RESUMEN
Lateral-shaking inducing neuro-degenerative agent virus (LindaV) is a novel member of the highly diverse genus Pestivirus within the family Flaviviridae. LindaV was first detected in Austria in 2015 and was associated with congenital tremor in piglets. Since then, the virus or specific antibodies have been found in a few further pig farms in Austria. However, the actual spatial distribution and the existence of reservoir hosts is largely unknown. Since other pestiviruses of pigs such as classical swine fever virus or atypical porcine pestivirus can also infect wild boar, the question arises whether LindaV is likewise present in the wild boar population. Therefore, we investigated the presence of neutralizing antibodies against LindaV in 200 wild boar samples collected in Southern Germany, which borders Austria. To establish a serological test system, we made use of the interchangeability of the surface glycoproteins and created a chimeric pestivirus using Bungowannah virus (species Pestivirus australiaense) as synthetic backbone. The E1 and E2 glycoproteins were replaced by the heterologous E1 and E2 of LindaV resulting in the chimera BV_E1E2_LV. Viable virus could be rescued and was subsequently applied in a neutralization test. A specific positive control serum generated against the E2 protein of LindaV gave a strong positive result, thereby confirming the functionality of the test system. All wild boar samples, however, tested negative. Hence, there is no evidence that LindaV has become highly prevalent in the wild boar population in Southern Germany.
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Anticuerpos Neutralizantes , Anticuerpos Antivirales , Infecciones por Pestivirus , Pestivirus , Sus scrofa , Enfermedades de los Porcinos , Animales , Alemania/epidemiología , Infecciones por Pestivirus/veterinaria , Infecciones por Pestivirus/epidemiología , Infecciones por Pestivirus/virología , Sus scrofa/virología , Anticuerpos Antivirales/sangre , Porcinos , Pestivirus/genética , Pestivirus/aislamiento & purificación , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/epidemiología , Anticuerpos Neutralizantes/sangre , Pruebas de NeutralizaciónRESUMEN
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.
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Anticuerpos Neutralizantes , Anticuerpos Antivirales , Infecciones por Papillomavirus , Vacunas contra Papillomavirus , Vacunas de Partículas Similares a Virus , Vacunas contra Papillomavirus/inmunología , Vacunas contra Papillomavirus/administración & dosificación , Vacunas contra Papillomavirus/genética , Infecciones por Papillomavirus/prevención & control , Infecciones por Papillomavirus/inmunología , Femenino , Vacunas de Partículas Similares a Virus/inmunología , Animales , Humanos , Ratones , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Papillomavirus Humano 18/inmunología , Papillomavirus Humano 18/genética , Virus del Papiloma HumanoRESUMEN
Tick-borne orthoflaviviruses (TBFs) are classified into three conventional groups based on genetics and ecology: mammalian, seabird and probable-TBF group. Recently, a fourth basal group has been identified in Rhipicephalus ticks from Africa: Mpulungu flavivirus (MPFV) in Zambia and Ngoye virus (NGOV) in Senegal. Despite attempts, isolating these viruses in vertebrate and invertebrate cell lines or intracerebral injection of newborn mice with virus-containing homogenates has remained unsuccessful. In this study, we report the discovery of Xinyang flavivirus (XiFV) in Haemaphysalis flava ticks from Xìnyáng, Henan Province, China. Phylogenetic analysis shows that XiFV was most closely related to MPFV and NGOV, marking the first identification of this tick orthoflavivirus group in Asia. We developed a reverse transcriptase quantitative PCR assay to screen wild-collected ticks and egg clutches, with absolute infection rates of 20.75â% in adult females and 15.19â% in egg clutches, suggesting that XiFV could be potentially spread through transovarial transmission. To examine potential host range, dinucleotide composition analyses revealed that XiFV, MPFV and NGOV share a closer composition to classical insect-specific orthoflaviviruses than to vertebrate-infecting TBFs, suggesting that XiFV could be a tick-only orthoflavivirus. Additionally, both XiFV and MPFV lack a furin cleavage site in the prM protein, unlike other TBFs, suggesting these viruses might exist towards a biased immature particle state. To examine this, chimeric Binjari virus with XIFV-prME (bXiFV) was generated, purified and analysed by SDS-PAGE and negative-stain transmission electron microscopy, suggesting prototypical orthoflavivirus size (~50 nm) and bias towards uncleaved prM. In silico structural analyses of the 3'-untranslated regions show that XiFV forms up to five pseudo-knot-containing stem-loops and a prototypical orthoflavivirus dumbbell element, suggesting the potential for multiple exoribonuclease-resistant RNA structures.
