover, the uAA-auxotrophy can prevent the vaccine from infecting other individuals or establishing the environment.

Δfur mutant as a potential live attenuated vaccine (LAV) candidate protects American eels (Anguilla rostrata) from Vibrio harveyi infection.

The eel farming industry is highly susceptible to Vibriosis. Although various types of vaccines against Vibriosis have been investigated, there is limited research on decreasing the virulence of Vibrions through gene knockout and utilizing it as live attenuated vaccines (LAV). In this study, we aim to develop a LAV candidate against Vibrio harveyi infection in American eels (Anguilla rostrata) using a ferric uptake regulator (fur) gene mutant strain of V. harveyi (Δfur mutant). After the eels were administrated with the Δfur mutant at the dose of 4 × 102 cfu/g body weight, the phagocytic activity of the leucocytes, plasma IgM antibody titers, activity of lysozyme and Superoxide Dismutase (SOD) enzyme, and gene expression levels of 18 immune related proteins were detected to evaluate the protection effect of the LAV. Preliminary findings suggest that the LAV achieved over 60% relative percent survival (RPS) after the American eels were challenged by a wild-type strain of V. harveyi infection on 28 and 42 days post the immunization (dpi). The protection was mainly attributed to increased plasma IgM antibody titers, higher levels of lysozyme, enhanced activity of SOD and some regulated genes encoded immune related proteins. Together, the Δfur mutant strain of V. harveyi, as a novel LAV vaccine, demonstrates promising protective effects against V. harveyi infection in American eels, thus presenting a potential candidate vaccine for fish farming.

Deep sequencing and variant frequency analysis for the quality control of a live bacterial vaccine against contagious bovine pleuropneumonia, strain T1.

Vaccination is the most cost-effective tool to control contagious bovine pleuropneumonia. The vaccines currently used in Africa are derived from a live strain called T1, which was attenuated by passage in embryonated eggs and broth culture. The number of passages is directly correlated to the degree of attenuation of the vaccinal strains and inversely correlated to their immunogenicity in cattle. Current quality control protocols applied to vaccine batches allow the assessment of identity, purity, and titers, but cannot assess the level of genetic drift form the parental vaccine strains. Deep sequencing was used to assess the genetic drift generated over controlled in vitro passages of the parental strain, as well as on commercial vaccine batches. Signatures of cloning procedures were detected in some batches, which imply a deviation from the standard production protocol. Deep sequencing is proposed as a new tool for the identity and stability control of T1 vaccines.

Recent progress and major gaps in the vaccine development for African swine fever.

The swine industry across the globe is recently facing a devastating situation imparted by a highly contagious and deadly viral disease, African swine fever. The disease is caused by a DNA virus, the African swine fever virus (ASFV) of the genus Asfivirus. ASFV affects both wild boars and domestic pigs resulting in an acute form of hemorrhagic fever. Since the first report in 1921, the disease remains endemic in some of the African countries. However, the recent occurrence of ASF outbreaks in Asia led to a fresh and formidable challenge to the global swine production industry. Culling of the infected animals along with the implementation of strict sanitary measures remains the only options to control this devastating disease. Efforts to develop an effective and safe vaccine against ASF began as early as in the mid-1960s. Different approaches have been employed for the development of effective ASF vaccines including inactivated vaccines, subunit vaccines, DNA vaccines, virus-vectored vaccines, and live attenuated vaccines (LAVs). Inactivated vaccines are a non-feasible strategy against ASF due to their inability to generate a complete cellular immune response. However genetically engineered vaccines, such as subunit vaccines, DNA vaccines, and virus vector vaccines, represent tailored approaches with minimal adverse effects and enhanced safety profiles. As per the available data, gene deleted LAVs appear to be the most potential vaccine candidates. Currently, a gene deleted LAV (ASFV-G-∆I177L), developed in Vietnam, stands as the sole commercially available vaccine against ASF. The major barrier to the goal of developing an effective vaccine is the critical gaps in the knowledge of ASFV biology and the immune response induced by ASFV infection. The precise contribution of various hosts, vectors, and environmental factors in the virus transmission must also be investigated in depth to unravel the disease epidemiology. In this review, we mainly focus on the recent progress in vaccine development against ASF and the major gaps associated with it.

The attenuated African swine fever vaccine HLJ/18-7GD provides protection against emerging prevalent genotype II variants in China.

