Molecular Evolution and Genetic Analysis of the Major Capsid Protein VP1 of Duck Hepatitis A Viruses: Implications for Antigenic Stability.
ABSTRACT: The duck hepatitis A virus (DHAV), a member of the family Picornaviridae, is the major cause of outbreaks with high mortality rates in young ducklings. It has three distinctive serotypes and among them, serotypes 1 (DHAV-1) and 3 (DHAV-3) were recognized in China. To investigate evolutionary and antigenic properties of the major capsid protein VP1 of these two serotypes, a primary target of neutralizing antibodies, we determined the VP1 coding sequences of 19 DHAV-1 (spanning 2000-2012) and 11 DHAV-3 isolates (spanning 2008-2014) associated with disease outbreaks. By bioinformatics analysis of VP1 sequences of these isolates and other DHAV strains reported previously, we demonstrated that DHAV-1 viruses evolved into two genetic lineages, while DHAV-3 viruses exhibited three distinct lineages. The rate of nucleotide substitution for DHAV-1 VP1 genes was estimated to be 5.57 x 10(-4) per site per year, which was about one-third times slower than that for DHAV-3 VP1 genes. The population dynamics analysis showed an upward trend for infection of DHAV-1 viruses over time with little change observed for DHAV-3 viruses. Antigenic study of representative DHAV-1 and DHAV-3 strains covering all observed major lineages revealed no detectable changes in viral neutralization properties within the serotype, despite the lack of cross-neutralization between serotypes 1 and 3 strains. Structural analysis identified VP1 mutations in DHAV-1 and DHAV-3 viruses that underpin the observed antigenic phenotypes. Results of our experiments described here shall give novel insights into evolution and antigenicity of duck picornaviruses.
Project description:As causative agents of duck viral hepatitis, duck hepatitis A virus type 1 (DHAV-1) and type 3 (DHAV-3) causes significant economic losses in the duck industry. However, a licensed commercial vaccine that simultaneously controls both pathogens is currently unavailable. Here, we generated duck enteritis virus recombinants (rC-KCE-2VP1) containing both VP1 from DHAV-1 (VP1/DHAV-1) and VP1 from DHAV-3 (VP1/DHAV-3) between UL27 and UL26. A self-cleaving 2A-element of FMDV was inserted between the two different types of VP1, allowing production of both proteins from a single open reading frame. Immunofluorescence and Western blot analysis results demonstrated that both VP1 proteins were robustly expressed in rC-KCE-2VP1-infected chicken embryo fibroblasts. Ducks that received a single dose of rC-KCE-2VP1 showed potent humoral and cellular immune responses and were completely protected against challenges of both pathogenic DHAV-1 and DHAV-3 strains. The protection was rapid, achieved as early as 3 days after vaccination. Moreover, viral replication was fully blocked in vaccinated ducks as early as 1 week post-vaccination. These results demonstrated, for the first time, that recombinant rC-KCE-2VP1 is potential fast-acting vaccine against DHAV-1 and DHAV-3.
Project description:Sixty-six human enterovirus serotypes have been identified by serum neutralization, but the molecular determinants of the serotypes are unknown. Since the picornavirus VP1 protein contains a number of neutralization domains, we hypothesized that the VP1 sequence should correspond with neutralization (serotype) and, hence, with phylogenetic lineage. To test this hypothesis and to analyze the phylogenetic relationships among the human enteroviruses, we determined the complete VP1 sequences of the prototype strains of 47 human enterovirus serotypes and 10 antigenic variants. Our sequences, together with those available from GenBank, comprise a database of complete VP1 sequences for all 66 human enterovirus serotypes plus additional strains of seven serotypes. Phylogenetic trees constructed from complete VP1 sequences produced the same four major clusters as published trees based on partial VP2 sequences; in contrast to the VP2 trees, however, in the VP1 trees strains of the same serotype were always monophyletic. In pairwise comparisons of complete VP1 sequences, enteroviruses of the same serotype were clearly distinguished from those of heterologous serotypes, and the limits of intraserotypic divergence appeared to be about 25% nucleotide sequence difference or 12% amino acid sequence difference. Pairwise comparisons suggested that coxsackie A11 and A15 viruses should be classified as strains of the same serotype, as should coxsackie A13 and A18 viruses. Pairwise identity scores also distinguished between enteroviruses of different clusters and enteroviruses from picornaviruses of different genera. The data suggest that VP1 sequence comparisons may be valuable in enterovirus typing and in picornavirus taxonomy by assisting in the genus assignment of unclassified picornaviruses.