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Flavivirus , Ixodidae , Filogenia , Animales , Flavivirus/genética , Flavivirus/clasificación , Flavivirus/aislamiento & purificación , China , Ixodidae/virología , FemeninoRESUMEN
The prevalence of porcine reproductive and respiratory syndrome virus 1 (PRRSV1) isolates has continued to increase in Chinese swine herds in recent years. However, no effective control strategy is available for PRRSV1 infection in China. In this study, we generated the first infectious cDNA clone (rHLJB1) of a Chinese PRRSV1 isolate and subsequently used it as a backbone to construct an ORF2-6 chimeric virus (ORF2-6-CON). This virus contained a synthesized consensus sequence of the PRRSV1 ORF2-6 gene encoding all the envelope proteins. The ORF2-6 consensus sequence shared > 90% nucleotide similarity with four representative strains (Amervac, BJEU06-1, HKEU16 and NMEU09-1) of PRRSV1 in China. ORF2-6-CON had replication efficacy similar to that of the backbone rHLJB1 virus in primary alveolar macrophages (PAMs) and exhibited cell tropism in Marc-145 cells. Piglet inoculation and challenge studies indicated that ORF2-6-CON is not pathogenic to piglets and can induce enhanced cross-protection against a heterologous SD1291 isolate. Notably, ORF2-6-CON inoculation induced higher levels of heterologous neutralizing antibodies (nAbs) against SD1291 than rHLJB1 inoculation, which was concurrent with a higher percentage of T follicular helper (Tfh) cells in tracheobronchial lymph nodes (TBLNs), providing the first clue that porcine Tfh cells are correlated with heterologous PRRSV nAb responses. The number of SD1291-strain-specific IFNγ-secreting cells was similar in ORF2-6-CON-inoculated and rHLJB1-inoculated pigs. Overall, our findings support that the Marc-145-adapted ORF2-6-CON can trigger Tfh cell and heterologous nAb responses to confer improved cross-protection and may serve as a candidate strain for the development of a cross-protective PRRSV1 vaccine.
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Virus del Síndrome Respiratorio y Reproductivo Porcino , Animales , Porcinos , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Células T Auxiliares Foliculares , Anticuerpos Neutralizantes , China , Secuencia de ConsensoRESUMEN
Numerous infectious diseases in cattle lead to reductions in body weight, milk production, and reproductive performance. Cattle are primarily vaccinated using inactivated vaccines due to their increased safety. However, inactivated vaccines generally result in weaker immunity compared with live attenuated vaccines, which may be insufficient in certain cases. Over the last few decades, there has been extensive research on the use of the Newcastle disease virus (NDV) as a live vaccine vector for economically significant livestock diseases. A single vaccination dose of NDV can sufficiently induce immunity; therefore, a booster vaccination dose is expected to yield limited induction of further immune response. We previously developed recombinant chimeric NDV (rNDV-2F2HN), in which its hemagglutinin-neuraminidase (HN) and fusion (F) proteins were replaced with those of avian paramyxovirus 2 (APMV-2). In vitro analysis revealed that rNDV-2F2HN expressing human interferon-gamma had potential as a cancer therapeutic tool, particularly for immunized individuals. In the present study, we constructed rNDV-2F2HN expressing the bovine rotavirus antigen VP6 (rNDV-2F2HN-VP6) and evaluated its immune response in mice previously immunized with NDV. Mice primarily inoculated with recombinant wild-type NDV expressing VP6 (rNDV-WT-VP6), followed by a booster inoculation of rNDV-2F2HN-VP6, showed a significantly stronger immune response than that in mice that received rNDV-WT-VP6 as both primary and booster inoculations. Therefore, our findings suggest that robust immunity could be obtained from the effects of chimeric rNDV-2F2HN expressing the same or a different antigen of a particular pathogen as a live attenuated vaccine vector.