Genetic changes have occurred in the genomes of prevalent African swine fever viruses (ASFVs) in the field in China, which may change their antigenic properties and result in immune escape. There is usually poor cross-protection between heterogonous isolates, and, therefore, it is important to test the cross-protection of the live attenuated ASFV vaccines against current prevalent heterogonous isolates. In this study, we evaluated the protective efficacy of the ASFV vaccine candidate HLJ/18-7GD against emerging isolates. HLJ/18-7GD provided protection against a highly virulent variant and a lower lethal isolate, both derived from genotype II Georgia07-like ASFV and isolated in 2020. HLJ/18-7GD vaccination prevented pigs from developing ASF-specific clinical signs and death, decreased viral shedding via the oral and rectal routes, and suppressed viral replication after challenges. However, HLJ/18-7GD vaccination did not provide solid cross-protection against genotype I NH/P68-like ASFV challenge in pigs. HLJ/18-7GD vaccination thus shows great promise as an alternative strategy for preventing and controlling genotype II ASFVs, but vaccines providing cross-protection against different ASFV genotypes may be needed in China.

Reverse Genetics System for Rift Valley Fever Virus.

Rift Valley fever virus (RVFV) is an important mosquito-borne virus that can cause severe disease manifestations in humans including ocular damage, vision loss, late-onset encephalitis, and hemorrhagic fever. In ruminants, RVFV can cause high mortality rates in young animals and high rates of abortion in pregnant animals resulting in an enormous negative impact on the economy of affected regions. To date, no licensed vaccines in humans or anti-RVFV therapeutics for animal or human use are available. The development of reverse genetics has facilitated the generation of recombinant infectious viruses that serve as powerful tools for investigating the molecular biology and pathogenesis of RVFV. Infectious recombinant RVFV can be rescued entirely from cDNAs containing predetermined mutations in their genomes to investigate virus-host interactions and mechanisms of pathogenesis and generate live-attenuated vaccines. In this chapter, we will describe the experimental procedures for the implementation of RVFV reverse genetics.

A recombination-resistant genome for live attenuated and stable PEDV vaccines by engineering the transcriptional regulatory sequences.

IMPORTANCE: Coronaviruses are important pathogens of humans and animals, and vaccine developments against them are imperative. Due to the ability to induce broad and prolonged protective immunity and the convenient administration routes, live attenuated vaccines (LAVs) are promising arms for controlling the deadly coronavirus infections. However, potential recombination events between vaccine and field strains raise a safety concern for LAVs. The porcine epidemic diarrhea virus (PEDV) remodeled TRS (RMT) mutant generated in this study replicated efficiently in both cell culture and in pigs and retained protective immunogenicity against PEDV challenge in pigs. Furthermore, the RMT PEDV was resistant to recombination and genetically stable. Therefore, RMT PEDV can be further optimized as a backbone for the development of safe LAVs.

A Novel Quadruple Gene-Deleted BoHV-1-Vectored RVFV Subunit Vaccine Induces Humoral and Cell-Mediated Immune Response against Rift Valley Fever in Calves.

Rift Valley fever virus (RVFV) is considered to be a high biodefense priority based on its threat to livestock and its ability to cause human hemorrhagic fever. RVFV-infected livestock are also a significant risk factor for human infection by direct contact with contaminated blood, tissues, and aborted fetal materials. Therefore, livestock vaccination in the affected regions has the direct dual benefit and one-health approach of protecting the lives of millions of animals and eliminating the risk of severe and sometimes lethal human Rift Valley fever (RVF) disease. Recently, we have developed a bovine herpesvirus type 1 (BoHV-1) quadruple gene mutant virus (BoHV-1qmv) vector that lacks virulence and immunosuppressive properties due to the deletion of envelope proteins UL49.5, glycoprotein G (gG), gE cytoplasmic tail, and US9 coding sequences. In the current study, we engineered the BoHV-1qmv further by incorporating a chimeric gene sequence to express a proteolytically cleavable polyprotein: RVFV envelope proteins Gn ectodomain sequence fused with bovine granulocyte-macrophage colony-stimulating factor (GMCSF) and Gc, resulting in a live BoHV-1qmv-vectored subunit vaccine against RVFV for livestock. In vitro, the resulting recombinant virus, BoHV-1qmv Sub-RVFV, was replicated in cell culture with high titers. The chimeric Gn-GMCSF and Gc proteins expressed by the vaccine virus formed the Gn-Gc complex. In calves, the BoHV-1qmv Sub-RVFV vaccination was safe and induced moderate levels of the RVFV vaccine strain, MP12-specific neutralizing antibody titers. Additionally, the peripheral blood mononuclear cells from the vaccinated calves had six-fold increased levels of interferon-gamma transcription compared with that of the BoHV-1qmv (vector)-vaccinated calves when stimulated with heat-inactivated MP12 antigen in vitro. Based on these findings, we believe that a single dose of BoHV-1qmv Sub-RVFV vaccine generated a protective RVFV-MP12-specific humoral and cellular immune response. Therefore, the BoHV-1qmv sub-RVFV can potentially be a protective subunit vaccine for cattle against RVFV.

