A variety of severe diseases with a broad range of symptoms, such as acute febrile respiratory disease, conjunctivitis, cystitis, gastroenteritis, myocarditis, and even life-threatening pneumonia are caused by HAdV infections in healthy adults and particularly in immunocompromised infants or young children2,4. HAdV species B (HAdV-B3, B7, B21, and B55) and C (HAdV-C1, C2, C5, and C6) are the most common viruses responsible for severe respiratory infections worldwide1,5C11. over 50% of the participants from China and nearly 70% of donors from Sierra Leone experienced detectable nAbs against HAdV-4 despite the few contamination cases officially reported in these regions. Furthermore, the prevalence of nAbs to HAdV-4 is lower than that to HAdV-5, and both varied by geographic location. In addition, the seropositive rates of both HAdV-4 and HAdV-5?nAbdominal muscles increased with age. However, the nAbs stimulated by HAdV-4 remained stable at low (200) levels among the different age groups, whereas moderate (201C1000) or high ( 1000) nAb levels were produced by HAdV-5 and tended to decrease with age. These results elucidate the human humoral immune response against HAdV-4 and revealed that this computer virus may be an underestimated causative agent of respiratory disease among adults in China and West Africa, demonstrating the importance of HAdV-4 surveillance 3-Hydroxydecanoic acid and providing useful insights for the future development of HAdV-4-based vaccines. Introduction Human adenoviruses (HAdVs) are nonenveloped, double-stranded DNA viruses belonging to the Adenoviridae family. To date, a total of 90 HAdV subtypes have been documented and classified into 7 species (ACG) according to their serological and molecular characteristics1C3. A variety of severe diseases with a broad range of symptoms, such as acute febrile respiratory disease, conjunctivitis, cystitis, gastroenteritis, myocarditis, and even life-threatening pneumonia are caused by HAdV infections in healthy adults and particularly in immunocompromised infants or young children2,4. HAdV species B (HAdV-B3, B7, B21, and B55) and C (HAdV-C1, C2, C5, and C6) are the most common viruses responsible for severe respiratory infections worldwide1,5C11. HAdV-4, the only member of human adenovirus species E, has been a 3-Hydroxydecanoic acid significant cause of acute respiratory disease in the US armed service recruits since its identification in the 1950s12. The high susceptibility to HAdV-4 contamination and the high communicability among incoming recruits during basic training might be attributable to the low level of pre-existing immunity in these individuals as well as the closed and crowded conditions in barracks12. However, outbreaks of severe acute febrile respiratory disease and even pneumonia due to HAdV-4 infections have recently occurred among populations of children in India, Taiwan, and South Korea1,2,5,13,14, as well as in healthy immunocompetent adults in the United States, Italy, and Singapore15C17. Increasing attention has been paid to HAdV-4-associated illnesses during the past 6 decades. However, although limited epidemiological data exists in the United States, no comparable reports are available elsewhere, especially in China and Africa. In this study, we established a specific HAdV-4 neutralization assay based on a recombinant replication-competent HAdV-4-luciferase (Ad4-Luc) virus to study the distribution of nAbs in relation to sex and age. In addition, we assessed the cross-reactivity and analyzed the differences in nAb levels between HAdV-4 and HAdV-5, the most representative serotype of prevalent adenovirus species C, which will greatly increase our understanding of the blood circulation of HAdV-4 and of human immunity to this pathogen. Materials and methods Generation of recombinant HAdV-4 and HAdV-5 reporter viruses The recombinant HAdV-5 3-Hydroxydecanoic acid vector-based luciferase-expressing computer virus was constructed using the AdMax system (Microbix Biosystems, ON, Canada). Human adenovirus type 4 strain RI-67 (GenBank No. “type”:”entrez-nucleotide”,”attrs”:”text”:”AY594253″,”term_id”:”51173196″,”term_text”:”AY594253″AY594253) 3-Hydroxydecanoic acid was purchased from your American Type Culture Collection (Manassas, VA, USA), and recombinant replication-competent adenovirus HAdV-4 expressing luciferase protein was constructed according to published protocols18. Briefly, the 3-Hydroxydecanoic acid terminal regions of the HAdV-4 genomes were polymerase chain reaction (PCR) amplified and subcloned into the pET-28a vector (conferring kanamycin resistance; Novagen, WI, USA) to obtain shuttle plasmids. Tlr4 The genomic plasmid pAd4 was generated by homologous recombination between the HAdV-4 genome and the shuttle plasmids in BJ5183 qualified cells (Agilent Technologies, CA, USA). The open reading frame of the ampicillin resistance gene and the fragments flanking the E3 region were then PCR amplified and subcloned into pET-28a to obtain pAd4-E3L-Amp-E3R. After sequencing, the E3L-Amp-E3R fragment was excised from pAd4-E3L-Amp-E3R and transformed into BJ5183 qualified cells made up of pAd4. The E3 region in pAd4 (conferring kanamycin resistance) was then deleted by homologous recombination with the E3L-Amp-E3R fragment using ampicillin screening to obtain pAd4E3-Amp (conferring kanamycin and ampicillin resistance). The ampicillin fragment was subsequently deleted using the restriction enzyme values of less than 0.05 were considered to be significant. Results Establishment and validation of neutralization.