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Avulavirus , Enfermedades de los Bovinos , Enfermedad de Newcastle , Enfermedades de los Roedores , Rotavirus , Vacunas Virales , Animales , Bovinos , Humanos , Ratones , Virus de la Enfermedad de Newcastle/genética , Pollos , Anticuerpos Antivirales , Vectores Genéticos , Avulavirus/genética , Proteínas Virales/genética , Vacunas de Productos Inactivados , InmunidadRESUMEN
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.
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Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Flavivirus , Vacunas contra la Encefalitis Japonesa , Humanos , Animales , Ratones , Vacunas Atenuadas , Encefalitis Japonesa/prevención & controlRESUMEN
Newcastle disease (ND) and infectious bursal disease (IBD) pose significant threats to the chicken industry, causing substantial economic losses. Currently, immunization through vaccination is the most effective strategy to prevent ND and IBD but currently used traditional vaccines, including inactivated or attenuated vaccines, face challenges in achieving a balance between immunogenicity and safety. To develop a green and efficient novel vaccine for ND and IBD, we developed a bivalent chimeric virus-like particle vaccine (ND-IBD cVLPs) displaying the ND virus (NDV) HN protein and the IBD virus (IBDV) VP2 protein based on the ND VLPs carrier platform and insect baculovirus expression system. This study aimed to evaluate the immunogenicity and protective efficacy of ND-IBD cVLPs in specific pathogen-free chickens. Chickens were immunized with 50 µg of purified ND-IBD cVLPs at 7 days old, boosted at 21 days old, and challenged at 42 days old. The results demonstrated that ND-IBD cVLPs stimulated highly effective hemagglutination inhibition antibody levels against NDV HN protein and enzyme-linked immunosorbent assay antibody levels against the IBDV VP2 protein. Furthermore, ND-IBD cVLPs provided complete protection against virulent NDV and IBDV challenges and mitigated pathological damage to the lung caused by NDV infection and the bursa of Fabricius caused by IBDV infection. These findings suggest that ND-IBD cVLPs hold promise as a safe and efficient novel vaccine candidate for the effective prevention of ND and IBD, extending the development of a foreign protein delivery platform of ND VLPs.
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Infecciones por Birnaviridae , Virus de la Enfermedad Infecciosa de la Bolsa , Enfermedad de Newcastle , Enfermedades de las Aves de Corral , Vacunas de Partículas Similares a Virus , Vacunas Virales , Animales , Pollos , Proteína HN , Anticuerpos Antivirales , Virus de la Enfermedad de Newcastle/genética , Enfermedad de Newcastle/prevención & control , Infecciones por Birnaviridae/prevención & control , Infecciones por Birnaviridae/veterinariaRESUMEN
IMPORTANCE: Human norovirus (HuNoV) is highly infectious and can result in severe illnesses in the elderly and children. So far, there is no effective antiviral drug to treat HuNoV infection, and thus, the development of HuNoV vaccines is urgent. However, NoV evolves rapidly, and currently, at least 10 genogroups with numerous genotypes have been found. The genetic diversity of NoV and the lack of cross-protection between different genotypes pose challenges to the development of broadly protective vaccines. In this study, guided by structural alignment between GI.1 and GII.4 HuNoV VP1 proteins, several chimeric-type virus-like particles (VLPs) were designed through surface-exposed loop grafting. Mouse immunization studies show that two of the designed chimeric VLPs induced cross-immunity against both GI.1 and GII.4 HuNoVs. To our knowledge, this is the first designed chimeric VLPs that can induce cross-immune activities across different genogroups of HuNoV, which provides valuable strategies for the development of cross-reactive HuNoV vaccines.
