Journal: Journal of virology
There are seven antigenically distinct serotypes of foot-and-mouth disease virus (FMDV), each of which has intra-typic variants. In the present study, we have developed methods to efficiently generate promising vaccines against seven serotypes or subtypes. The capsid-coding gene (P1) of the vaccine strain O1/Manisa/Turkey/69 was replaced with the amplified or synthetic genes from the O, A, Asia1, C, SAT 1, SAT 2, and SAT 3 serotypes. The seven serotype viruses were rescued successfully. Each chimeric FMDV with replacing P1 showed its serotype-specific antigenicity and varied in terms of pathogenesis in pigs and mice. Pigs vaccinated with an experimental trivalent vaccine containing the inactivated recombinants based on the main serotypes O, A, and Asia1 effectively protected them from virus challenge. This technology could be a potential strategy for customized vaccine with challenge tool to protect against epizootic disease from specific serotypes or subtypes of FMDV.IMPORTANCE Foot-and-mouth disease virus (FMDV) causes significant economic losses. For the vaccine preparation, the selection of vaccine strains was complicated by its high antigenic variation. In the present study, we suggested that an effective strategy can be rapidly prepare and evaluate a mass-producing customized vaccines against epidemic strains. The P1 gene encoding the structural proteins of the well-known vaccine virus was replaced by the synthetic or amplified genes of seven representative serotype viruses. These chimeric viruses generally replicated readily in cell culture and had the similar particle size as the original vaccine strain. Their antigenicity mirrored that of the original serotype from which their P1 gene was derived. Animal infection experiments revealed that the recombinants varied in terms of pathogenicity. This strategy will be a useful tool for rapidly generating customized FMD vaccines or challenge viruses against all serotypes, especially for FMD-free countries which have prohibited import of FMDVs.
The foot-and-mouth disease virus (FMDV) afflicts livestock in more than 80 countries limiting food production and global trade. Production of foot-and-mouth disease (FMD) vaccines requires cytosolic expression of the FMDV 3C protease to cleave the P1 polyprotein into mature capsid proteins, but the FMDV 3C protease is toxic to host cells. To identify less toxic isoforms of the FMDV 3C protease, we screened 3C mutants for increased transgene output over wild-type 3C using a Gaussia luciferase reporter system. The novel point-mutation 3C(L127P) increased yields of recombinant FMDV subunit proteins in mammalian and bacterial cells expressing P1-3C transgenes and retained the ability to process P1 polyproteins from multiple FMDV serotypes. The 3C(L127P) mutant produced crystalline-arrays of FMDV virus-like particles in mammalian and bacterial cells, potentially providing a practical method of rapid, inexpensive FMD vaccine production in bacteria.Importance: The mutant FMDV 3C protease L127P significantly increased yields of recombinant FMDV subunit antigens and produced virus-like particles in mammalian and bacterial cells. The L127P mutation provides a novel advancement for economical FMD vaccine production.
Chikungunya virus (CHIKV), a mosquito-borne human pathogen, causes a disabling disease characterized by severe joint pain that can persist for weeks, months or even years in patients. The non-structural protein 3 (nsP3) plays essential roles during acute infection, but little is known about the function of nsP3 during chronic disease. Here, we used sub-diffraction multi-color microscopy for spatial and temporal analysis of CHIKV nsP3 within human cells that persistently replicate replicon RNA. Round cytoplasmic granules of various sizes (i) contained nsP3 and stress granule assembly factors 1 and 2 (G3BP1/2); (ii) were next to double-stranded RNA foci and nsP1-positive structures; and (iii) were close to the nuclear membrane and the nuclear pore complex protein Nup98. Analysis of protein turnover and mobility by live-cell microscopy revealed that granules could persist for hours to days, accumulated newly synthesized protein, and moved through the cytoplasm at varying speeds. Granules also had a static internal architecture and were stable in cell lysates. Refractory cells that had cleared the non-cytotoxic replicon regained the ability to respond to arsenite-induced stress. In summary, nsP3 can form uniquely stable granular structures that persist long-term within the host cell. This continued presence of viral and cellular protein-complexes has implications for the study of the pathogenic consequences of lingering CHIKV infection and the development of strategies to mitigate the burden of chronic musculoskeletal disease brought about by a medically important arthropod-borne virus (arbovirus).ImportanceChikungunya virus (CHIKV) is a re-emerging alphavirus transmitted by mosquitos and causes transient sickness but also chronic disease affecting muscles and joints. No approved vaccines or antivirals are available. Thus, a better understanding of the viral life cycle and the role of viral proteins can aid in identifying new therapeutic targets. Advances in microscopy and development of non-cytotoxic replicons (Utt, Das, Varjak, Lulla, Lulla, Merits, J Virol 89:3145-62, 2015, doi:10.1128/JVI.03213-14) have allowed researchers to study viral proteins within controlled laboratory environments over extended durations. Here we established human cells that stably replicate replicon RNA and express tagged non-structural protein 3. The ability to track nsP3 within the host cell and during persistent replication can benefit fundamental research efforts to better understand long-term consequences of the persistence of viral protein complexes and thereby provide the foundation for new therapeutic targets to control CHIKV infection and treat chronic disease symptoms.