Genomic similarity and antibody-dependent enhancement of immune serum potentially affect the protective efficacy of commercial MLV vaccines against NADC30-like PRRSV.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant diseases affecting the pig industry worldwide. The PRRSV mutation rate is the highest among the RNA viruses. To date, NADC30-like PRRSV and highly pathogenic PRRSV (HP-PRRSV) are the dominant epidemic strains in China; however, commercial vaccines do not always provide sufficient cross-protection, and the reasons for insufficient protection are unclear. This study isolated a wild-type NADC30-like PRRSV, SX-YL1806, from Shaanxi Province. Vaccination challenge experiments in piglets showed that commercial modified live virus (MLV) vaccines provided good protection against HP-PRRSV. However, it could not provide sufficient protection against the novel strain SX-YL1806. To explore the reasons for this phenomenon, we compared the genomic homology between the MLV strain and HP-PRRSV or NADC30-like PRRSV and found that the MLV strain had a lower genome similarity with NADC30-like PRRSV. Serum neutralization assay showed that MLV-immune serum slightly promoted the homologous HP-PRRSV replication and significantly promoted the heterologous NADC30-like PRRSV strain replication in vitro, suggesting that antibody-dependent enhancement (ADE) might also play a role in decreasing MLV protective efficacy. These findings expand our understanding of the potential factors affecting the protective effect of PRRSV MLV vaccines against the NADC30-like strains.

Deletion of a 7-amino-acid region in the porcine epidemic diarrhea virus envelope protein induces higher type I and III interferon responses and results in attenuation in vivo.

Porcine epidemic diarrhea virus (PEDV) leads to enormous economic losses for the pork industry. However, the commercial vaccines failed to fully protect against the epidemic strains. Previously, the rCH/SX/2016-SHNXP strain with the entire E protein and the rCH/SX/2015 strain with the deletion of 7-amino-acid (7-aa) at positions 23-29 in E protein were constructed and rescued. The pathogenicity assay indicated that rCH/SX/2015 is an attenuated strain, but rCH/SX/2016-SHNXP belongs to the virulent strains. Then, the recombination PEDV (rPEDV-EΔaa23-aa29)strain with a 7-aa deletion in the E protein was generated, using the highly virulent rCH/SX/2016-SHNXP strain (rPEDV-Ewt) as the backbone. Compared with the rPEDV-Ewt strain, the release and infectivity of the rPEDV-EΔaa23-aa29 strain were significantly reduced in vitro, but stronger interferon (IFN) responses were triggered both in vitro and in vivo. The pathogenicity assay showed that the parental strain resulted in severe diarrhea (100%) and death (100%) in all piglets. Compared with the parental strain group, rPEDV-EΔaa23-aa29 caused lower mortality (33%) and diminished fecal PEDV RNA shedding. At 21 days, all surviving pigs were challenged orally with rPEDV-Ewt. No pigs died in the two groups. Compared with the mock group, significantly delayed and milder diarrhea and reduced fecal PEDV RNA shedding were detected in the rPEDV-EΔaa23-aa29 group. In conclusion, the deletion of a 7-aa fragment in the E protein (EΔaa23-aa29) attenuated PEDV but retained its immunogenicity, which can offer new ideas for the design of live attenuated vaccines and provide new insights into the attenuated mechanism of PEDV. IMPORTANCE Porcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets and remains a large challenge to the pork industry. Unfortunately, no safe and effective vaccines are available yet. The pathogenesis and molecular basis of the attenuation of PEDV remain unclear, which seriously hinders the development of PEDV vaccines. This study found that the rPEDV carrying EΔaa23-aa29 mutation in the E protein induced significantly higher IFN responses than the parental virus, partially attenuated, and remained immunogenic in piglets. For the first time, PEDV E was verified as an IFN antagonist in the infection context and identified as a virulence factor of PEDV. Our data also suggested that EΔaa23-aa29 mutation can be a good target for the development of live attenuated vaccines for PEDV and also provide new perspectives for the attenuated mechanism of PEDV.

