The Unique Glycosylation at Position 986 on the E2 Glycoprotein of Classical Swine Fever Virus Is Responsible for Viral Attenuation and Protection against Lethal Challenge

Classical swine fever (CSF) is an economically important disease of pigs caused by the classical swine fever virus (CSFV). The live attenuated vaccine C-strain (also called HCLV strain) against CSF was produced by multiple passages of a highly virulent strain in rabbits. However, the molecular determinants for its attenuation and protection remain unclear. In this study, we identified a unique glycosylation at position 986 ( 986 NYT 988 ) on the E2 glycoprotein Domain IV of C-strain but not ( 986 NYA 988 ) the highly virulent CSFV Shimen strain. We evaluated the infectivity, virulence, and protective efficacy of the C-strain-based mutant rHCLV-T988A lacking the glycosylation and Shimen strain mutant rShimen-A988T acquiring an additional glycosylation at position 986. rShimen-A988T showed a significantly decreased viral replication ability in SK6 cells, while rHCLV-T988A exhibited a growth kinetics indistinguishable from that of C-strain. Removal of the C-strain glycosylation site does not affect viral replication in rabbits and the attenuated phenotype in pigs. However, rShimen-A988T was attenuated and protected the pigs from a lethal challenge at 14 days post-inoculation. In contrast, the rHCLV-T988A-inoculated pigs showed transient fever, a few clinical signs, and pathological changes in the spleens upon challenge with the Shimen strain. Mechanistic investigations revealed that the unique glycosylation at position 986 influences viral spreading, alters the formation of E2 homodimers, and leads to increased production of neutralizing antibodies. Collectively, our data for the first time demonstrate that the unique glycosylation at position 986 on the E2 glycoprotein is responsible for viral attenuation and protection. IMPORTANCE Viral glycoproteins involve in infectivity, virulence, and host immune responses. Deglycosylation on the E rns , E1, or E2 glycoprotein of highly virulent classical swine fever virus (CSFV) attenuated viral virulence in pigs, indicating that the glycosylation contributes to the pathogenicity of highly virulent strain. However, the effects of the glycosylation on the C-strain E2 glycoprotein on viral infectivity in cells, viral attenuation, and protection in pigs have not been elucidated. This study demonstrates the unique glycosylation at position 986 on the C-strain E2 glycoprotein. C-strain mutant removing the glycosylation at the site provides only partial protection against CSFV challenge. Remarkably, the addition of the glycan to E2 of the highly virulent Shimen strain attenuates the viral virulence and confers complete protection against the lethal challenge in pigs. Our findings provide a new insight into the contribution of the glycosylation to the virus attenuation and protection.

A Clinical Comparison of Imported or Locally Acquired COVID-19 Suggests Declining Severity after Viral Passages at the Early Stage of the Pandemic in China

Objective: This retrospective, two-center study aimed to provide solid clinical evidence to support the viral attenuation theory after passages of SARS-CoV-2 during the early stages of the pandemic. Methods: Clinical characteristics and outcomes of 150 COVID-19 patients hospitalized in Wenzhou, China between January and April 2020 were compared. These patients had similar demographic characteristics. Infections of 77 patients who visited Wuhan within 14 days before symptom onset were categorized as imported cases and that of the remaining 73 patients who had no recent travel history were categorized as locally transmitted cases. Results: COVID-19 cases in Wenzhou appeared to be mostly mild at the early stages of the pandemic. There were no differences in clinical manifestations, laboratory testing results, and radiographic presentation between imported and locally transmitted cases in Wenzhou, except that a higher proportion of lymphopenia was found in the imported case group. Assessment of infection severity showed that severe conditions were observed in 10.7% of the patients, with the imported case group having a significantly higher rate (15.6%) than the locally transmitted case group (5.5%, x2 = 4.016, p = 0.045). Conclusion: Although, the clinical manifestations of locally acquired infections were indistinguishable from those imported from Wuhan, they were less likely to develop into severe medical conditions, suggesting the possibility of virulence attenuation after viral passages during the early stages of the pandemic.