Project description:BACKGROUND:Duck viral hepatitis (DVH) is a highly contagious viral disease affecting ducks. It can be caused by five agents, including duck hepatitis A virus genotypes 1 (DHAV-1), 2 (DHAV-2), and 3 (DHAV-3), as well as duck hepatitis virus 2 and duck hepatitis virus 3. Since 2007, DHAV-3 has been known to be the most prevalent in East and South Asia. So far, the information regarding the propagation of DHAV-3 in cultured cells is limited. In this study, we describe the comparative studies on the growth properties of DHAV-3 in primary duck embryo fibroblast (DEF) cells using two different strains: a virulent strain C-GY and an attenuated strain YDF120. The effect of fetal calf serum (FCS) and chick serum (CS) on DHAV-3 replication and the mechanism of the inhibitory effect conferred by FCS were also investigated. RESULTS:Following serial passages, both C-GY and YDF120 failed to produce cytopathic effect and plaques. The combined quantitative real-time PCR and indirect immunofluorescence staining methods showed that the two viruses could be propagated productively in DEF cells. Investigation of the viral growth kinetics revealed that the two viruses replicated in DEF cells with similar efficiencies, while the viral load of the virulent C-GY strain peaked more rapidly when compared with the attenuated YDF120 strain. Neutralization assay and time-of-drug-addition study indicated that FCS displayed inhibitory effect on DHAV-3 replication. Analysis on the mechanism of action of FCS against DHAV-3 demonstrated that the inhibitory effect was reflected at three steps of the DHAV-3 life cycle including adsorption, replication, and release. CONCLUSIONS:Both virulent and attenuated DAHV-3 strains can establish noncytocidal, productive infections in DEF cells. The virulent strain replicates more rapidly than the attenuated strain in early infection period. FCS has an inhibitory effect on DHAV-3 replication, which may be attributed to action of a non-specific inhibitory factor present in FCS directly on the virus. These findings may provide new insights into the development of potential antiviral agents.
Project description:The VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized.To characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlapping 12-mer peptides, we found that mAb 2D10 recognized phages displaying peptides with the consensus motif LPAPTS. Sequence alignment showed that the epitope 173LPAPTS178 is highly conserved among the DHAV-1 genotypes. Moreover, the six amino acid peptide LPAPTS was proven to be the minimal unit of the epitope with maximal binding activity to mAb 2D10. DHAV-1-positive duck serum reacted with the epitope in dot blotting assay, revealing the importance of the six amino acids of the epitope for antibody-epitope binding. Competitive inhibition assays of mAb 2D10 binding to synthetic LPAPTS peptides and truncated VP1 protein fragments, detected by Western blotting, also verify that LPAPTS was the VP1 epitope.We identified LPAPTS as a VP1-specific linear B-cell epitope recognized by the neutralizing mAb 2D10. Our findings have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against DHAV-1.
Project description:We report here the complete genome sequence of a duck hepatitis A virus 1 (DHAV-1), strain FJ1220, isolated from a dead pigeon in eastern China. DHAV-1 FJ1220 has high homology of up to 99.6% to the DHAV-1 strain Du/CH/LGD/111238 but relatively low homology to strains FFZ05 and FZ05. An amino acid hypervariable region in the VP1 protein of FJ1220 has the motif (180)TPSGR(184) replaced by (180)ALSRG(184) compared to strains FFZ05 and FZ05.
