Higher affinities of fibers with cell receptors increase the infection capacity and virulence of human adenovirus type 7 and type 55 compared to type 3.

IMPORTANCE: HAdV-3, -7, and -55 are the predominant types causing acute respiratory disease outbreaks and can lead to severe and fatal pneumonia in children and adults. In recent years, emerging or re-emerging strains of HAdV-7 and HAdV-55 have caused multiple outbreaks globally in both civilian and military populations, drawing increased attention. Clinical studies have reported that HAdV-7 and HAdV-55 cause more severe pneumonia than HAdV-3. This study aimed to investigate the mechanisms explaining the higher severity of HAdV-7 and HAdV-55 infection compared to HAdV-3 infection. Our findings provided evidence linking the receptor-binding protein fiber to stronger infectivity of the strains mentioned above by comparing several fiber-chimeric or fiber-replaced adenoviruses. Our study improves our understanding of adenovirus infection and highlights potential implications, including in novel vector and vaccine development.

SARS-CoV-2 mRNA vaccine requires signal peptide to induce antibody responses.

New SARS-CoV-2 variants continue to prevail worldwide, and effective vaccines are needed to prevent an epidemic. mRNA vaccines are gradually being applied to the prevention and control of infectious diseases with significant safety and effectiveness. The spike (S) protein of SARS-CoV-2 is the main target of mRNA vaccine design, but the impact of the signal peptide (SP), transmembrane region (TM), and cytoplasmic tail (CT) on mRNA vaccine remains unclear. In this study, we constructed three forms of mRNA vaccines related to the S protein: full-length, deletion of the TM and CT, and simultaneous deletion of the SP, TM and CT, and compared their immunogenicity. Our experimental data show that full-length S protein and deletion of the TM and CT could effectively induce neutralizing antibody production in mice, while S protein without the SP and TM could not. This indicates that the S protein SP is necessary for the design of mRNA vaccine.

Identification of conserved linear epitopes in the SARS-CoV-2 receptor-binding region using monoclonal antibodies.

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused millions of cases of infections, leading to a global health emergency. The SARS-CoV-2 spike (S) protein plays the most important role in viral infection, and S1 subunit and its receptor-binding domain (RBD) are widely considered the most attractive vaccine targets. The RBD is highly immunogenic and its linear epitopes are important for vaccine development and therapy, but linear epitopes on the RBD have rarely been reported. In this study, 151 mouse monoclonal antibodies (mAbs) against the SARS-CoV-2 S1 protein were characterized and used to identify epitopes. Fifty-one mAbs reacted with eukaryotic SARS-CoV-2 RBD. Sixty-nine mAbs reacted with the S proteins of Omicron variants B.1.1.529 and BA.5, indicating their potential as rapid diagnostic materials. Three novel linear epitopes of RBD, R6 (391CFTNVYADSFVIRGD405), R12 (463PFERDISTEIYQAGS477), and R16 (510VVVLSFELLHAPAT523), were identified; these were highly conserved in SARS-CoV-2 variants of concern and could be detected in the convalescent serum of COVID-19 patients. From pseudovirus neutralization assays, some mAbs including one detecting R12 were found to possess neutralizing activity. Together, from the reaction of mAbs with eukaryotic RBD (N501Y), RBD (E484K), and S1 (D614G), we found that a single amino acid mutation in the SARS-CoV-2 S protein may cause a structural alteration, exerting substantial impact on mAb recognition. Our results could, therefore, help us better understand the function of the SARS-CoV-2 S protein and develop diagnostic tools for COVID-19.

Characterization and application of a series of monoclonal antibodies against SARS-CoV-2 nucleocapsid protein.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has a significant global social and economic impact, and the emergence of new and more destructive mutant strains highlights the need for accurate virus detection. Here, 90 monoclonal antibodies (MAbs) that exclusively reacted with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP) were generated. These MAbs did not cross-react with NPs of common human coronaviruses (HCoVs, i.e., 229E, OC43, HKU1, and NL63) and Middle East Respiratory Syndrome Coronavirus. Subsequently, overlapped peptides in individual fragments (N1-N4) of NP were synthesized. N1-3 (25-GSNQNGERSGARSKQ-39), N3-1 (217-AALALLLLDRLNQL-230), and N4-8 (393-TLLPAADLDDFSKQL-407) were identified as major epitopes using enzyme-linked immunoassay (ELISA) and recognized by 47, 1, and 18 MAbs, respectively. The 24 remaining MAbs exhibited no reactivity with all synthetic peptides. Among MAb-epitope pairs, only MAbs targeting epitope N1-3 displayed no cross-reaction with NPs of SARS-CoV-1 and other SARS-related CoVs. All Omicron variants contained a three-amino acid deletion (31ERS33) in the N1-3 region. Thus, MAbs targeting N1-3 failed to recognize these variants. Furthermore, a double-antibody sandwich ELISA for antigen detection was established using the optimal MAbs. Overall, a series of MAbs targeting SARS-CoV-2 NP was prepared, characterized with epitope mapping, and applied for the detection of SARS-CoV-2 antigens, and some novel B-cell epitopes of the viral NP were identified.