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) has a narrow host cell tropism, limited to cells of the monocyte/macrophage lineage. CD163 protein is expressed at high levels on the surface of specific macrophage types and a soluble form is circulating in blood. CD163 has been described as a fusion receptor for PRRSV, with the scavenger receptor cysteine-rich domain 5 (SRCR5) region having been shown to be the interaction site for the virus.As reported earlier, we have generated pigs in which Exon 7 of the CD163 gene has been deleted using CRISPR/Cas9 editing in pig zygotes. These pigs express CD163 protein lacking SRCR5 (ΔSRCR5 CD163) and show no adverse effects when maintained under standard husbandry conditions. The ΔSRCR5 CD163 was not only detected on the surface of macrophage subsets, but the secreted, soluble protein can also detected in the serum of the edited pigs, as shown here by porcine soluble CD163-specific ELISA. Previous results showed that primary macrophage cells from ΔSRCR5 CD163 animals are resistant to PRRSV-1, subtypes 1, 2, and 3, as well as PRRSV-2 infection in vitro Here, ΔSRCR5 pigs were challenged with a highly virulent PRRSV-1, subtype 2 strain. In contrast to the wildtype control group, ΔSRCR5 pigs showed no signs of infection and no viremia or antibody response indicative of a productive infection. Histopathological analysis of lung and lymph node tissue showed no presence of virus replicating cells in either tissue. This shows that ΔSRCR5 pigs are fully resistant to infection by the virus.Importance Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is the etiological agent of PRRS, causing late-term abortions, stillbirths, and respiratory disease in pigs, incurring major economic losses to the world-wide pig industry. The virus is highly mutagenic and can be divided into two species, PRRSV-1 and PRRSV-2, each containing several subtypes. Current control strategies mainly involve biosecurity measures, depopulation, and vaccination. Vaccines are at best only partially protective against infection with heterologous subtypes and sublineages and modified-live vaccines have frequently been reported to revert to virulence. Here we demonstrate that a genetic control approach results in complete resistance to PRRSV infection in vivo CD163 is edited such as to remove the viral interaction domain while maintaining protein expression and biological function, averting any potential adverse effect associated with protein knock-out. This research demonstrates a genetic control approach with potential benefits in animal welfare as well as to the pork industry.
Poliomyelitis is a highly infectious disease caused by poliovirus (PV). It can result in paralysis and may be fatal. Integrated global immunisation programmes using live-attenuated oral (OPV) and/or inactivated PV vaccines (IPV) have systematically reduced its spread and paved the way for eradication. Immunisation will continue post-eradication to ensure against reintroduction of the disease, but there are biosafety concerns for both OPV and IPV. These could be addressed by the production and use of virus-free virus-like particle (VLP) vaccines which mimic the ‘empty’ capsids (ECs) normally produced in viral infection. Although ECs are antigenically indistinguishable from mature virus particles, they are less stable and readily convert to an alternative conformation unsuitable for vaccine purposes. Stabilised ECs, expressed recombinantly as VLPs, could be ideal candidate vaccines for a polio-free world. However, although genome-free PV ECs have been expressed as VLPs in a variety of systems, their inherent antigenic instability has proved a barrier to further development. In this study, we have selected thermally-stable ECs of type-1 PV (PV-1). The ECs are antigenically stable at temperatures above the conversion temperature of wild type (wt) virion. We have identified mutations on the capsid surface and internal networks that are responsible for the EC stability. With reference to the capsid structure, we speculate on the roles of these residues in capsid stability and postulate that such stabilised VLPs could be used as novel vaccines.