Project description:Among the causative agents of duck viral hepatitis, duck hepatitis A virus genotype 1 (DHAV-1) is the most common virus reported in most outbreaks worldwide. How to propagate DHAV-1 in cell cultures efficiently remains a problem to be explored. Here, we aimed to test the effect of serum type on DHAV-1 replication in duck embryo fibroblast (DEF) cells. Comparative studies involved virus culture and passage, observation of cytopathic effect (CPE), virus quantification, and plaque formation assay. From the results of these investigations, we conclude that use of chicken serum (CS) in maintenance medium allows DHAV-1 to establish productive, cytocidal infection in DEF cells, whereas FCS exerts inhibitory effects on DHAV-1 replication, CPE development, and plaque formation. By using a neutralization test, we found that the direct action of FCS on virions is likely to play a key role in inhibiting DHAV-1 replication in DEF cells. Mechanism analyses revealed that FCS inhibits DHAV-1 replication at virus adsorption and reduces extracellular virus yields. The present work may shed light on a new perspective for antiviral agent development, and have provided a virus-host cell system for further studies on molecular mechanism involved DHAV-1 replication and pathogenesis.
Project description:Monoclonal-antibody (MAb)-resistant mutants were used to map antigenic sites on foot-and-mouth disease virus (FMDV), which resulted in the identification of neutralizing epitopes in the flexible ?G-?H loop in VP1. For FMDV SAT2 viruses, studies have shown that at least two antigenic sites exist. By use of an infectious SAT2 cDNA clone, 10 structurally exposed and highly variable loops were identified as putative antigenic sites on the VP1, VP2, and VP3 capsid proteins of SAT2/Zimbabwe (ZIM)/7/83 (topotype II) and replaced with the corresponding regions of SAT2/Kruger National Park (KNP)/19/89 (topotype I). Virus neutralization assays using convalescent-phase antisera raised against the parental virus, SAT2/ZIM/7/83, indicated that the mutant virus containing the TQQS-to-ETPV mutation in the N-terminal part of the ?G-?H loop of VP1 showed not only a significant increase in the neutralization titer but also an increase in the index of avidity to the convalescent-phase antisera. Furthermore, antigenic profiling of the epitope-replaced and parental viruses with nonneutralizing SAT2-specific MAbs led to the identification of two nonneutralizing antigenic regions. Both regions were mapped to incorporate residues 71 to 72 of VP2 as the major contact point. The binding footprint of one of the antigenic regions encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 48 to 50 of VP1, and the second antigenic region encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 84 to 86 and 109 to 11 of VP1. This is the first time that antigenic regions encompassing residues 71 to 72 of VP2 have been identified on the capsid of a SAT2 FMDV.Monoclonal-antibody-resistant mutants have traditionally been used to map antigenic sites on foot-and-mouth disease virus (FMDV). However, for SAT2-type viruses, which are responsible for most of the FMD outbreaks in Africa and are the most varied of all seven serotypes, only two antigenic sites have been identified. We have followed a unique approach using an infectious SAT2 cDNA genome-length clone. Ten structurally surface-exposed, highly varied loops were identified as putative antigenic sites on the VP1, VP2, and VP3 capsid proteins of the SAT2/ZIM/7/83 virus. These regions were replaced with the corresponding regions of an antigenically disparate virus, SAT2/KNP/19/89. Antigenic profiling of the epitope-replaced and parental viruses with SAT2-specific MAbs led to the identification of two unique antibody-binding footprints on the SAT2 capsid. In this report, evidence for the structural engineering of antigenic sites of a SAT2 capsid to broaden cross-reactivity with antisera is provided.
