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A previous study proved that vGPE− mainly maintains the properties of classical swine fever (CSF) virus, which is comparable to the GPE− vaccine seed and is a potentially valuable backbone for developing a CSF marker vaccine. Chimeric viruses were constructed based on an infectious cDNA clone derived from the live attenuated GPE− vaccine strain as novel CSF vaccine candidates that potentially meet the concept of differentiating infected from vaccinated animals (DIVA) by substituting the glycoprotein Erns of the GPE− vaccine strain with the corresponding region of non-CSF pestiviruses, either pronghorn antelope pestivirus (PAPeV) or Phocoena pestivirus (PhoPeV). High viral growth and genetic stability after serial passages of the chimeric viruses, namely vGPE−/PAPeV Erns and vGPE−/PhoPeV Erns, were confirmed in vitro. In vivo investigation revealed that two chimeric viruses had comparable immunogenicity and safety profiles to the vGPE− vaccine strain. Vaccination at a dose of 104.0 TCID50 with either vGPE−/PAPeV Erns or vGPE−/PhoPeV Erns conferred complete protection for pigs against the CSF virus challenge in the early stage of immunization. In conclusion, the characteristics of vGPE−/PAPeV Erns and vGPE−/PhoPeV Erns affirmed their properties, as the vGPE− vaccine strain, positioning them as ideal candidates for future development of a CSF marker vaccine.

Usutu virus (USUV) is currently emerging in Europe, where it has caused numerous mass bird die-off events and neuroinvasive disease in humans. Multiple strains of USUV are circulating throughout Europe, but only some of them have been associated with severe disease in humans. The USUV proteins responsible for and the mechanisms through which they cause severe disease are unknown; however, this information could be invaluable in evaluating disease potential of specific strains and the creation of anti-viral therapies. Here, we swapped genes between USUV strains that cause mild and severe disease and were able to identify a viral protein that mediates virulence. We also discovered that the mild strain of USUV takes significantly longer to complete fusion during viral entry into host cells than the severe strain. This delayed fusion could have impacts on cellular tropism, viral kinetics, susceptibility of the virus to immune responses, and, ultimately, disease severity.

Mpox, the disease caused by monkeypox virus (MPXV) is increasing in Africa and in 2022 spread to more than 100 countries sickening more than 100,000 individuals. Four clades of MPXV have been recognized with differences in severity of disease and extent of human-to-human transmission. Determination of the genetic basis for these differences could help to develop improved therapeutics and vaccines. The Castaneous (CAST) mouse is highly susceptible to MPXV and virulence differences of MPXV clades are statistically significant mimicking their relative severities in humans. The present study was intended to evaluate the CAST mouse as a model for investigating genomic differences by replacing genes of Zaire-1979 005 (Z-79) clade Ia MPXV with homologous gene sequences of the less virulent and less transmissible USA-2003 clade IIa MPXV. The expectation was that some gene replacements would reduce the virulence of Z-79 chimera. Recombinant viruses expressing firefly luciferase were constructed in which partially overlapping Z-79 DNA segments of 5,000 to 13,000 bp containing ∼ 40 genes in total from the two ends of MPXV-79 were replaced with corresponding segments of USA-2003. Virulence was determined by live animal imaging in addition to weight loss and survival. Although there were statistical differences in survival and viral luminescence between the clade Ia and clade IIa MPXVs, no significant difference was found by replacing individual or multiple genes of clade Ia Z-79 with corresponding genes of clade IIa USA-2003. The absence of a significant reduction in virulence can have several explanations that would inform future experiments.