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Infecciones por Caliciviridae , Epítopos , Genotipo , Norovirus , Vacunas Virales , Virión , Animales , Humanos , Ratones , Infecciones por Caliciviridae/inmunología , Infecciones por Caliciviridae/prevención & control , Infecciones por Caliciviridae/virología , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Inmunización , Norovirus/química , Norovirus/clasificación , Norovirus/genética , Norovirus/inmunología , Vacunas Virales/química , Vacunas Virales/genética , Vacunas Virales/inmunología , Quimera/genética , Quimera/inmunología , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Virión/química , Virión/genética , Virión/inmunologíaRESUMEN
The US Centers for Disease Control and Prevention (CDC), as part of the Federal Select Agent Program, and under the purview of 42 CFR §73.3, has the ability to regulate chimeric viruses that contain portions of pathogens that are part of the select agents and toxins list. In addition, the CDC is responsible for excluding pathogens from regulation, including chimeric viruses, that are sufficiently attenuated. Since 2003, the CDC has excluded over 20 chimeric viruses that contain portions of select agents. But in late 2021, the CDC proposed a regulatory first-the addition of a chimeric virus to the select agents and toxins list. To better understand the importance and applicability of this action, we surveyed the landscape of previous exclusions from select agent regulation. First, we reviewed the exclusion criteria used by the Intragovernmental Select Agents and Toxins Technical Advisory Committee in their advisement of the Federal Select Agent Program. We then reviewed the literature on chimeric viruses that contain portions of select agents and that have been excluded from regulation due to sufficient attenuation, focusing on chimeric alphaviruses and chimeric avian influenza viruses. By analyzing biological commonalities and patterns in the structure and methodology of the development of previously excluded chimeric viruses, we provide insight into how the CDC has used exclusion criteria in the past to regulate chimeric viruses. We conclude by contrasting previous exclusions with the recent addition of SARS-CoV-1/SARS-CoV-2 chimeric viruses to the select agents and toxins list, demonstrating that this addition strays from established, effective regulatory processes, and is thus a regulatory misstep.
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Virus de la Influenza A , Humanos , Animales , BiologíaRESUMEN
In late 2021, the US Centers for Disease Control and Prevention (CDC) posted an interim final rule (86 FR 64075) to the federal register regulating the possession, use, and transfer of SARS-CoV-1/SARS-CoV-2 chimeric viruses. In doing so, the CDC provided the reasoning that viral chimeras combining the transmissibility of SARS-CoV-2 with the pathogenicity and lethality of SARS-CoV-1 pose a significant risk to public health and should thus be placed on the select agents and toxins list. However, 86 FR 64075 lacked clarity in its definitions and scope, some of which the CDC addressed in response to public comments in the final rule, 88 FR 13322, in early 2023. To evaluate these regulatory actions, we reviewed the existing select agent regulations to understand the landscape of chimeric virus regulation. Based on our findings, we first present clear definitions for the terms "chimeric virus," "viral chimera," and "virulence factor" and provide a list of SARS-CoV-1 virulence factors in an effort to aid researchers and federal rulemaking for these agents moving forward. We then provide suggestions for a combination of similarity and functional characteristic cutoffs that the government could use to enable researchers to distinguish between regulated and nonregulated chimeras. Finally, we discuss current select agent regulations and their overlaps with 86 FR 64075 and 88 FR 13322 and make suggestions for how to address chimera concerns within and/or without these regulations. Collectively, we believe that our findings fill important gaps in current federal regulations and provide forward-looking philosophical and practical analysis that can guide future decisionmaking.