Genetic signatures associated with the virulence of porcine epidemic diarrhea virus AH2012/12.

IMPORTANCE: Porcine epidemic diarrhea (PED) caused by PED virus (PEDV) remains a big threat to the swine industry worldwide. Vaccination with live attenuated vaccine is a promising method to prevent and control PED, because it can elicit a more protective immunity than the killed vaccine, subunit vaccine, and so on. In this study, we found two obvious deletions in the genome of a high passage of AH2012/12. We further confirmed the second deletion which contains seven amino acids at the carboxy-terminus of the S2 gene and the start codon of ORF3 can reduce its pathogenicity in vivo. Animal experiments indicated that the recombinant PEDV with deleted carboxy-terminus of S gene showed higher IgG, IgA, neutralization antibodies, and protection effects against virus challenge than the killed vaccine. These data reveal that the engineering of the carboxy-terminus of the S2 gene may be a promising method to develop live attenuated vaccine candidates of PEDV.

A novel chimeric vaccine candidate for porcine reproductive and respiratory syndrome virus (PRRSV) I and II elicits neutralizing antibodies against both types.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important virus within the swine industry. The virus causes respiratory disease and reproductive failure. Two species of PRRSV-I and II are co-dominant, yet no effective vaccination strategy has been developed to protect against these two types. With an aim to develop a chimeric vaccine strain to protect against both types, in this study, a chimeric porcine reproductive and respiratory syndrome virus (PRRSV) type I and II was rescued using reverse genetics for the first time. Four chimeric infectious clones were designed based on the genomic arrangement of the structural proteins. However, only the clone carrying the transcriptional regulatory sequence (TRS) and ORF6 of a PRRSV-I and ORF6 of a PRRSV-II generated a viable recombinant virus, suggesting that concurrent expression of ORF6 from both parental viruses is essential for the recovery of type I and II chimeric PRRSV. The chimeric virus showed significantly lower replication ability than its parental strains in vitro, which was improved by serial passaging. In vivo, groups of pigs were inoculated with either the chimeric virus, one of the parental strains, or PBS. The chimeric virus replicated in pig tissue and was detected in serum 7 days post-inoculation. Serum neutralization tests indicated that pigs inoculated with the chimeric virus elicited neutralizing antibodies that inhibited infection with strains of both species and with greater coverage than the parental viruses. In conclusion, the application of this technique to construct a chimeric PRRSV holds promise for the development of a highly effective modified live vaccine candidate. This is particularly significant since there are currently no approved commercial divalent vaccines available to combat PRRSV-I and II co-infections.

A novel HRM-based gap-qRT-PCR for identification and quantitation of the vaccine and field strain(s) of lumpy skin disease virus.

Lumpy skin disease (LSD) has become the most important animal health problem in India due to high morbidity, mortality and production losses caused by it. A homologous live-attenuated LSD vaccine (Lumpi-ProVacInd) was recently developed by using a local LSD virus (LSDV) strain (LSDV/2019/India/Ranchi) in India which is likely to replace the existing practice of vaccinating cattle with goatpox vaccine. It is essential to differentiate the vaccine and field strains, if a live-attenuated vaccine has been used for control and eradication of the disease. As compared to the prevailing vaccine and field/virulent strains, the Indian vaccine strain (Lumpi-ProVacInd) has a unique deletion of 801 nucleotides in its inverted terminal repeat (ITR) region. We exploited this unique feature and developed a novel high resolution melting-based gap quantitative real-time PCR (HRM-gap-qRT-PCR) for rapid identification and quantitation of the vaccine and field strain(s) of LSDV.

A one-health approach to design an mRNA-based vaccine candidate against the lumpy skin disease virus as an alternative to live-attenuated vaccines.

