Mt10-CVB3 Vaccine Virus Protects against CVB4 Infection by Inducing Cross-Reactive, Antigen-Specific Immune Responses

Group B coxsackieviruses (CVB) containing six serotypes, B1–B6, affect various organs, and multiple serotypes can induce similar diseases such as myocarditis and pancreatitis. Yet, no vaccines are currently available to prevent these infections. Translationally, the derivation of vaccines that offer protection against multiple serotypes is highly desired. In that direction, we recently reported the generation of an attenuated strain of CVB3, termed Mt10, which completely protects against both myocarditis and pancreatitis induced by the homologous wild-type CVB3 strain. Here, we report that the Mt10 vaccine can induce cross-protection against multiple CVB serotypes as demonstrated with CVB4. We note that the Mt10 vaccine could induce cross-reactive neutralizing antibodies (nABs) against both CVB1 and CVB4. In challenge studies with CVB4, the efficacy of the Mt10 vaccine was found to be 92%, as determined by histological evaluation of the heart and pancreas. Antibody responses induced in Mt10/CVB4 challenged animals indicated the persistence of cross-reactive nABs against CVB1, CVB3, and CVB4. Evaluation of antigen-specific immune responses revealed viral protein 1 (VP1)-reactive antibodies, predominantly IgG2a, IgG2b, IgG3, and IgG1. Similarly, by using major histocompatibility complex class II tetramers, we noted induction of VP1-specific CD4 T cells capable of producing multiple T cell cytokines, with interferon-γ being predominant. Finally, none of the vaccine recipients challenged with CVB4 revealed the presence of viral nucleic acid in the heart or pancreas. Taken together, our data suggest that the Mt10 vaccine can prevent infections caused by multiple CVB serotypes, paving the way for the development of monovalent CVB vaccines to prevent heart and pancreatic diseases of enteroviral origin.

Enterovirus 71 structural viral protein 1 promotes autophagy by inducing an m6A-mediated PMP22 overexpression in mouse Schwann cells

Objective Enterovirus 71 (EV71), one of the enteroviruses responsible for the hand, foot and mouth disease (HFMD), can cause severe neurologic diseases such as brainstem encephalitis and demyelination. The molecular mechanism of demyelination is still not fully understood. This study aims to investigate the mechanism of how the EV71 structural viral protein 1, VP1 can act on host cellular pathways in mouse Schwann cells. Methods An EV71 VP1-expressing vector was generated and transfected into mouse Schwann cells (MSCs). Selective mRNA methylation inhibitor (DAA) was employed to identify key members of m6A pathway that are targeted by VP1. To investigate the role of METTL14 and YTHDF1 in PMP22 expression, small interfering RNA against METTL14 and YTHDF1 was employed to knockdown the METTL14 and YTHDF1 expression in MSCs. Real-time PCR and Western blot analysis were performed to determine the expression of PMP22 and m6A modification-associated proteins. Results Our results demonstrated VP1 upregulated m6A pathway by targeting METTL14 and YTHDF1. The expression of PMP22 was decreased by inhibiting the expression of METTL14 and YTHDF1. Conclusions VP1 upregulates m6A modification, which in turn causes the hypermethylation of PMP22 in MSCs, resulting in a higher level of PMP22. Ultimately, this VP1-induced PMP22 overexpression leads to MSC autophagy.

Functional Insights into Silymarin as an Antiviral Agent against Enterovirus A71 (EV-A71)

Enterovirus A71 (EV-A71) is a major neurovirulent agent capable of causing severe hand, foot and mouth disease (HFMD) associated with neurological complications and death. Currently, no FDA-approved antiviral is available for the treatment of EV-A71 infections. The flavonoid silymarin was shown to exert virucidal effects, but the binding site on the capsid was unknown. In this study, the ligand interacting site of silymarin was determined in silico and validated in vitro. Moreover, the potential of EV-A71 to develop resistance against silymarin was further evaluated. Molecular docking of silymarin with the capsid of EV-A71 indicated that silymarin binds to viral protein 1 (VP1) of EV-A71, specifically at the GH loop of VP1. The in vitro binding of silymarin with VP1 of EV-A71 was validated using recombinant VP1 through ELISA competitive binding assay. Continuous passaging of EV-A71 in the presence of silymarin resulted in the emergence of a mutant carrying a substitution of isoleucine by threonine (I97T) at position 97 of the BC loop of EV-A71. The mutation was speculated to overcome the inhibitory effects of silymarin. This study provides functional insights into the underlying mechanism of EV-A71 inhibition by silymarin, but warrants further in vivo evaluation before being developed as a potential therapeutic agent.