A QP509L/QP383R-deleted African swine fever virus is highly attenuated in swine but does not confer protection against parental virus challenge

African swine fever (ASF), a devastating infectious disease in swine, severely threatens the global pig farming industry. Disease control has been hampered by the unavailability of vaccines. Here, we report that deletion of the QP509L and QP383R genes (ASFV-ΔQP509L/QP383R) from the highly virulent ASFV CN/GS/2018 strain results in complete viral attenuation in swine. Animals inoculated with ASFV-ΔQP509L/QP383R at a 10 4 50% hemadsorbing dose (HAD 50 ) remained clinically normal during the 17-day observational period. All ASFV-ΔQP509L/QP383R-infected animals had low viremia titers and developed a low-level p30-specific antibody response. However, ASFV-ΔQP509L/QP383R did not induce protection against challenge with the virulent parental ASFV CN/GS/2018 isolate. RNA-sequencing analysis revealed that innate immune-related genes ( Ifnb , Traf2 , Cxcl10 , Isg15 , Rantes , and Mx1 ) were significantly lower in ASFV-ΔQP509L/QP383R-infected than in ASFV-infected porcine alveolar macrophages. In addition, ASFV-ΔQP509L/QP383R-infected pigs had low levels of IFN-β based on ELISA. These data suggest that deletion of ASFV QP509L/383R reduces virulence but does not induce protection against lethal ASFV challenge. Importance African swine fever (ASF) is endemic to several parts of the word, with outbreaks of the disease devastating the swine farming industry; currently, no commercially available vaccine exists. Here, we report that deletion of the previously uncharacterized QP509L and QP383R viral genes completely attenuates virulence in the ASFV CN/GS/2018 isolate. However, ASFV-ΔQP509L/QP383R-infected animals were not protected from developing an ASF infection after challenge with the virulent parental virus. ASFV-ΔQP509L/QP383R induced lower levels of innate immune-related genes and IFN-β than the parental virus. Our results increase our knowledge on developing an effective and live ASF attenuated vaccine.

The long-lasting enigma of polycytidine (polyC) tract

Long polycytidine (polyC) tracts varying in length from 50 to 400 nucleotides were first described in the 5′-noncoding region (NCR) of genomes of picornaviruses belonging to the Cardio- and Aphthovirus genera over 50 years ago, but the molecular basis of their function is still unknown. Truncation or complete deletion of the polyC tracts in picornaviruses compromises virulence and pathogenicity but do not affect replicative fitness in vitro, suggesting a role as “viral security” RNA element. The evidence available suggests that the presence of a long polyC tract is required for replication in immune cells, which impacts viral distribution and targeting, and, consequently, pathogenic progression. Viral attenuation achieved by reduction of the polyC tract length has been successfully used for vaccine strategies. Further elucidation of the role of the polyC tract in viral replication cycle and its connection with replication in immune cells has the potential to expand the arsenal of tools in the fight against cancer in oncolytic virotherapy (OV). Here, we review the published data on the biological significance and mechanisms of action of the polyC tract in viral pathogenesis in Cardio- and Aphthoviruses.

In vitro adaptation and characterization of attenuated hypervariable region 1 swap chimeras of hepatitis C virus

Hepatitis C virus (HCV) chronically infects 70 million people worldwide with an estimated annual disease-related mortality of 400,000. A vaccine could prevent spread of this pervasive human pathogen, but has proven difficult to develop, partly due to neutralizing antibody evasion mechanisms that are inherent features of the virus envelope glycoproteins, E1 and E2. A central actor is the E2 motif, hypervariable region 1 (HVR1), which protects several non-overlapping neutralization epitopes through an incompletely understood mechanism. Here, we show that introducing different HVR1-isolate sequences into cell-culture infectious JFH1-based H77 (genotype 1a) and J4 (genotype 1b) Core-NS2 recombinants can lead to severe viral attenuation. Culture adaptation of attenuated HVR1-swapped recombinants permitted us to identify E1/E2 substitutions at conserved positions both within and outside HVR1 that increased the infectivity of attenuated HVR1-swapped recombinants but were not adaptive for original recombinants. H77 recombinants with HVR1 from multiple other isolates consistently acquired substitutions at position 348 in E1 and position 385 in HVR1 of E2. Interestingly, HVR1-swapped J4 recombinants primarily acquired other substitutions: F291I (E1), F438V (E2), F447L/V/I (E2) and V710L (E2), indicating a different adaptation pathway. For H77 recombinants, the adaptive E1/E2 substitutions increased sensitivity to the neutralizing monoclonal antibodies AR3A and AR4A, whereas for J4 recombinants, they increased sensitivity to AR3A, while having no effect on sensitivity to AR4A. To evaluate effects of the substitutions on AR3A and AR4A binding, we performed ELISAs on extracted E1/E2 protein and performed immunoprecipitation of relevant viruses. However, extracted E1/E2 protein and immunoprecipitation of HCV particles only reproduced the neutralization phenotypes of the J4 recombinants. Finally, we found that the HVR1-swap E1/E2 substitutions decrease virus entry dependency on co-receptor SR-BI. Our study identifies E1/E2 positions that could be critical for intra-complex HVR1 interactions while emphasizing the need for developing novel tools for molecular studies of E1/E2 interactions.

