A New Bat-HKU2-like Coronavirus in Swine, China, 2017.

We identified from suckling piglets with diarrhea in China a new bat-HKU2-like porcine coronavirus (porcine enteric alphacoronavirus). The GDS04 strain of this coronavirus shares high aa identities (>90%) with the reported bat-HKU2 strains in Coronaviridae-wide conserved domains, suggesting that the GDS04 strain belongs to the same species as HKU2.

A flagellin-adjuvanted PED subunit vaccine improved protective efficiency against PEDV variant challenge in pigs.

PEDV variants, causing severe diarrhea in neonatal suckling piglets with a mortality rate up to 100%, have being epidemic since late 2010 in china. To meet the pressing need of safe and cost-efficient PED maternal vaccines against PEDV variant, we vaccinated growing piglets with a flagellin-adjuvanted PED vaccine rSF-COE-3D by injection at Houhai acupoint. The vaccination not only enhanced the antibody responses of serum IgG/IgA, mucosal IgA and serum neutralizing antibody, but also improved the production of IFN-γ and IL-4. Moreover, rSF-COE-3D could provide a better protection against the challenge of a high pathogenic PEDV variant, with less diarrhea pigs, less pigs with detectable PEDV shed, lower rank values of feces and less apparent lesions and inflammation in duodenum of the PEDV infected pigs. The improved protective efficiency of rSF-COE-3D compared with COE was mostly benefited from the enhanced production of serum IgA, mucosal IgA in feces and serum neutralizing antibody titers. Taken together, our data suggest that rSF-COE-3D would be a novel efficient PED vaccine.

PED subunit vaccine based on COE domain replacement of flagellin domain D3 improved specific humoral and mucosal immunity in mice.

Porcine epidemic diarrhea (PED) is an important re-emergent infectious disease and inflicts huge economic losses to the swine industry worldwide. To meet the pressing need of developing a safe and cost-efficient PED maternal vaccine, we generated three PED subunit vaccine candidates, using recombined Salmonella flagellin (rSF) as a mucosal molecular adjuvant. Domain D3 in rSF was replaced with COE domain of PEDV to generate rSF-COE-3D. COE fused to the flanking C'/N' terminal of rSF yielded rSF-COE-C and rSF-COE-N. As a result, rSF-COE-3D could significantly improve COE specific antibody production including serum IgG, serum IgA, mucosal IgA and PEDV neutralizing antibody. Furthermore, rSF-COE-3D elicited more CD3+CD8+ T cell and cytokine production of IFN-γ and IL-4 in mouse splenocytes. In summary, our data showed that rSF-COE-3D could improve specific humoral and mucosal immunity in mice, thus suggesting that rSF-COE-3D could be applied as a novel efficient maternal PED vaccine.

Oral administration of coated PEDV-loaded microspheres elicited PEDV-specific immunity in weaned piglets.

Porcine epidemic diarrhea virus (PEDV) infects pigs of all ages by invading villous epithelial cells of the small intestine causing severe diarrhea with high mortality rate in suckling piglets. Mucosal immunity is believed to play an important role in PEDV control and mucosal delivery of vaccines induces mucosal immunity more efficiently than parenteral vaccination. In this study, coated PEDV-loaded microspheres with the size range of 700-900 µm in diameter were developed by centrifugal granulation-fluidized bed coating and demonstrated as an effective oral delivery system to protect PEDV antigens against the complex gastrointestinal environment by detecting the live virus particles in microspheres after the simulated gastric fluid treatment and the PEDV RNA in fecal swabs collected from all weaned piglets (100%) orally inoculated with coated PEDV-loaded microspheres. Weaned piglets orally immunized with coated PEDV-loaded microspheres developed higher levels of PEDV-specific antibodies (IgG and IgA) in their sera and saliva than those negative control groups (p < 0.001 or p < 0.01). Furthermore, neutralization assays demonstrated that serum antibodies in coated PEDV-loaded microspheres groups could significantly inhibit virus infection in Vero cells, compared to PEDV only group (p < 0.05). Overall, our results indicate that the coated PEDV-loaded microspheres might serve as an effective way to induce PEDV-specific mucosal immunity in pigs against PEDV.

Evaluation of purified recombinant spike fragments for assessment of the presence of serum neutralizing antibodies against a variant strain of porcine epidemic diarrhea virus.

Since 2010, variant strains of porcine epidemic diarrhea virus (PEDV) have caused disasters in the pork industry. The spike (S) protein, as the major immunity-eliciting antigen, has previously been used for serological testing and has been found to correlate significantly with the results of the serum neutralization (SN) test. However, further evaluation of this method is needed as new epidemic strains of PEDV emerge. Hence, the main objective of this study was to assess sow sera and determine the correlation between enzyme-linked immunosorbent assay (ELISA) results (involving a newly isolated GDS01 virus-based ELISA and ELISAs based on seven recombinant fragments comprising overlapping S1 and partial S2 sequences) and SN titers. Furthermore, we determined the reliability of the ELISAs based on receiver operating characteristics (ROC) curve analyses. For the most promising ELISA, i.e., the SP4 ELISA, the correlation coefficient (r) and the area under curve (AUC) were determined to be 0.6113 and 0.8538, respectively. In addition, we analyzed the homology of the SP4 sequences obtained from different strains (including vaccine strains) and found that various strains showed a high degree of homology in this region. Thus, we conclude that SP4 is a promising serological testing protein for use in the field.

A recombinant H7N9 influenza vaccine with the H7 hemagglutinin transmembrane domain replaced by the H3 domain induces increased cross-reactive antibodies and improved interclade protection in mice.

Influenza A H7N9 virus is the latest emerging pandemic threat, and has rapidly diverged into three clades, demanding a H7N9 virus vaccine with broadened protection against unmatched strains. Hemagglutinin (HA)-based structural design approaches for stabilizing HA proteins have provided excitingly promising results. However, none of the HA-based structural design approaches has been applied to a recombinant replicative influenza virus. Here we report that our HA-based structural design approach is a first in the field to generate a recombinant replicative H7N9 virus (H7N9-53TM) showing broadened protection. The H7N9-53TM contains a replaced H3 HA transmembrane domain (TM) in its HA protein. In mice, the inactivated H7N9-53TM vaccine induced significantly higher HI titers, HA-specific IgG titers, and IFN-γ production than the corresponding H7N9-53WT inactivated virus vaccine containing wild-type HA. More excitingly, mice immunized with the H7N9-53TM showed full protection against homologous (H7N9-53) and interclade (H7N9-MCX) challenges with minimal weight loss, no detectable lung viral loads, and no apparent pulmonary lesions and inflammation, while mice immunized with the H7N9-53WT showed partial protection (only 60% against H7N9-MCX) with severe weight loss, detectable lung viral loads, and severe pulmonary lesions and inflammation. In summary, this study presents a better vaccine candidate (H7N9-53TM) against H7N9 pandemics. Furthermore, our HA-based structural design approach would be conceivably applicable to other subtype influenza viruses, especially the viruses from emerging pandemic and epidemic influenza viruses such as H5N1 and H1N1.