In recent years, the rabbit hemorrhagic disease virus 2(RHDV GI.2) has rapidly spread worldwide due to its broad natural host range, strong pathogenicity, and significant antigenic differences from the traditional RHDV1 (GI.1). The widespread prevalence of both GI.1 and GI.2 RHDV poses a serious threat to the healthy development of the global rabbit industry. Despite this, there is still a notable absence of effective multivalent or broad-spectrum vaccines for controlling RHD(GI.1 and GI.2). In the present study, we developed a broad-spectrum chimeric antigen vaccine using a substitution strategy targeting the surface loop of the main antigen protein VP60 of RHDV. The chimeric VP60 antigen, expressed by a recombinant baculovirus expression system, was successfully assembled into virus-like particles (VLPs). The VLPs exhibited typical natural virus size and morphology under an electron microscope. Immunization with chimeric VLPs effectively protected rabbits from lethal challenged by both virulent strains of RHDV, GI.1 and GI.2, showing an effect comparable to that of a mixture vaccine containing two wild-type VLPs. These findings demonstrate a promising strategy for developing a cost-effective and straightforward preparation process for broad-spectrum vaccine against RHD. •The Loop region of the VP60 protein, found in the rabbit hemorrhagic disease virus and its variant strains, plays a pivotal role in generating protective antibodies.•The sequence substitution in the loop region is prominently exhibited on the surface of the heterotypic VP60 protein, without impeding the formation of chimeric virus-like particle (VLP).•The chimeric virus-like particles have the ability to simultaneously elicit hemagglutination inhibition antibodies against GI.1 and GI.2 VP60 protein, demonstrating effective protection against GI.1 and GI.2 rabbit hemorrhagic disease virus.

Persistent infection with high-risk human papillomavirus (HPV) types can lead to the development of cancer in HPV-infected tissues, including the cervix, oropharynx, anus, penis, vagina, and vulva. While current HPV vaccines cover approximately 90 % of cervical cancers, nearly 10 % of cases associated with HPV types not included in the vaccines remain unaddressed, notably HPV59. This study describes the development of a chimeric virus-like particle (VLP) targeting HPV18/45/59, proposed as a vaccine candidate for high-risk HPV type (HPV59) currently lacking commercial vaccines. Given that the majority of neutralizing antibody epitopes are located on the surface loops, we engineered a strategic swap of these loops between the closely related HPV18 and HPV45. This methodology was then extended to incorporate surface loops of HPV59, resulting in the lead candidate construct of the H18-45BCEF-59HI chimeric VLP with two surface loops swapping from HPV45 to HPV18. Characterization confirmed that H18-45BCEF-59HI closely resembled the wild-type (WT) backbone types in particle size and morphology, as verified by Transmission Electron Microscopy (TEM), High-Performance Size-Exclusion Chromatography (HPSEC), and Analytical Ultracentrifugation (AUC), and demonstrated similar thermal stability as evidenced by Differential Scanning Calorimetry (DSC). Immunization studies in mice with the chimeric VLPs assessed their immunogenicity, revealing that the H18-45EF-59HI chimeric VLP exhibited optimal cross-neutralization. Additionally, when produced in a Good Manufacturing Practice (GMP)-like facility, the H18-45BCEF-59HI VLP was selected as a promising vaccine candidate for the prevention of HPV18/45/59 infection. This study not only offers a potential solution to the current vaccination gap but also provides a foundational approach for the design of vaccines targeting viruses with multiple subtypes or variants.

Human papilloma virus type 16 (HPV16) is the most prevalent etiologic agent associated with cervical cancer, and its early proteins E5, E6 and E7 play important roles in cervical epithelium transformation to cervical intraepithelial neoplasia and even cervical cancer. Hence, these oncoproteins are ideal target antigens for developing immunotherapeutic vaccines against HPV-associated infection and cervical cancer. Currently, multi-epitope vaccines have been a promising strategy for immunotherapy for viral infection or cancers. In this study, the E5aa28-46, E6aa37-57 and E7aa26-57 peptides were selected and linked to form a novel multi-epitopes vaccine (E765m), which was inserted into the major immune dominant region (MIR) of hepatitis B virus core antigen (HBc) to construct a HBc-E765m chimeric virus-like particles (cVLPs). The immunogenicity and immunotherapeutic effect of the cVLPs vaccine was evaluated in immunized mice and a tumor-bearing mouse model. The results showed that HBc-E765m cVLPs elicited high E5-, E6- and E7- specific CTL and serum IgG antibody responses, and also relatively high levels of the cytokines IFN-γ, IL-4 and IL-5. More importantly, the cVLPs vaccine significant suppressed tumor growth in mice bearing E5-TC-1 tumors. Our findings provide strong evidence that this novel HBc-E765m cVLPs vaccine could be a candidate vaccine for specific immunotherapy in HPV16-associated cervical intraepithelial neoplasia or cervical cancer.

