A 176 amino acid polypeptide derived from the mumps virus HN ectodomain shows immunological and biological properties similar to the HN protein.
BACKGROUND
The hemagglutinin-neuraminidase (HN) protein is the major antigenic determinant of the Mumps virus (MuV) and plays an important role in the viral infectious cycle through its hemagglutination/hemadsorption (HA/HD) and neuraminidase (NA) activities.


OBJECTIVE
analyze the biological and immunological properties of a polypeptide derived from a highly conserved region of the HN ectodomain.


METHODS
a highly conserved region of the HN gene among several MuV genotypes was chosen to be cloned in a eukaryotic expression vector. The pcDNAHN176-construct was transfected into Vero cells and RNA expression was detected by RT-PCR, while the corresponding polypeptide was detected by immunofluorescence and immunochemistry techniques. The HD and NA activities were also measured. The immunogenic properties of the construct were evaluated using two systems: rabbit immunization to obtain sera for detection of the HN protein and neutralization of MuV infection, and hamster immunization to evaluate protection against MuV infection.


RESULTS
A 567 nucleotide region from the HN gene was amplified and cloned into the plasmid pcDNA3.1. Vero cells transfected with the construct expressed a polypeptide that was recognized by a MuV-hyperimmune serum. The construct-transfected cells showed HD and NA activities. Sera from immunized rabbits in vitro neutralized two different MuV genotypes and also detected both the HN protein and the HN176 polypeptide by western blot. Hamsters immunized with the pcDNAHN176-construct and challenged with MuV showed a mild viral infection in comparison to non-immunized animals, and Th1 and Th2 cytokines were detected in them.


CONCLUSIONS
The pcDNAHN176-construct was capable of expressing a polypeptide in Vero cells that was identified by a hyperimmune serum anti Mumps virus, and these cells showed the HD and NA activities of the complete MuV HN protein. The construct also elicited a specific immune response against MuV infection in hamsters.
Herrera
Emma
E
Lab Virología, ENCB-IPN Carpio y Plan de Ayala S/N Casco de Santo Tomás, México D,F, 11340 México.
Barcenas
Patricia
P
Hernández
Rubicela
R
Méndez
Alfonso
A
Pérez-Ishiwara
Guillermo
G
Barrón
Blanca
B
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D017124:Conserved Sequence; D006224:Cricetinae; D016207:Cytokines / Q000097:blood; D015870:Gene Expression; D020869:Gene Expression Profiling; D015707:HN Protein / Q000235:genetics / Q000276:immunology*; D015151:Immunoblotting; D007150:Immunohistochemistry; D009109:Mumps virus / Q000276:immunology*; D009500:Neutralization Tests; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014709:Vero Cells
D000914:Antibodies, Viral; D016207:Cytokines; D015707:HN Protein; D012333:RNA, Messenger; D011994:Recombinant Proteins