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COVID-19 , Toxinas Biológicas , Humanos , SARS-CoV-2 , Salud PúblicaRESUMEN
Mosquito-borne dengue viruses (DENVs) have evolved to four serotypes with 69%-78% amino acid identities, resulting in incomplete immunity, where one serotype's infection does not cross-protect against secondary infections by other serotypes. Despite the amino acid differences, structural and nonstructural (NS) proteins among serotypes play similar functions. NS3 is an enzyme complex: NS3 has N-terminal protease (PRO) and C-terminal helicase (HEL) activities in addition to 5' RNA triphosphatase (5'RTP), which is involved in the RNA capping process. In this study, the effects of NS3 replacements among serotypes were tested. The replacement of NS3 full-length (FULL), PRO or HEL region suppressed viral replication in BHK-21 mammalian cells, while the single compensatory mutation improved the viral replications; P364S mutation in HEL revived PRO (DENV3)-replaced DENV1, while S68T alteration in NS2B recovered HEL (DENV1)-replaced DENV2. The results suggest that the interactions between PRO and HEL as well as HEL and NS2B are required for replication competence. Lower-frequency mutations also appeared at various locations in viral proteins, although after infecting C6/36 mosquito cells, the mutations' frequencies changed, and/or new mutations appeared. In contrast, the inter-domain region (INT, 12 amino acids)-replaced chimera quickly replicated without mutation in BHK-21 cells, although extended cell culture accumulated various mutations. These results suggest that NS3 variously interacts with DENV proteins, in which the chimeric NS3 domain replacements induced amino acid mutations, irrespective of replication efficiency. However, the viral sequences are further adjusted for replication efficiency, to fit in both mammalian cells and mosquito cells. IMPORTANCE Enzyme activities for replicating DENV 5' cap positive (+) sense RNA have been shown to reside in NS3 and NS5. However, it remains unknown how these enzymes coordinately synthesize negative (-) sense RNA, from which abundant 5' cap (+) sense RNA is produced. We previously revealed that NS5 dimerization and NS5 methyltransferase(MT)-NS3HEL interaction are important for DENV replication. Here, we found that replication incompetence due to NS3PRO or HEL replacement was compensated by a mutation at HEL or NS2B, respectively, suggesting that the interactions among NS2B, NS3PRO, and HEL are critical for DENV replication.
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Virus del Dengue , Animales , Aminoácidos/genética , Culicidae/metabolismo , ADN Helicasas/genética , Mamíferos/genética , Mutación , Péptido Hidrolasas/genética , ARN , ARN Helicasas/metabolismo , Serina Endopeptidasas/metabolismo , Serogrupo , Proteínas no Estructurales Virales/metabolismoRESUMEN
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.
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Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Pestivirus , Vacunas Virales , Porcinos , Animales , Vacunas Marcadoras , Anticuerpos Antivirales , Vacunas Atenuadas , Virus de la Fiebre Porcina Clásica/genética , Pestivirus/genéticaRESUMEN
Porcine epidemic diarrhea virus (PEDV) is a re-emerging enteropathogenic coronavirus that causes high mortality in neonatal piglets. The addition of trypsin plays a crucial role in the propagation of PEDV, but also increases the complexity of vaccine production and increases its cost. Previous studies have suggested that the S2' site and Y976/977 of the PEDV spike (S) protein might be the determinants of PEDV trypsin independence. In this study, to achieve a recombinant trypsin-independent PEDV strain, we used trypsin-dependent genotype 2 (G2) PEDV variant AJ1102 to generate three recombinant PEDVs with mutations in S (S2' site R894G and/or Y976H). The three recombinant PEDVs were still trypsin dependent, suggesting that the S2' site R894 and Y976 of AJ1102 S are not key sites for PEDV trypsin dependence. Therefore, we used AJ1102 and the classical trypsin-independent genotype 1 (G1) PEDV strain JS2008 to generate a recombinant PEDV carrying a chimeric S protein, and successfully obtained trypsin-independent PEDV strain rAJ1102-S2'JS2008, in which the S2 (amino acids 894-1386) domain was replaced with the corresponding JS2008 sequence. Importantly, immunization with rAJ1102-S2'JS2008 induced neutralizing antibodies against both AJ1102 and JS2008. Collectively, these results suggest that rAJ1102-S2'JS2008 is a novel vaccine candidate with significant advantages, including no trypsin requirement for viral propagation to high titers and the potential provision of protection for pigs against G1 and G2 PEDV infections.