OBJECTIVE: Recently, lumpy skin disease (LSD) has been spread over the Asian, European, and Middle Eastern regions making it a significant hazard to the chain of cattle production, milk production, and human milk consumption, requiring prompt attention. Lumpy skin disease virus has high morbidity and low fatality rates, but its infections have led to terrible economic and agricultural consequences. Although live-attenuated vaccines have been commercialized, farmers in different regions have not taken them well because of the allergic responses against the vaccines. The study aims to develop an mRNA-based vaccine candidate for LSDV, using immunoinformatic approaches to minimize allergenicity and homology while maximizing immunogenic potential. MATERIALS AND METHODS: The study used extensive immunoinformatic approaches to shortlist five proteins from the LSDV genome that belong to the transmembrane region and are crucial in early viral interaction with host cells. The B-cell and T-cell-specific epitopes were chosen based on non-allergenicity, antigenicity, non-homology, surface accessibility, and lower IC50 inhibition values. The construct's stability, hydrophilicity, and antigenic potential were analyzed using the instability index, Grand Average of Hydropathicity (GRAVY) index, and antigenicity, respectively. RESULTS: We selected a total of 34 epitopes, consisting of 12 B-cell-specific epitopes and 22 T-cell-specific epitopes. These epitopes were chosen based on their characteristics such as non-allergenicity, antigenicity, non-homology, surface accessibility, and lower IC50 inhibition values. Specifically, 11 epitopes were selected for Major Histocompatibility Complex-I, and another 11 epitopes were chosen for Major Histocompatibility Complex-II. The inclusion of the RS09 adjuvant enhanced the immunogenic potential of the vaccine. The instability index was found to be 38.60. Additionally, the GRAVY index, indicating hydrophilicity, was calculated as -0.151. Furthermore, the antigenicity value of 0.6073 confirmed its potential to elicit an immune response. Further supporting its immunogenic potential, strong immune stimulation was observed, with IgM+IgG titers reaching 6,000 (arbitrary units) and IFNg titers measuring 400,000 ng/mL. These results provide additional evidence of the vaccine's ability to stimulate a robust immune response. CONCLUSIONS: The study results indicate that the developed mRNA-based vaccine candidate for LSDV has high immunogenic potential and could serve as an effective alternative to live-attenuated vaccines. Further experimental validations are required to test its efficacy. The study also highlights the potential of the One-Health approach to tackle non-zoonotic diseases that have significant consequences for the environment and humanity.

Rescue of a Live-Attenuated Porcine Epidemic Diarrhea Virus HSGP Strain Using a Virulent Strain and a Partially Attenuated Strain.

In South Korea in 2013, the G1-based vaccine failed to prevent an outbreak of G2b-type porcine epidemic diarrhea virus (PEDV), which is more pathogenic than the traditional G1-type strain, thereby allowing the virus to spread. In 2017 and 2018, field samples were cultured sequentially on Vero cells to isolate HS (virulent) and SGP-M1 (partially attenuated) strains, respectively, of the G2b type. The HS strain harbors a single amino acid (aa) change and two aa deletions in the N-terminal domain of S1 (55I56G57E→55K56Δ57Δ). The SGP-M1 strain harbors a seven aa deletion in the C-terminal domain of S2 (1380~1386ΔFEKVHVQ). By co-infecting various animal cells with these two strains (HS and SGP-M1), we succeeded in cloning strain HSGP, which harbors the mutations present in the two original viruses. The CPE pattern of the HSGP strain was different from that of the HS and SGP-M1 strains, with higher viral titers. Studies in piglets showed attenuated pathogenicity of the HSGP strain, with no clinical symptoms or viral shedding, and histopathologic lesions similar to those in negative controls. These findings confirm that deletion of specific sequences from the S gene attenuates the pathogenicity of PEDV. In addition, HSGP strains created by combining two different strains have the potential for use as novel attenuated live vaccine candidates.

The attenuated live vaccine strain W2512 provides protection against novel variant infectious bursal disease virus.

Infectious bursal disease virus (IBDV) causes an acute and highly contagious infectious disease characterized by severe immunosuppression, causing great economic losses to the poultry industry globally. Over the past 30 years, this disease has been well controlled through vaccination and strict biosafety measures. However, novel variant IBDV strains have emerged in recent years, posing a new threat to the poultry industry. Our previous epidemiological survey showed that few novel variant IBDV strains had been isolated from chickens immunized with the attenuated live vaccine W2512-, suggesting that this vaccine is efficacious against novel variant strains. Here, we report the protective effect of the W2512 vaccine against novel variant strains in SPF chickens and commercial yellow-feathered broilers. We found that W2512 causes severe atrophy of the bursa of Fabricius in SPF chickens and commercial yellow-feathered broilers, induces high levels of antibodies against IBDV, and protects chickens from infection with the novel variant strains via a placeholder effect. This study highlights the protective effect of commercial attenuated live vaccines against the novel IBDV variant and provides guidance for the prevention and control of this disease.