Retrospective and prospective studies of transmissible viral proventriculitis in broiler chickens in Brazil

We investigated the occurrence and pathologic findings of transmissible viral proventriculitis (TVP) associated with the chicken proventricular necrosis virus (CPNV) in commercial broiler chickens in southeastern Brazil. Seventy-three broilers, 25–36 d old, with a history of reduced growth, were referred to our veterinary pathology services from 2013 to 2017. Broilers were clinically examined, weighed, and euthanized for postmortem examination. Broilers of different ages with proventricular histologic lesions were positive for CPNV by RT-PCR; however, the intensity of histologic lesions was higher among 33-d-old animals, and viral RNA detection was more frequent among those that were 28 d old. In the proventriculi of 35 of 73 (48%) broilers, lesions were characterized by glandular epithelial necrosis, lymphoplasmacytic and histiocytic infiltrates, and metaplasia of glandular epithelium to ductal epithelium. In 24 of 73 (36%) broilers with histologic TVP-compatible lesions, CPNV was detected by RT-PCR for the viral protein 1 ( VP1) gene. Broilers with histologic lesions were lighter than expected compared to the Cobb 500 standard weight. TVP has not been reported previously in broiler chickens in Brazil, to our knowledge.

Genomic Sequencing and Comparison of Sacbrood Viruses from Apis cerana and Apis mellifera in Taiwan

Sacbrood virus (SBV) was the first identified bee virus and shown to cause serious epizootic infections in the population of Apis cerana in Taiwan in 2015. Herein, the whole genome sequences of SBVs in A. cerana and A. mellifera were decoded and designated AcSBV-TW and AmSBV-TW, respectively. The whole genomes of AcSBV-TW and AmSBV-TW were 8776 and 8885 bp, respectively, and shared 90% identity. Each viral genome encoded a polyprotein, which consisted of 2841 aa in AcSBV-TW and 2859 aa in AmSBV-TW, and these sequences shared 95% identity. Compared to 54 other SBVs, the structural protein and protease regions showed high variation, while the helicase was the most highly conserved region among SBVs. Moreover, a 17-amino-acid deletion was found in viral protein 1 (VP1) region of AcSBV-TW compared to AmSBV-TW. The phylogenetic analysis based on the polyprotein sequences and partial VP1 region indicated that AcSBV-TW was grouped into the SBV clade with the AC-genotype (17-aa deletion) and was closely related to AmSBV-SDLY and CSBV-FZ, while AmSBV-TW was grouped into the AM-genotype clade but branched independently from other AmSBVs, indicating that the divergent genomic characteristics of AmSBV-TW might be a consequence of geographic distance driving evolution, and AcSBV-TW was closely related to CSBV-FZ, which originated from China. This 17-amino-acid deletion could be found in either AcSBV or AmSBV in Taiwan, indicating cross-infection between the two viruses. Our data revealed geographic and host specificities between SBVs. The amino acid difference in the VP1 region might serve as a molecular marker for describing SBV cross-infection.

The VP1u of Human Parvovirus B19: A Multifunctional Capsid Protein with Biotechnological Applications

The viral protein 1 unique region (VP1u) of human parvovirus B19 (B19V) is a multifunctional capsid protein with essential roles in virus tropism, uptake, and subcellular trafficking. These functions reside on hidden protein domains, which become accessible upon interaction with cell membrane receptors. A receptor-binding domain (RBD) in VP1u is responsible for the specific targeting and uptake of the virus exclusively into cells of the erythroid lineage in the bone marrow. A phospholipase A2 domain promotes the endosomal escape of the incoming virus. The VP1u is also the immunodominant region of the capsid as it is the target of neutralizing antibodies. For all these reasons, the VP1u has raised great interest in antiviral research and vaccinology. Besides the essential functions in B19V infection, the remarkable erythroid specificity of the VP1u makes it a unique erythroid cell surface biomarker. Moreover, the demonstrated capacity of the VP1u to deliver diverse cargo specifically to cells around the proerythroblast differentiation stage, including erythroleukemic cells, offers novel therapeutic opportunities for erythroid-specific drug delivery. In this review, we focus on the multifunctional role of the VP1u in B19V infection and explore its potential in diagnostics and erythroid-specific therapeutics.

Recombinant Enterovirus 71 Viral Protein 1 Fused to a Truncated Newcastle Disease Virus NP (NPt) Carrier Protein

Enterovirus 71 (EV71) is the major causative agent in hand, foot, and mouth disease (HFMD), and it mainly infects children worldwide. Despite the risk, there is no effective vaccine available for this disease. Hence, a recombinant protein construct of truncated nucleocapsid protein viral protein 1 (NPt-VP1198–297), which is capable of inducing neutralizing antibody against EV71, was evaluated in a mouse model. Truncated nucleocapsid protein Newcastle disease virus that was used as immunological carrier fused to VP1 of EV71 as antigen. The recombinant plasmid carrying corresponding genes was constructed by recombinant DNA technology and the corresponding protein was produced in Escherichia coli expression system. The recombinant NPt-VP1198–297 protein had elicited neutralizing antibodies against EV71 with the titer of 1:16, and this result is higher than the titer that is elicited by VP1 protein alone (1:8). It was shown that NPt containing immunogenic epitope(s) of VP1 was capable of inducing a greater functional immune response when compared to full-length VP1 protein alone. It was capable to carry larger polypeptide compared to full-length NP protein. The current study also proved that NPt-VP1198–297 protein can be abundantly produced in recombinant protein form by E. coli expression system. The findings from this study support the importance of neutralizing antibodies in EV71 infection and highlight the potential of the recombinant NPt-VP1198–297 protein as EV71 vaccine.