Project description:Human enteroviruses (family Picornaviridae) are the major cause of aseptic meningitis and also cause a wide range of other acute illnesses, including neonatal sepsis-like disease, acute flaccid paralysis, and acute hemorrhagic conjunctivitis. The neutralization assay is usually used for enterovirus typing, but it is labor-intensive and time-consuming and standardized antisera are in limited supply. We have developed a molecular typing system based on reverse transcription-PCR and nucleotide sequencing of the 3' half of the genomic region encoding VP1. The standard PCR primers amplify approximately 450 bp of VP1 for most known human enterovirus serotypes. The serotype of an "unknown" may be inferred by comparison of the partial VP1 sequence to those in a database containing VP1 sequences for the prototype strains of all 66 human enterovirus serotypes. Fifty-one clinical isolates of known serotypes from the years 1991 to 1998 were amplified and sequenced, and the antigenic and molecular typing results agreed for all isolates. With one exception, the nucleotide sequences of homologous strains were at least 75% identical to one another (>88% amino acid identity). Strains with homologous serotypes were easily discriminated from those with heterologous serotypes by using these criteria for identification. This method can greatly reduce the time required to type an enterovirus isolate and can be used to type isolates that are difficult or impossible to type with standard immunological reagents. The technique may also be useful for the rapid determination of whether viruses isolated during an outbreak are epidemiologically related.
Project description:BACKGROUND:The cocirculation of duck hepatitis A virus subtypes 1 (DHAV-1) and 3 (DHAV-3) in ducklings has resulted in significant economic losses. Ducklings with DHAV-1 or DHAV-3 infection show similar clinical signs and gross lesions; hence, it is important to identify the viral subtypes in infected ducklings as early as possible for better clinical management. METHODS AND RESULTS:Based on multiple 5' noncoding region (5'-NCR) sequences of DHAV-1 and DHAV-3 strain alignments, universal and type-specific primers were designed and synthesized. With three primers in one-tube reverse transcription-PCR (RT-PCR), reference DHAV-1 and DHAV-3 isolates ranging over 60 years and across many different countries were successfully amplified, indicating that the primer sequences were completely conserved. The sequence results and the sizes of amplicons from reference DHAV-1 and DHAV-3 isolates are completely correlated with their subtypes. Moreover, with this one-tube RT-PCR system, amplicon sizes from liver samples of reference DHAV-1- or DHAV-3-infected birds fit closely with their subtypes, which was determined by virus isolation and neutralization testing. No other duck-origin RNA viruses were detected. The sensitivity of viral RNA detection was 10 pg. With this system, 20% subtype 1, 45% subtype 3, and 9% coinfection of two subtypes were detected in 55 clinical samples. CONCLUSIONS AND SIGNIFICANCE:This novel approach could be used for rapidly typing DHAV-1 or DHAV-3 infection in routine clinical surveillance or epidemiological screening.
Project description:Members of the family Picornaviridae are the most common viruses infecting humans, and species in several genera also infect a wide variety of other mammals. Picornaviruses have traditionally been classified by antigenic type, based on a serum neutralization assay. However, this method is time-consuming and labor-intensive, is sensitive to virus aggregation and antigenic variation, and requires a large number of antisera to identify all serotypes, even when antiserum pools are used. We developed generic reverse transcription (RT)-PCR primers that will amplify all human enterovirus serotypes, as well as many rhinoviruses and other picornaviruses, and used RT-PCR amplification of the VP1 gene and amplicon sequencing to identify enteroviruses that were refractory to typing by neutralization with pooled antisera. Enterovirus serotypes determined by sequencing were confirmed by neutralization with monospecific antisera. Of 55 isolates tested, 49 were of known enterovirus serotypes, two were rhinoviruses, and four were clearly picornaviruses but did not match any known picornavirus sequence. All four untyped picornaviruses were closely related to one another in sequence, suggesting that they are of the same serotype. RT-PCR, coupled with amplicon sequencing, is a simple and rapid method for the typing and classification of picornaviruses and may lead to the identification of many new picornavirus serotypes.