Seroepidemiology of astrovirus MLB1.

Seroepidemiology of Astrovirus MLB1 Lori R. Holtz,a Irma K. Bauer,a Hongbing Jiang,a Robert Belshe,b Pamela Freiden,c Stacey L. Schultz-Cherry,c David Wanga Washington University School of Medicine, St. Louis, Missouri, USAa; Saint Louis University, St. Louis, Missouri, USAb; St. Jude Children’s Research Hospital, Memphis, Tennessee, USAc To determine the seroprevalence of astrovirus MLB1 (MLB1), an indirect enzyme-linked immunosorbent assay (ELISA) was established. MLB1 seropositivity was high in children <6 months old, decreased to a nadir at 12 to 23 months old, and increased to 100% by adulthood. MLB1 infection is common, and primary exposure occurs in childhood. A stroviruses were first discovered in humans in 1975. These “classic astroviruses” consist of eight closely related serotypes that are responsible for about 10% of sporadic nonbacterial diarrhea in children. The extent of human infection by these viruses varies, depending on serotype. For example, 90% of the general population has antibodies to serotype 1, while only 10% has antibodies to serotype 7 (1). Recently a new clade of astroviruses has been identified. This clade currently consists of astroviruses MLB1, MLB2, and MLB3. All three viruses were initially discovered in stool samples from children with diarrhea (2–4) and have since been detected in additional diarrhea cases as well as control stools (3, 5, 6). MLB1, MLB2, and MLB3 have not yet been associated with any human disease, although we have detected MLB2 in the plasma of a febrile child (7). A recent case control study of MLB1 in a cohort of Indian children found no association of MLB1 with diarrhea (5). Currently it is unclear if the presence of these novel astroviruses in human stool samples represents true human infection. Here we describe the first evidence of human antibody response to MLB1 and demonstrate that infection by MLB1 is common. An enzyme-linked immunosorbent assay (ELISA) targeting MLB1 open reading frame 2 (ORF2) was established and validated using rabbit antibody raised against an MLB1 peptide as a positive control and preimmune serum as a negative control. Polyclonal MLB1 capsid peptide antibody (DW60) was generated by the immunization of rabbits with TNNQRARSTRPNFTPAPKF (positions 22 to 40) and ARRAMFDALVKTGVSVEEASR (positions 697 to 717) (21st Century Biochemicals). MLB2 capsid peptide (ASNQRTRSAGPGPKC) (positions 22 to 35) and CPLPHRTIEW DESSD (positions 646 to 659) (Genscript) were synthesized and injected into rabbits to produce antibodies DW57 and DW58, respectively. Open reading frames 2 (capsid protein) of MLB1, MLB2, and MLB3 were cloned as follows. MLB1 (nucleotides 3843 to 6113 from GenBank accession no. NC_011400.1) flanked with NotI and PstI restriction sites, MLB2 (nucleotides 3843 to 6080 from GenBank accession no. NC_016155.1) flanked with NotI and XhoI restriction sites, and MLB3 (nucleotides 3843 to 6086 from GenBank accession no. NC_019028.1) flanked with NotI and XhoI were amplified from stool RNA by reverse transcription-PCR with the following primers: SF0118F (5=ATAGCGGCCGCATGGCTAATGCCAGTAAAGGTGT-3=) and SF0111R (5=-ATAGACGTCTTATTTACAATTGTATGCTTCCT CGT-3=) for MLB1, LG0166 (5=-ATAGCGGCCGCATGGCTAAT GCCAATAAGGGTGT-3=) and LG0168 (5=-ATACTCGAGTTAT TTGTCTAAAAGACATGCTTCGC-3=) for MLB2, and LG0205 908 cvi.asm.org Clinical and Vaccine Immunology FIG 1 (A) Coomassie blue staining of a sodium dodecyl sulfate-polyacrylamide gel showing baculovirus-expressed His-tagged MLB1 (expected size, 88 kDa), MLB2 (expected size, 87 kDa), and MLB3 (expected size, 88 kDa) capsid proteins. (B) Western blot using polyclonal MLB1 peptide antibody (DW60) (1:500) against recombinant His-MLB1 capsid (5 ng) and polyclonal MLB2 peptide antibody (DW58) (1:2,500) against recombinant His-MLB2 capsid (100 ng). Mock, protein lysate from uninfected Sf9 cells. (5=-ATAGCGGCCGCATGGCTAATGCCAATAAGGGTGTG3=) and LG0206 (5=-ATACTCGAGTTATTTGTCTAAAAGACA TGCCTCA-3=) for MLB3. Recombinant baculoviruses were generated by the Bac-to-Bac system (Invitrogen) per the manufacturer’s protocol at the St. Jude’s Protein Production Core Facility. Human astrovirus serotype 1 capsid protein was produced as previously described (8) with the following modification: an N-terminal His tag was added to the previous construct. Baculovirus-expressed His-tagged RANK protein was kindly provided by Daved Fremont (9). MLB1, MLB2, and MLB3 capsid proteins were expressed in a baculovirus system as N-terminal 6⫻His-tagged proteins and af- Received 21 February 2014 Returned for modification 27 March 2014 Accepted 21 April 2014 Published ahead of print 30 April 2014 Editor: R. L. Hodinka Address correspondence to David Wang, . Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/CVI.00100-14 p. 908 –911 June 2014 Volume 21 Number 6 Seroepidemiology of Astrovirus MLB1 FIG 2 ELISA results for MLB1. A total of 395 serum samples were assayed for antibodies against MLB1 capsid protein. finity purified using HiTrap chelating high-performance (HP) columns (GE Healthcare). SDS-polyacrylamide gel electrophoresis and Coomassie staining were used to assess the purity of the protein (Fig. 1A). Also, mass spectrometry was used to confirm the mass of the major product. Additionally, a Western blot assay using the polyclonal rabbit antibodies against MLB1 and MLB2 capsid gave signals of approximately the expected size (88 kDA for MLB1 and 87 kDA for MLB2) (Fig. 1B). We analyzed 395 deidentified sera from subjects 2 months to 95 years of age, which were obtained from healthy volunteers in North America for previous studies conducted at the Saint Louis University Center for Vaccine Development collected between 1984 and 2009 (examples of studies include references 10 and 11). The ages of the subjects and numbers of sera were as follows: 0 to 6 months, n ⫽ 22; 7 to 11 months, n ⫽ 23; 12 to 23 months, n ⫽ 35; 2 years, n ⫽ 13; 3 years, n ⫽ 20; 4 to 6 years, n ⫽ 47; 7 to 17 years, n ⫽ 75; 18 to 32 years, n ⫽ 38; 33 to 64 years, n ⫽ 42; and ⬎65 years, n ⫽ 80. The institutional review boards at Saint Louis University and Washington University School of Medicine approved this study. ELISAs were optimized and performed as described previously (12) with the following changes: horseradish peroxidase-conjugated protein A/G (Thermo-Scientific) (which binds IgG, IgM, and IgA) diluted 1:10,000 was used, the His-MLB1 capsid concentration was 25 ng/well, and the His-MLB2 capsid concentration was 75 ng/well. For the preincubation assays, serum samples were diluted in blocking buffer and preincubated overnight at 4°C with 10⫻ the coating amount (250 ng for MLB1 plates or 750 ng for MLB2 plates) of recombinant His-MLB1 capsid, His-MLB2 capsid, His-MLB3 capsid, His-human astrovirus 1 capsid, or HisRANK (an irrelevant protein). To establish the cutoff value, we calculated the average of the 30 lowest serum samples and set the cutoff 3 standard deviations (SD) above (...truncated)