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Virus de la Diarrea Epidémica Porcina , Enfermedades de los Porcinos , Vacunas Virales , Animales , Porcinos , Virus de la Diarrea Epidémica Porcina/genética , Vacunas Virales/genética , Enfermedades de los Porcinos/prevención & control , Mutación , Anticuerpos Neutralizantes/genéticaRESUMEN
Background: On December 31, 2019, the World Health Organization's China Country Office was alerted to cases of pneumonia of unknown cause detected in Wuhan City, Hubei Province of China. Methods: Due to the fact that to date, the question of the origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has not been resolved yet, the author analyzed the main advances in the development of genetic engineering of viruses that took place before the onset of the COVID-19 pandemic. Results: The first artificial genetically modified viruses could appear in nature in the mid-1950s. The technique of nucleic acid hybridization was developed by the end-1960s. In the late 1970s, a method called the "reverse genetics" emerged to synthesize ribonucleic acid and deoxyribonucleic acid molecules. In the early 1980-s, it became possible to combine the genes of different viruses and insert the genes of one virus into the genome of another virus. Since that time, the production of vector vaccines began. At present, by modern technologies one can assemble any virus based on the nucleotide sequence available in the virus database or designed by a computer as a virtual model. Conclusion: Scientists around the world are invited to answer the call of Neil Harrison and Jeffrey Sachs of Columbia University, for a thorough and independent investigation into the origin of SARS-CoV-2. Only a full understanding of the origin of the new virus can minimize the likelihood of a similar pandemic in the future.
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Chimeric virus-like particles (cVLPs) show great potential in improving public health as they are safe and effective vaccine candidates. The capsid protein of caliciviruses has been described previously as a self-assembling, highly immunogenic delivery platform. The ability to significantly induce cellular and humoral immunity can be used to boost the immune response to low immunogenic foreign antigens displayed on the surface of VLPs. Capsid proteins of caliciviruses despite sequence differences share similar architecture with structural loops that can be genetically modified to present foreign epitopes on the surface of cVLPs. Here, based on the VP1 protein of norovirus (NoV), we investigated the impact of the localization of the epitope in different structural loops of the P domain on the immunogenicity of the presented epitope. In this study, three distinct loops of NoV VP1 protein were genetically modified to present a multivalent influenza virus epitope consisting of a tandem repeat of M2/NP epitopes. cVLPs presenting influenza virus-conserved epitopes in different localizations were produced in the insect cells and used to immunize BALB/c mice. Specific reaction to influenza epitopes was compared in sera from vaccinated mice to determine whether the localization of the foreign epitope has an impact on the immunogenicity.
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The therapeutic potential of Newcastle disease virus (NDV) has been reported as both an oncolytic agent and a vaccine vector against many antigens. However, in the individuals already immunized with NDVs, second and subsequent administration does not provide substantial benefits. In this study, two types of recombinant chimeric NDVs using APMV-2 F and HN genes were generated. In rNDV-2HN, the wild-type NDV HN gene was replaced with the APMV-2 HN gene, and in rNDV-2F/2HN, both wild-type F and HN genes were replaced with APMV-2 F and HN genes, respectively. We enhanced the immune responses of these chimeric viruses by inserting the human IFN-γ gene. To examine the escape from NDV antiserum, each virus was treated with diluted NDV antiserum, and HEp-2 cells were infected with these virus particles. The two constructed chimeric viruses indicated notably lower virus-neutralizing titer compared to wild-type NDV and escaped the action of NDV antiserum. These two chimeric viruses infected both respiratory and colon cancer cell lines, indicating their potential as a cancer treatment tool. Chimeric viruses with enhanced immune responses can be considered a novel therapeutic strategy in cancer treatment that can be administered multiple times and used to enhance immune cells interaction.