Epitope mapping of severe acute respiratory syndrome-related coronavirus nucleocapsid protein with a rabbit monoclonal antibody.

The emergency SARS-CoV-2, a member of severe acute respiratory syndrome-related coronaviruses (SARSr-CoV), is still greatly harming the health of mankind. SARS-CoV-2-specific monoclonal antibodies (MAbs), which can identify SARS-CoV-2 from common human coronaviruses, are considered to extensively apply to developing rapid and reliable antigen assays. In this study we generated a rabbit MAb (RAb) detecting SARS-CoV-2 nucleocapsid protein (NP), which has cross-reaction with SARS-CoV-1 NP, but not with NPs of MERS and common human CoVs (OC43, NL63, 229E, and HKU1). With truncated NP fragments and synthesized peptides, the linear epitope detected by RAb was mapped in peptide N4-8, 393-407 amino acid residue (TLLPAADLDDFSKQL) of SARS-CoV-2 NP. This epitope N4-8 was highly conserved in SARSr-CoVs, including SARS-CoV-2, SARS-CoV-1, and bat CoV RaTG13 strain. However, the corresponding peptide of bat SARSr-CoV BtKY72 strain could not be recognized by RAb, which indicates amino acid D399 may be critical for N4-8 epitope detected by RAb. The present study will be conducive to developing reliable diagnosis for SARS-CoV-2 and gaining insights into the function of the SARS-CoV-2 N protein.

Seroprevalence of Neutralizing Antibodies against Six Human Adenovirus Types Indicates the Low Level of Herd Immunity in Young Children from Guangzhou, China.

Human adenoviruses (HAdVs) commonly cause many diseases such as respiratory diseases, gastroenteritis, cystitis worldwide. HAdV-3, -7, -4 and emergent HAdV-55 and HAdV-14 are the most important types causing severe respiratory diseases. There is no effective drug available for clinical treatment, and no vaccine available for the general population. Therefore, it is important to investigate the seroprevalence against HAdV for developing novel vaccines and vectors. In this study, we investigated the seroprevalence and titer levels of neutralizing antibodies (NAb) against HAdV-3, -4, -7, -14, -55, and -11 in total 278 healthy populations between 0 months and 49 years of age (228 children and 50 adults) from Guangzhou. In children under the age of 18 years, the seropositive rates were significantly increased against HAdV-3 at 12.07%, 33.96%, and 64.29% and against HAdV-7 at 0%, 18.87%, and 19.05% in age groups of 1-2, 3-5, and 6-17 years, respectively. The seroprevalence was very low (0% ~ 8.1%) for all other four types. In adults aged between 18 and 49 years, HAdV-3, -4, and -7 (> 50.00%) were the most common types, followed by HAdV-14 (38.00%), -55 (34.00%), and -11 (24.00%). Adults tended to have high NAb titers against HAdV-4 and -55. HAdV-55-seropositive donors tended to be HAdV-11- and HAdV-14-seropositive. These results indicated the low level of herd immunity against all six HAdV types in young children, and HAdV-14, -55, -11 in adults from Guangzhou City. Our findings demonstrate the importance of monitoring HAdV types and developing vaccines against HAdV for children and adults.

Probing the nuclear import signal and nuclear transport molecular determinants of PRV ICP22.

BACKGROUND: Herpes simplex virus 1 (HSV-1) ICP22 is a multifunctional protein and important for HSV-1 replication. Pseudorabies virus (PRV) ICP22 (P-ICP22) is a homologue of HSV-1 ICP22 and is reported to be able to selectively modify the transcription of different kinetic classes of PRV genes, however, the subcellular localization, localization signal and molecular determinants for its transport to execute this function is less well understood. RESULTS: In this study, by utilizing live cells fluorescent microscopy, P-ICP22 fused to enhanced yellow fluorescent protein (EYFP) gene was transient expressed in live cells and shown to exhibit a predominantly nucleus localization in the absence of other viral proteins. By transfection of a series of P-ICP22 deletion mutants fused to EYFP, a bona fide nuclear localization signal (NLS) and its key amino acids (aa) of P-ICP22 was, for the first time, determined and mapped to aa 41-60 (PASTPTPPKRGRYVVEHPEY) and aa 49-50 (KR), respectively. Besides, the P-ICP22 was demonstrated to be targeted to the nucleus via Ran-, importin α1-, and α7-mediated pathway. CONCLUSIONS: Our findings reported herein disclose the NLS and molecular mechanism for nuclear transport of P-ICP22, these results will uncover new avenues for depicting the biological roles of P-ICP22 during PRV infection.

