The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome (MERS)-CoV underscores the threat of cross-species transmission events leading to outbreaks in humans. Here we examine the disease potential of a SARS-like virus, SHC014-CoV, which is currently circulating in Chinese horseshoe bat populations. Using the SARS-CoV reverse genetics system, we generated and characterized a chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse-adapted SARS-CoV backbone. The results indicate that group 2b viruses encoding the SHC014 spike in a wild-type backbone can efficiently use multiple orthologs of the SARS receptor human angiotensin converting enzyme II (ACE2), replicate efficiently in primary human airway cells and achieve in vitro titers equivalent to epidemic strains of SARS-CoV. Additionally, in vivo experiments demonstrate replication of the chimeric virus in mouse lung with notable pathogenesis. Evaluation of available SARS-based immune-therapeutic and prophylactic modalities revealed poor efficacy; both monoclonal antibody and vaccine approaches failed to neutralize and protect from infection with CoVs using the novel spike protein. On the basis of these findings, we synthetically re-derived an infectious full-length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo. Our work suggests a potential risk of SARS-CoV re-emergence from viruses currently circulating in bat populations.
Contemporary H3N2 influenza viruses have a glycosylation site that alters binding of antibodies elicited by egg-adapted vaccine strains
- Proceedings of the National Academy of Sciences of the United States of America
- Published over 3 years ago
H3N2 viruses continuously acquire mutations in the hemagglutinin (HA) glycoprotein that abrogate binding of human antibodies. During the 2014-2015 influenza season, clade 3C.2a H3N2 viruses possessing a new predicted glycosylation site in antigenic site B of HA emerged, and these viruses remain prevalent today. The 2016-2017 seasonal influenza vaccine was updated to include a clade 3C.2a H3N2 strain; however, the egg-adapted version of this viral strain lacks the new putative glycosylation site. Here, we biochemically demonstrate that the HA antigenic site B of circulating clade 3C.2a viruses is glycosylated. We show that antibodies elicited in ferrets and humans exposed to the egg-adapted 2016-2017 H3N2 vaccine strain poorly neutralize a glycosylated clade 3C.2a H3N2 virus. Importantly, antibodies elicited in ferrets infected with the current circulating H3N2 viral strain (that possesses the glycosylation site) and humans vaccinated with baculovirus-expressed H3 antigens (that possess the glycosylation site motif) were able to efficiently recognize a glycosylated clade 3C.2a H3N2 virus. We propose that differences in glycosylation between H3N2 egg-adapted vaccines and circulating strains likely contributed to reduced vaccine effectiveness during the 2016-2017 influenza season. Furthermore, our data suggest that influenza virus antigens prepared via systems not reliant on egg adaptations are more likely to elicit protective antibody responses that are not affected by glycosylation of antigenic site B of H3N2 HA.
Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.
Since it emerged in Brazil in May 2015, the mosquito-borne Zika virus (ZIKV) has raised global concern due to its association with a significant rise in the number of infants born with microcephaly and neurological disorders such as Guillain-Barré syndrome. We developed prototype subunit and adenoviral-based Zika vaccines encoding the extracellular portion of the ZIKV envelope gene (E) fused to the T4 fibritin foldon trimerization domain (Efl). The subunit vaccine was delivered intradermally through carboxymethyl cellulose microneedle array (MNA). The immunogenicity of these two vaccines, named Ad5.ZIKV-Efl and ZIKV-rEfl, was tested in C57BL/6 mice. Prime/boost immunization regimen was associated with induction of a ZIKV-specific antibody response, which provided neutralizing immunity. Moreover, protection was evaluated in seven-day-old pups after virulent ZIKV intraperitoneal challenge. Pups born to mice immunized with Ad5.ZIKV-Efl were all protected against lethal challenge infection without weight loss or neurological signs, while pups born to dams immunized with MNA-ZIKV-rEfl were partially protected (50%). No protection was seen in pups born to phosphate buffered saline-immunized mice. This study illustrates the preliminary efficacy of the E ZIKV antigen vaccination in controlling ZIKV infectivity, providing a promising candidate vaccine and antigen format for the prevention of Zika virus disease.