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A short mouse-specific peptide from zona pellucida 3 (mZP3, amino acids 328-342) has been shown to be associated with antibody-mediated contraception. In this study, we investigated the production of mZP3 in the plant, as an orally applicable host, and examined the immunogenicity of this small peptide in the BALB/c mouse model. The mZP3 peptide was inserted into the major immunodominant region of the hepatitis B core antigen and was produced in Nicotiana benthamiana plants via Agrobacterium-mediated transient expression. Soluble HBcAg-mZP3 accumulated at levels up to 2.63 mg/g leaf dry weight (LDW) containing ~172 µg/mg LDW mZP3 peptide. Sucrose gradient analysis and electron microscopy indicated the assembly of the HBcAg-mZP3 virus-like particles (VLPs) in the soluble protein fraction. Subcutaneously administered mZP3 peptide displayed on HBcAg VLPs was immunogenic in BALB/c mice at a relatively low dosage (5.5 µg mZP3 per dose) and led to the generation of mZP3-specific antibodies that bound to the native zona pellucida of wild mice. Oral delivery of dried leaves expressing HBcAg-mZP3 also elicited mZP3-specific serum IgG and mucosal IgA that cross-reacted with the zona pellucida of wild mice. According to these results, it is worthwhile to investigate the efficiency of plants producing HBcAg-mZP3 VLPs as immunogenic edible baits in reducing the fertility of wild mice through inducing antibodies that cross-react to the zona pellucida.
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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.
Asunto(s)
Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Virus de la Diarrea Viral Bovina , Vacunas Virales , Animales , Porcinos , Conejos , Peste Porcina Clásica/prevención & control , ADN Complementario , Vacunas Virales/genética , Virus de la Fiebre Porcina Clásica/genética , Vacunación , Anticuerpos Antivirales , Proteínas del Envoltorio Viral/genéticaRESUMEN
@#Objective To construct recombinant chimeric vesicular stomatitis virus(VSV)expressing G protein of rabies virus(RV)using VSV as vector.MethodsTo rescue the recombinant virus,G gene of VSV antigenome was replaced with G gene of RV vaccine CTN-1 strain,and co-transfected into 293T cells with helper plasmids coding T7 RNA polymerase and proteins N,P and L of VSV. The expression of RV G gene and G protein was detected by RT-PCR,immunofluorescence assay and Western blot. The recombinant virus was subcultured in Vero cells,the virus titer of different generations was detected and the virus growth curve was drawn.ResultsThe recombinant virus VSV-RVG was successfully rescued. RTPCR results demonstrated that the RV G gene was successfully inserted into the genome of the recombinant virus,and the expression of RVG protein was detected by immunofluorescence assay and Western blot. The recombinant virus was continuously passaged for 5 generations,and the virus titer was stable within 7. 5 ～ 8. 5 lgTCID50/mL.ConclusionThe recombinant chimeric VSV expressing RV G protein was successfully constructed with good genetic stability,which lays a foundation of the construction of reverse genetics technology platform based on VSV vector.
RESUMEN
Cucumber mosaic virus (CMV), which has great impact on agronomic production worldwide, is both aphid and seed transmitted. Although the mechanisms of aphid transmission have been widely studied, those underlying the ability of CMV to survive and remain infectious during the passage from one generation to the next through the seeds are still to be clarified. Moreover, the viral determinants of seed transmission rate are poorly understood. Three viral genotypes produced from same RNA 1 and 2 components of CMV-Fny but differing in RNA 3 (the wild type CMV-Fny, a pseudorecombinant CMV-Fny/CMV-S and a chimeric CMV previously obtained by our group, named F, FS and CS, respectively) were propagated in Nicotiana tabacum cv Xanthi plants in order to assess differences in tobacco seed transmission rate and persistence through plant generations in the absence of aphid transmission. Seed-growth tests revealed CMV infection in the embryos, but not in the integuments. Seedlings from seed-growth tests showed the presence of all considered viruses but at different rates: from 4% (F, FS) to 16% (CS). Electron microscopy revealed absence (CS) of viral particles or virions without the typical central hole (F and FS). In agreement, structural characteristics of purified CMV particles, assessed by circular dichroism spectroscopy, showed anomalous spectra of nucleic acids rather than the expected nucleoproteins. These alterations resulted in no seed transmission beyond the first plant generation. Altogether, the results show for the first time that correct virion assembly is needed for seed infection from the mother plant but not to seedling invasion from the seed. We propose that incorrect virion formation, self-assembly and architecture stability might be explained if during the first stages of germination and seedling development some tobacco seed factors target viral regions responsible for protein-RNA interactions.