ABSTRACT
Based on findings in small RNA-sequencing (Seq) data analysis, we developed highly sensitive and specific real-time reverse transcription (RT)-PCR assays with locked nucleic acid probes targeting the abundantly expressed leader sequences of Middle East respiratory syndrome coronavirus (MERS-CoV) and other human coronaviruses. Analytical and clinical evaluations showed their noninferiority to a commercial multiplex PCR test for the detection of these coronaviruses.
Subject(s)
Coronavirus Infections/diagnosis , Coronavirus/classification , Coronavirus/isolation & purification , Oligonucleotide Probes/genetics , Oligonucleotides/genetics , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Child , Child, Preschool , Coronavirus/genetics , Coronavirus Infections/virology , Female , Humans , Infant , Male , Middle Aged , Sensitivity and Specificity , Young AdultABSTRACT
A novel avian influenza A(H7N9) virus has emerged to infect humans in eastern China since 2013. An effective vaccine is needed because of the high mortality despite antiviral treatment and intensive care. We sought to develop an effective vaccine for A(H7N9) virus. The HA2 subunit was chosen as the vaccine antigen because it is highly conserved among the human A(H7N9) virus strains. Moreover, in silico analysis predicted two immunogenic regions within the HA2 subunit that may contain potential human B-cell epitopes. The HA2 fragment was readily expressed in Escherichia coli. In BALB/c mice, intraperitoneal immunization with two doses of HA2 with imiquimod (2-dose-imiquimod) elicited the highest geometric mean titer (GMT) of anti-HA2 IgG (12699), which was greater than that of two doses of HA2 without imiquimod (2-dose-no-adjuvant) (6350), one dose of HA2 with imiquimod (1-dose-imiquimod) (2000) and one dose of HA2 without imiquimod (1-dose-no-adjuvant) (794). The titer of anti-HA2 IgG was significantly higher in the 1-dose-imiquimod group than the 1-dose-no-adjuvant group. Although both hemagglutination inhibition titers and microneutralization titers were below 10, serum from immunized mice showed neutralizing activity in a fluorescent focus microneutralization assay. In a viral challenge experiment, the 2-dose-imiquimod group had the best survival rate (100 %), followed by the 2-dose-no-adjuvant group (90 %), the 1-dose-imiquimod group (70 %) and the 1-dose-no-adjuvant group (40 %). The 2-dose-imiquimod group also had significantly lower mean pulmonary viral loads than the 1-dose-imiquimod, 1-dose-no-adjuvant and non-immunized groups. This recombinant A(H7N9)-HA2 vaccine should be investigated as a complement to egg- or cell-based live attenuated or subunit influenza vaccines.
Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza A Virus, H7N9 Subtype/immunology , Influenza Vaccines/immunology , Orthomyxoviridae Infections/prevention & control , Adjuvants, Immunologic/administration & dosage , Aminoquinolines/administration & dosage , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Disease Models, Animal , Escherichia coli/genetics , Gene Expression , Hemagglutination Inhibition Tests , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Imiquimod , Immunoglobulin G/blood , Influenza Vaccines/administration & dosage , Influenza Vaccines/genetics , Injections, Intraperitoneal , Mice, Inbred BALB C , Neutralization Tests , Protein Subunits/genetics , Protein Subunits/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Survival Analysis , Vaccination/methods , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/genetics , Vaccines, Subunit/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunologyABSTRACT
OBJECTIVES: A severe acute respiratory syndrome (SARS)-like disease due to a novel betacoronavirus, human coronavirus EMC (HCoV-EMC), has emerged recently. HCoV-EMC is phylogenetically closely related to Tylonycteris-bat-coronavirus-HKU4 and Pipistrellus-bat-coronavirus-HKU5 in Hong Kong. We conducted a seroprevalence study on archived sera from 94 game-food animal handlers at a wild life market, 28 SARS patients, and 152 healthy blood donors in Southern China to assess the zoonotic potential and evidence for intrusion of HCoV-EMC and related viruses into humans. METHODS: Anti-HCoV-EMC and anti-SARS-CoV antibodies were detected using screening indirect immunofluorescence (IF) and confirmatory neutralizing antibody tests. RESULTS: Two (2.1%) animal handlers had IF antibody titer of ≥ 1:20 against both HCoV-EMC and SARS-CoV with neutralizing antibody titer of <1:10. No blood donor had antibody against either virus. Surprisingly, 17/28 (60.7%) of SARS patients had significant IF antibody titers with 7/28 (25%) having anti-HCoV-EMC neutralizing antibodies at low titers which significantly correlated with that of HCoV-OC43. Bioinformatics analysis demonstrated a significant B-cell epitope overlapping the heptad repeat-2 region of Spike protein. Virulence of SARS-CoV over other betacoronaviruses may boost cross-reactive neutralizing antibodies against other betacoronaviruses. CONCLUSIONS: Convalescent SARS sera may contain cross-reactive antibodies against other betacoronaviruses and confound seroprevalence study for HCoV-EMC.