Strategies are needed to improve the immunogenicity of HIV-1 envelope (Env) antigens for more long lived, efficacious HIV-1 vaccine induced B-cell responses. HIV-1 Env gp140 (native or un-cleaved molecules) or gp120 monomeric proteins elicit relatively poor B-cell responses which are short-lived. We hypothesized that Env engagement of the CD4 receptor on T-helper cells may result in anergic effects on T-cell recruitment and consequently a lack of strong robust and durable B-memory responses. To test this hypothesis we occluded the CD4 binding site (CD4bs) of gp140 by stable cross-linking with a 3kD CD4 miniprotein mimetic serving to block ligation of gp140 on CD4+T-cells while preserving CD4 inducible (CDi) neutralizing and epitopes targeted by antibody dependent cellular cytotoxic (ADCC) effector responses. Importantly immunization of rhesus macaques consistently gave superior B-cell (p<0.001) response kinetics and superior ADCC (p<0.014) in a group receiving the CD4bs-occluded vaccine compared to those animals immunized with gp140. Of the cytokines examined, Ag-specific IL-4 T-helper ELISpots in the CD4bs-occluded group increased earlier (p=0.025) during the inductive phase. Importantly CD4bs-occluded gp140 antigen not only induced superior B-cell and ADCC responses, the elevated B-cell responses proved to be remarkably durable lasting more than 60 weeks post-immunization.IMPORTANCE Attempts to develop HIV vaccines capable of inducing potent and durable B-cell responses have until now been unsuccessful. Antigen specific B-cell development and affinity maturation occurs in germinal centers in lymphoid follicles through a critical interaction between B-cells and T follicular helper cells. The HIV envelope binds the CD4 receptor on T-cells as soluble shed antigen or as antigen antibody complexes causing impairment in the activation of these specialized CD4 positive T-cells. We proposed that CD4-binding impairment may in part be responsible for the relatively poor B-cell responses to HIV envelope based vaccines. To test this hypothesis we blocked the CD4 binding site of the envelope antigen and compared it to currently used unblocked envelope protein. We found superior and durable B-cell responses in macaques vaccinated with an occluded CD4 binding site on the HIV envelope antigen, demonstrating a potentially important new direction in future design of new HIV vaccines.
Evaluation of the epitope specificities, location (systemic, mucosal) and effector function of antibodies elicited by novel HIV-1 immunogens engineered to improve exposure of specific epitopes is critical for HIV-1 vaccine development. Utilizing an array of humoral assays, we evaluated the magnitude, epitope specificity, avidity and function of systemic and mucosal immune responses elicited by a vaccine regimen containing Env cross-linked to a CD4 mimetic miniprotein (gp140-M64U1) in rhesus macaques. Crosslinking of gp140 Env with M64U1 resulted in an earlier increase in both the magnitude and avidity of the IgG binding response compared to Env protein alone. Notably, binding IgG responses at an early time point correlated with Antibody Dependent Cellular Cytotoxicity (ADCC) function at the peak immunity time point, which was higher for the crosslinked Env group compared to the Env group alone. In addition, the crosslinked Env group developed higher IgG responses against a linear epitope in the C1 gp120 region of the HIV-1 envelope glycoprotein. These data demonstrate that structural modification of the HIV-1 envelope immunogen by crosslinking gp140 with the CD4 mimetic M64U1 elicited an earlier increase of binding antibody responses and altered the specificity of the IgG responses that correlated with the rise of subsequent antibody-mediated antiviral functions.IMPORTANCE The development of an efficacious HIV-1 vaccine remains a global priority to prevent new cases of HIV-1 infection. Of the six HIV-1 efficacy trials to date, only one has demonstrated partial efficacy, and the immune correlates analysis of this trial revealed a role for binding antibodies and antibody Fc mediated effector functions. New HIV-1 envelope immunogens are being engineered to selectively expose the most vulnerable and conserved sites on the HIV-1 envelope with the goal of eliciting antiviral antibodies. Evaluation of the humoral responses elicited by these novel immunogen designs in nonhuman primates is critical for understanding how to improve upon immunogen design to inform further testing in human clinical trials. Our results demonstrate that Env structural modifications that aim to mimic the CD4 bound conformation can result in earlier antibody elicitation, altered epitope specificity and increased antiviral function post immunization.