• Previous studies have found a correlation between the abilities of PVX vector-expressed HCPro variants to bind small RNAs (sRNAs), and to suppress silencing. Moreover, HCPro preferred to bind viral sRNAs of 21–22 nucleotides (nt) containing 5′-terminal adenines. This would require such viral sRNAs to have either different access to the suppressor than those of plant sequences, or different molecular properties. • To investigate this preference further, we have used suppressor-competent or suppressor-deficient HCPro variants, expressed from either T-DNAs or potyvirus constructs. Then, the sRNAs generated in plants and associated with the purified HCPro variants were characterized. • Marked differences were observed in the ratios of sRNAs of plant vs nonplant origin that bound to suppressor-competent HCPro, depending on the mode of its expression. Regardless of the means of expression, HCPro retained the same preference among the nonplant sRNAs of 21–22 nt for those with 5′-terminal adenines. Relative methylation levels of individual sRNAs were assessed, and the nonplant sRNAs were found to be significantly less methylated in the presence of the suppressor. • Targeted binding of sRNAs based on size, 5′-terminal sequence and origin, together with affecting their methylation, could explain how HCPro counteracts silencing.

To develop the new classical swine fever (CSF) vaccine candidate with differentiating infected vaccinated animals (DIVA) characteristics, a chimeric CSF virus (CSFV) was constructed based on an infectious cDNA clone of the CSF vaccine C-strain. The 5’- and 3’-untranslated regions (UTRs) and partial E2 region (residues 690-860) of the C-strain were substituted with the corresponding regions of bovine viral diarrhoea virus (BVDV) to construct the chimeric cDNA clone pC/bUTRs-tE2. The chimeric virus rC/bUTRs-tE2 was generated by several passages of pC/bUTRs-tE2-transfected PK15 cells. Stable growth and genetic properties of rC/bUTRs-tE2 were obtained after 30 serial passages. Compared to parental rC/bUTRs-tE2 (1st passage), two residue mutations (M834K and M979K) located in E2 in rC/bUTRs-tE2 P30 were observed. Compared to the C-strain, rC/bUTRs-tE2 exhibited unchanged cell tropism and decreased plaque-forming ability. Substituting the C-strain UTRs with the BVDV UTRs resulted in significantly increased viral replication in PK15 cells. Compared to CSFV Erns-positive and BVDV tE2-negative antibody responses induced by the CSF vaccine C-strain, immunization of rabbits and piglets with rC/bUTRs-tE2 resulted in serological profiles of CSFV Erns- and BVDV tE2-positive antibodies, which are used to serologically discriminate pigs that are clinically infected and vaccinated. Vaccination of piglets with rC/bUTRs-tE2 conferred complete protection against lethal CSFV challenge. Our results suggest that rC/bUTRs-tE2 is a promising new CSF marker vaccine candidate.

Usutu virus (USUV) is an emerging arthropod-borne flavivirus that has expanded into multiple European countries during the past several decades. USUV infection in human has been linked to severe neurological complications, and no vaccine is now available against USUV. In this work, we develop a live-attenuated chimeric USUV vaccine (termed ChinUSUV) based on the full-length infectious cDNA clone of the licensed Japanese encephalitis virus (JEV) vaccine strain SA14-14-2. In vitro studies demonstrate that ChinUSUV replicates efficiently and maintains its genetic stability. Remarkably, ChinUSUV exhibits a significant attenuation phenotype in multiple mouse models even compared with the licensed JEV vaccine. A single immunization with ChinUSUV elicits potent IgG and neutralizing antibody responses as well as T cell response. Passive transfer of sera from ChinUSUV-immunized mice confers significant protection against lethal homologous challenge in suckling mice. Taken together, our results suggest that ChinUSUV represents a potential USUV vaccine candidate that merits further development.