Altering Compositional Properties of Viral Genomes to Design Live-Attenuated Vaccines

Live-attenuated vaccines have been historically used to successfully prevent numerous diseases caused by a broad variety of RNA viruses due to their ability to elicit strong and perdurable immune-protective responses. In recent years, various strategies have been explored to achieve viral attenuation by rational genetic design rather than using classic and empirical approaches, based on successive passages in cell culture. A deeper understanding of evolutionary implications of distinct viral genomic compositional aspects, as well as substantial advances in synthetic biology technologies, have provided a framework to achieve new viral attenuation strategies. Herein, we will discuss different approaches that are currently applied to modify compositional features of viruses in order to develop novel live-attenuated vaccines.

Codon Usage and Adenovirus Fitness: Implications for Vaccine Development

Vaccination is the most effective method to date to prevent viral diseases. It intends to mimic a naturally occurring infection while avoiding the disease, exposing our bodies to viral antigens to trigger an immune response that will protect us from future infections. Among different strategies for vaccine development, recombinant vaccines are one of the most efficient ones. Recombinant vaccines use safe viral vectors as vehicles and incorporate a transgenic antigen of the pathogen against which we intend to generate an immune response. These vaccines can be based on replication-deficient viruses or replication-competent viruses. While the most effective strategy involves replication-competent viruses, they must be attenuated to prevent any health hazard while guaranteeing a strong humoral and cellular immune response. Several attenuation strategies for adenoviral-based vaccine development have been contemplated over time. In this paper, we will review them and discuss novel approaches based on the principle that protein synthesis from individual genes can be modulated by codon usage bias manipulation. We will summarize vaccine approaches that consider recoding of viral proteins to produce adenoviral attenuation and recoding of the transgene antigens for both viral attenuation and efficient viral epitope expression.

Implications of SARS-CoV-2 mutations for genomic RNA structure and host microRNA targeting

The SARS-CoV-2 virus is a recently-emerged zoonotic pathogen already well adapted to transmission and replication in humans. Although the mutation rate is limited, recently introduced mutations in SARS-CoV-2 have the potential to alter viral fitness. In addition to amino acid changes, mutations could affect RNA secondary structure critical to viral life cycle, or interfere with sequences targeted by host miRNAs. We have analysed subsets of genomes from SARS-CoV-2 isolates from around the globe and show that several mutations introduce changes in Watson-Crick pairing, with resultant changes in predicted secondary structure. Filtering to targets matching miRNAs expressed in SARS-CoV-2 permissive host cells, we identified twelve separate target sequences in the SARS-CoV-2 genome; eight of these targets have been lost through conserved mutations. A genomic site targeted by the highly abundant miR-197-5p, overexpressed in patients with cardiovascular disease, is lost by a conserved mutation. Our results are compatible with a model that SARS-CoV-2 replication within the human host could be constrained by host miRNA defence. The impact of these and further mutations on secondary structures, miRNA targets or potential splice sites offers a new context in which to view future SARS-CoV-2 evolution, and a potential platform for engineered viral attenuation and antigen presentation.