Next-generation sequencing was used for discovery and de novo assembly of a novel, highly divergent DNA virus at the interface between the Parvoviridae and Circoviridae. The virus, provisionally named “parvovirus-like” hybrid virus (PHV), is nearly identical by sequence to another DNA virus, NIH-CQV, previously reported in Chinese patients with seronegative (non-A-E) hepatitis. Although we initially detected PHV in a wide range of clinical samples, with all strains sharing ∼99% nucleotide and amino acid identity with each other and with NIH-CQV, the exact origin of the virus was eventually traced to contaminated silica-binding spin columns used for nucleic acid extraction. Definitive confirmation of the origin of PHV, and presumably NIH-CQV, was obtained by in-depth analyses of water eluted through contaminated spin columns. Analysis of environmental metagenome libraries detected PHV sequences in coastal marine waters of North America, suggesting that a potential association between PHV and diatoms (algae) that generate the silica matrix used in the spin columns may have resulted in inadvertent viral contamination during manufacture. The confirmation of PHV/NIH-CQV as laboratory reagent contaminants and not bona fide infectious agents of humans underscores the rigorous approach needed to establish the validity of new viral genomes discovered by next-generation sequencing.
Avian influenza virus (AIV) surveillance in Antarctica during 2013 revealed the prevalence of evolutionarily distinct influenza viruses of H11N2 subtype in Adélie penguins. Here we present results from the continued surveillance of AIV on the Antarctic Peninsula during 2014 and 2015. In addition to the continued detection of H11 subtype viruses during 2014 in a snowy sheathbill, we isolated a novel H5N5 subtype virus during 2015 in a chinstrap penguin. Gene sequencing and phylogenetic analysis revealed that the H11 virus detected in 2014 had a >99.1% nucleotide similarity to the H11N2 viruses isolated in 2013, suggesting continued prevalence of this virus over multiple years in Antarctica. However, phylogenetic analysis of the H5N5 virus showed that their genome segments were recently introduced into the continent, except for the NP gene that was similar to that in the endemic H11N2 viruses. Our analysis indicates geographically diverse origins for the H5N5 virus genes; with the majority of its genome segments derived from North American lineage viruses, but the neuraminidase gene derived from a Eurasian lineage virus. In summary, we show the persistence of AIV lineages over multiple years in Antarctica; recent introduction of gene segments from diverse regions; and reassortment between different AIV lineages in Antarctica, which together, significantly increases our understanding of AIV ecology in this fragile and pristine environment.
Budding of filoviruses, arenaviruses, and rhabdoviruses is facilitated by subversion of host proteins, such as Nedd4 E3 ubiquitin ligase, by viral PPxY late (L) budding domains expressed within the matrix proteins of these RNA viruses. As L domains are important for budding and are highly conserved in a wide array of RNA viruses, they represent potential broad-spectrum targets for the development of antiviral drugs. To identify potential competitive blockers, we used the known Nedd4 WW-domain/PPxY interaction interface as the basis of an in silico screen. Using PPxY-dependent budding of Marburg (MARV) VP40 virus-like particles (VLPs) as our model system, we identified small molecule hit 1: that inhibited Nedd4-PPxY interaction and PPxY-dependent budding. This lead candidate was subsequently improved with additional structure-activity relationship (SAR) analog testing which enhanced anti-budding activity into the nanomolar range. Current leads 4: and 5: exhibit on-target effects by specifically blocking the MARV VP40 PPxY-host Nedd4 interaction and subsequent PPxY-dependent egress of MARV VP40 VLPs. In addition, leads 4: and 5: exhibited anti-budding activity against Ebola and Lassa fever VLPs, as well as vesicular stomatitis (VSV) and rabies (RABV) viruses. These data provide target validation and suggest that inhibition of the PPxY-Nedd4 interaction can serve as the basis for the development of a novel class of broad-spectrum, host-oriented antivirals targeting viruses that depend on a functional PPxY L domain for efficient egress.