Structural Analysis of Merkel Cell Polyomavirus (MCPyV) Viral Capsid Protein 1 (VP1) in HIV-1 Infected Individuals

Merkel cell polyomavirus (MCPyV) viral protein 1 (VP1) is the capsid protein that mediates virus attachment to host cell receptors and is the major immune target. Given the limited data on MCPyV VP1 mutations, the VP1 genetic variability was examined in 100 plasma and 100 urine samples from 100 HIV+ individuals. Sequencing of VP1 DNA in 17 urine and 17 plasma specimens, simultaneously MCPyV DNA positive, revealed that 27 samples displayed sequences identical to VP1 of MCC350 strain. VP1 from two urine specimens had either Thr47Ser or Ile115Phe substitution, whereas VP1 of one plasma contained Asp69Val and Ser251Phe substitutions plus deletion (∆) of Tyr79. VP1 DNA in the remaining samples had mutations encoding truncated protein. Three-dimensional prediction models revealed that Asp69Val, Ser251Phe, and Ile115Phe caused neutral effects while Thr47Ser and Tyr79∆ produced a deleterious effect reducing VP1 stability. A549 cells infected with urine or plasma samples containing full-length VP1 variants with substitutions, sustained viral DNA replication and VP1 expression. Moreover, medium harvested from these cells was able to infect new A549 cells. In cells infected by samples with truncated VP1, MCPyV replication was hampered. In conclusion, MCPyV strains with unique mutations in the VP1 gene are circulating in HIV+ patients. These strains display altered replication efficiency compared to the MCC350 prototype strain in A549 cells.

Enterovirus infections in pediatric patients hospitalized with acute gastroenteritis in Chiang Mai, Thailand, 2015–2018

Background Infection with viruses especially rotavirus, norovirus, astrovirus, and adenovirus has been known to be a major cause of acute gastroenteritis in children under 5 years of age globally, particularly in developing countries. Also, some genotypes of enteroviruses (EVs) have been reported to be associated with gastroenteritis. This study is aimed to investigate the prevalence and genotype diversity of EV in children admitted to hospitals with acute gastroenteritis. Methods A total of 1,736 fecal specimens were collected from children hospitalized with diarrhea in Chiang Mai, Thailand from 2015 to 2018. All specimens were tested for the presence of EV by RT-PCR of the 5′ untranslated region. The genotypes of EV were further identified by nucleotide sequencing and phylogenetic analysis of the viral protein 1 (VP1) gene. Results EV was detected in 154 out of 1,736 specimens (8.9%) throughout the study period. The prevalence of EV detected in 2015, 2016, 2017, and 2018 was 7.2%, 9.0%, 11.2%, and 8.6%, respectively. EV was detected all year round with a high prevalence during rainy season in Thailand. Overall, 37 genotypes of EV were identified in this study. Among these, coxsackievirus (CV)-A24 and CV-B5 (7.5% each), and EV-C96 (6.8%) were the common genotypes detected. Conclusion This study demonstrates the prevalence, seasonal distribution, and genotype diversity of EV circulating in children hospitalized with acute gastroenteritis in Chiang Mai, Thailand during the period 2015 to 2018.

Genetic diversity of enterovirus G detected in faecal samples of wild boars in Japan: identification of novel genotypes carrying a papain-like cysteine protease sequence

The genetic diversity of enterovirus G (EV-G) was investigated in the wild-boar population in Japan. EV-G-specific reverse transcription PCR demonstrated 30 (37.5 %) positives out of 80 faecal samples. Of these, viral protein 1 (VP1) fragments of 20 samples were classified into G1 (3 samples), G4 (1 sample), G6 (2 samples), G8 (4 samples), G11 (1 sample), G12 (7 samples), G14 (1 sample) and G17 (1 sample), among which 11 samples had a papain-like cysteine protease (PL-CP) sequence, believed to be the first discoveries in G1 (2 samples) or G17 (1 sample) wild-boar EV-Gs, and in G8 (2 samples) or G12 (6 samples) EV-Gs from any animals. Sequences of the non-structural protein regions were similar among EV-Gs possessing the PL-CP sequence (PL-CP EV-Gs) regardless of genotype or origin, suggesting the existence of a common ancestor for these strains. Interestingly, for the two G8 and two G12 samples, the genome sequences contained two versions, with or without the PL-CP sequence, together with the homologous 2C/PL-CP and PL-CP/3A junction sequences, which may explain how the recombination and deletion of the PL-CP sequences occured in the PL-CP EV-G genomes. These findings shed light on the genetic plasticity and evolution of EV-G.