In a previous in vitro study, the SupT1 cell line was explored as a decoy target for HIV-1, proposing SupT1 cell infusion as a possible cell-based therapy for HIV. In the present work, the previous in vitro model was translated into an in vivo setting. Specifically, Hu-PBMC BRGS mice were infected with a high input of HIV-1 LAI (100,000 TCID50), and 40 million 30 Gy-irradiated SupT1 cells were infused weekly for 4 weeks as a therapy. Blood samples were taken to monitor CD4+ T cell count and viral load, and mice were monitored daily for signs of illness. At the earliest time point analyzed (Week 1), there was a significantly lower plasma viral load (~10-fold) in all animals treated with SupT1 cell infusion, associated with a higher CD4+ T cell count. At later time points, infection proceeded with robust viral replication and evident CD4+ T cell depletion, except in one mouse that showed complete suppression of viral replication and preservation of CD4+ T cell count. No morbidity or mortality was associated with SupT1 cell infusion. The interesting tendencies observed in the generated data suggest that this approach should be further investigated as a possible cell-based HIV therapy.
The evolution of new and reemerging historic virulent strains of respiratory viruses from animal reservoirs is a significant threat to human health. Inefficient human-to-human transmission of zoonotic strains may initially limit the spread of transmission, but an infection may be contracted by touching contaminated surfaces. Enveloped viruses are often susceptible to environmental stresses, but the human coronaviruses responsible for severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) have recently caused increasing concern of contact transmission during outbreaks. We report here that pathogenic human coronavirus 229E remained infectious in a human lung cell culture model following at least 5 days of persistence on a range of common nonbiocidal surface materials, including polytetrafluoroethylene (Teflon; PTFE), polyvinyl chloride (PVC), ceramic tiles, glass, silicone rubber, and stainless steel. We have shown previously that noroviruses are destroyed on copper alloy surfaces. In this new study, human coronavirus 229E was rapidly inactivated on a range of copper alloys (within a few minutes for simulated fingertip contamination) and Cu/Zn brasses were very effective at lower copper concentration. Exposure to copper destroyed the viral genomes and irreversibly affected virus morphology, including disintegration of envelope and dispersal of surface spikes. Cu(I) and Cu(II) moieties were responsible for the inactivation, which was enhanced by reactive oxygen species generation on alloy surfaces, resulting in even faster inactivation than was seen with nonenveloped viruses on copper. Consequently, copper alloy surfaces could be employed in communal areas and at any mass gatherings to help reduce transmission of respiratory viruses from contaminated surfaces and protect the public health.
Porcine circovirus type 2 (PCV2) is considered to be the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), which has become a serious economic problem for the swine industry worldwide. The major genotypes, PCV2a and PCV2b, are highly prevalent in the pig population and are present worldwide. However, another newly emerging PCV2b genotype mutant, which has a mutation in its ORF2-encoded capsid protein, has been sporadically present in China, as well as in other countries. It is therefore important to determine the relative virulence of the newly emerging PCV2b genotype mutant, compared with the existing PCV2a and PCV2b genotypes, and to investigate whether the newly emerging mutant virus induces more severe illness.
Understanding the mechanisms of attenuation and virulence of foot-and-mouth disease virus (FMDV) in the natural host species is critical for development of next-generation countermeasures such as live-attenuated vaccines. Functional genomics analyses of FMDV have identified few virulence factors of which the leader proteinase (Lpro) is the most thoroughly investigated. Previous work from our laboratory has characterized host factors in cattle inoculated with virulent FMDV and attenuated mutant strains with transposon insertions within Lpro.
A newly discovered iridescent virus that causes severe disease and high mortality in farmed Litopenaeus vannamei in Zhejiang, China, has been verified and temporarily specified as shrimp hemocyte iridescent virus (SHIV). Histopathological examination revealed basophilic inclusions and pyknosis in hematopoietic tissue and hemocytes in gills, hepatopancreas, periopods and muscle. Using viral metagenomics sequencing, we obtained partial sequences annotated as potential iridoviridae. Phylogenetic analyses using amino acid sequences of major capsid protein (MCP) and ATPase revealed that it is a new iridescent virus but does not belong to the five known genera of Iridoviridae. Transmission electron microscopy showed that the virus exhibited a typical icosahedral structure with a mean diameter of 158.6 ± 12.5 nm (n = 30)(v-v) and 143.6 ± 10.8 nm (n = 30)(f-f), and an 85.8 ± 6.0 nm (n = 30) nucleoid. Challenge tests of L. vannamei via intermuscular injection, per os and reverse gavage all exhibited 100% cumulative mortality rates. The in situ hybridization showed that hemopoietic tissue, gills, and hepatopancreatic sinus were the positively reacting tissues. Additionally, a specific nested PCR assay was developed. PCR results revealed that L. vannamei, Fenneropenaeus chinensis, and Macrobrachium rosenbergii were SHIV-positive, indicating a new threat existing in the shrimp farming industry in China.
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.