Identification of molecular determinants for the nuclear import of pseudorabies virus UL31.

Herpes simplex virus 1 (HSV-1) UL31 is a multifunctional protein and important for HSV-1 infection. Pseudorabies virus (PRV) UL31 is a late protein homologous to HSV-1 UL31. Previous studies showed that PRV UL31 is predominantly localized to nucleus, however, the molecular determinants for its nuclear import were unclear to date. Here, by utilizing live cells fluorescent microscopy, UL31 fused with enhanced yellow fluorescent protein was transiently expressed in live cells and confirmed to exclusively target to the nucleus in the absence of other viral proteins. Furthermore, the nuclear import of UL31 was found to be dependent on the Ran-, importin α1-, α3-, α5-, α7-, ß1-and transportin-1-mediated pathway. Therefore, these results would open up new avenues for depicting the biological functions of UL31 during PRV infection.

Characterization of the nuclear import and export signals of pseudorabies virus UL31.

The pseudorabies virus (PRV) UL31 protein (pUL31) is a homologue of the herpes simplex virus 1 pUL31, which is a multifunctional protein that is important for HSV-1 infection. However, little is known concerning the subcellular localization signal of PRV UL31. Here, by transfection with a series of PRV UL31 deletion mutants fused to an enhanced yellow fluorescent protein (EYFP) gene, a bipartite nuclear localization signal (NLS) and a PY motif NLS of UL31 were identified and mapped to amino acids (aa) 4 to 20 (RRRLLRRKSSAARRKTL) and aa 21 to 34 (TRAARDRYAPYFAY), respectively. Additionally, the predicted nuclear export signal (NES) was shown to be nonfunctional. Taken together, this information opens up new avenues for investigating the biological functions of UL31 during PRV infection.

Molecular cloning and characterization of pseudorabies virus EP0 gene.

The pseudorabies virus (PRV) early protein EP0 is a homologue of the herpes simplex virus 1 (HSV-1) immediate-early protein ICP0, which is a multifunctional protein and important for HSV-1 infection. However, the exact function of EP0 is not clear. In this study, using polymerase chain reaction, a 1,104 base-pair sequence of the EP0 gene was amplified from the PRV Becker strain genome and identification of the EP0gene was confirmed by further cloning and sequencing. Bioinformatics analysis indicated that the PRV EP0 gene encoded a putative polypeptide with 367 amino acids. The encoded protein, designated as EP0 contained a conserved RING-finger superfamily domain and was found to be closely related with the herpes virus RING-finger superfamily and was highly conserved among the counterparts encoded by RING-finger genes. Multiple nucleic acid sequence and amino-acid sequence alignments suggested that PRV EP0 showed a relatively higher similarity with EP0-like proteins of genus Varicellovirus than with those of other genera of Alphaherpesvirinae. In addition, phylogenetic analysis showed that PRV EP0 had a close evolutionary relationship with members of genus Varicellovirus, especially bovine herpesvirus 1 (BoHV-1) and BoHV-5. Antigen prediction indicated that several potential B-cell epitopes were located in EP0. Also, subcellular localization analysis demonstrated that EP0 was predominantly localized in the nucleus, suggesting that it might function as a nuclear-targeted protein.

Cloning, expression, purification, antiserum preparation and its characteristics of the truncated UL6 protein of herpes simplex virus 1.

The herpes simplex virus 1 (HSV-1) portal protein UL6 is important for HSV-1 replication, however, its precise functions in the virus life cycle are poorly understood. As we known, a relatively important tool for disclosing these functions is the antiserum specifically detecting UL6 in the HSV-1-infected cell. To this end, a recombinant protein consisting of C-terminal 297-676 amino acids of UL6 fused to His-tag was expressed in E. coli and purified from inclusion body by the Ni(2+)-NTA affinity chromatography under denaturing conditions, which was then refolded and used for the preparation of antiserum in rabbit. As results, western blot and immunofluorescence assay showed that this antiserum could specifically detect the purified truncated UL6 as well as native UL6 in the HSV-1 infected cells, indicating that the prepared antiserum could serve as a valuable tool for further exploring the functions of UL6.