Impairment of the DeISGylation Activity of Foot-and-Mouth Disease Virus Lpro Causes Attenuation In Vitro and In Vivo

ABSTRACT Foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) affects several pathways of the host innate immune response. Previous studies in bovine cells demonstrated that deletions (leaderless [LLV]) or point mutations in Lpro result in increased expression of interferon (IFN) and IFN-stimulated genes (ISGs), including, among others, the ubiquitin-like protein modifier ISG15 and the ubiquitin specific peptidase USP18. In addition to its conventional papain-like protease activity, Lpro acts as a deubiquitinase (DUB) and deISGylase. In this study, we identified a conserved residue in Lpro that is involved in its interaction with ISG15. Mutation W105A rendered Escherichia coli-expressed Lpro unable to cleave the synthetic substrate pro-ISG15 while preserving cellular eIF4G cleavage. Interestingly, mutant FMDV W105A was viable. Overexpression of ISG15 and the ISGylation machinery in porcine cells resulted in moderate inhibition of FMDV replication, along with a decrease of the overall state of ISGylation in wild-type (WT)-infected cells. In contrast, reduced deISGylation was observed upon infection with W105A and leaderless virus. Reduction in the levels of deubiquitination was also observed in cells infected with the FMDV LproW105A mutant. Surprisingly, similarly to WT, infection with W105A inhibited IFN/ISG expression despite displaying an attenuated phenotype in vivo in mice. Altogether, our studies indicate that abolishing/reducing the deISGylase/DUB activity of Lpro causes viral attenuation independently of its ability to block the expression of IFN and ISG mRNA. Furthermore, our studies highlight the potential of ISG15 to be developed as a novel biotherapeutic molecule against FMD. IMPORTANCE In this study, we identified an aromatic hydrophobic residue in foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) (W105) that is involved in the interaction with ISG15. Mutation in Lpro W105 (A12-LproW105A) resulted in reduced deISGylation in vitro and in porcine-infected cells. Impaired deISGylase activity correlated with viral attenuation in vitro and in vivo and did not affect the ability of Lpro to block expression of type I interferon (IFN) and other IFN-stimulated genes. Moreover, overexpression of ISG15 resulted in the reduction of FMDV viral titers. Thus, our study highlights the potential use of Lpro mutants with modified deISGylase activity for development of live attenuated vaccine candidates, and ISG15 as a novel biotherapeutic against FMD.

A Molecular Determinant of West Nile Virus Secretion and Morphology as a Target for Viral Attenuation

ABSTRACT West Nile virus (WNV), a member of the Flavivirus genus and currently one of the most common arboviruses worldwide, is associated with severe neurological disease in humans. Its high potential to reemerge and rapidly disseminate makes it a bona fide global public health problem. The surface membrane glycoprotein (M) has been associated with Flavivirus-induced pathogenesis. Here, we identified a key amino acid residue at position 36 of the M protein whose mutation impacts WNV secretion and promotes viral attenuation. We also identified a compensatory site at position M-43 whose mutation stabilizes M-36 substitution both in vitro and in vivo. Moreover, we found that introduction of the two mutations together confers a full attenuation phenotype and protection against wild-type WNV lethal challenge, eliciting potent neutralizing-antibody production in mice. Our study thus establishes the M protein as a new viral target for rational design of attenuated WNV strains. IMPORTANCE West Nile virus (WNV) is a worldwide (re)emerging mosquito-transmitted Flavivirus causing fatal neurological diseases in humans. However, no human vaccine has been yet approved. One of the most effective live-attenuated vaccines was empirically obtained by serial passaging of wild-type yellow fever Flavivirus. However, such an approach is not acceptable nowadays, and the development of a rationally designed vaccine is necessary. Generating molecular infectious clones and mutating specific residues known to be involved in Flavivirus virulence constitute a powerful tool to promote viral attenuation. WNV membrane glycoprotein is thought to carry such essential determinants. Here, we identified two residues of this protein whose substitutions are key to the full and stable attenuation of WNV in vivo, most likely through inhibition of secretion and possible alteration of morphology. Applied to other flaviviruses, this approach should help in designing new vaccines against these viruses, which are an increasing threat to global human health.