Regular ArticleGenomic Organization and Mapping of Transcription and Translation Products of the NADL-2 Strain of Porcine Parvovirus
Abstract
The NADL-2 strain of PPV was cloned into pUC19 and independent infectious clones were sequenced. This permitted a correction of published sequences and to predict a cruciform structure as an alternative to the 5′-hairpin of the "-" strand. This 5′-end structural covariance is shared with other parvoviruses of the same group and two alternative sequences ("flip" and "flop") were present in the region of the cruciform. Transcript and translation product mapping allowed the prediction of the location of the different expression signals. The 5′-startpoints of the transcripts were located at nucleotides 225 and 2035, respectively, and the polyadenylation site at nucleotides 4829-4833. This indicated that the TATA boxes at 196-TATA and 2004-AATA and the 4813-AATAAA polyadenylation sequence would be functional. Alternative splicing of capsid gene (VP) transcripts (either 2280-AG/GT or 2313-AG/GT spliced with 2386-AG/GA), to maintain or remove the first AUG (at 2287) in the ORF, yielded two 2.9-kb mRNAs containing a nested set of protein-coding sequences (VP-1 and VP-2 with predicted molecular mass 80.9 and 64.3 kDa, respectively). Three nonstructural (NS) protein gene transcripts were identified. The 4.7-kb transcript was not spliced in the NS gene and was predicted to code for a 75.5-kDa protein (NS-1; published value of phosphorylated form 84 kDa). The splicing sites of two different 3.3-kb NS transcripts were analyzed. These transcripts were predicted to code for the NS-2 protein (18.1 kDa). Of the two NS-2 transcripts, one had also the VP-intron removed downstream of the NS-2 coding sequences. A 2.9-kb transcript would code for an NS-3 protein (12.4 kDa) although such a protein has not been described before. A flow chart of the information from the viral DNA to the viral proteins is presented and several differences, both for the NS and the VP genes, with closely related parvoviruses are noted.
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Isolation, characterization, and phylogenetic analysis of two new porcine parvovirus 1 isolates from Northern China
2024, Virus ResearchPorcine parvovirus (PPV) is a pathogen of infectious reproductive disease, which can cause stillbirth, mummification, embryo death, and infertility (SMEDI) syndrome in pigs. The objective of this study was to gain new insights into the evolution and phylogeny of the PPV1 genome. In this study, we isolated two new PPV1 (HLJ202108-Y and SDLC202109) from northern China and sequenced their whole genomes. The new isolates were found to have three amino acid substitutions (K195R, K562R, and S578P) in nonstructural protein 1. The VP2 amino acid site contained nine nonsynonymous substitutions, including six substitutions of the Kresse strain corresponding to the NADL-2 strain and three substitutions of A414S, S436T, and N555K. Genetic evolution analysis was conducted on 107 reference sequences available in the GenBank database, and 4–5 PPV1 taxa were defined. The new isolates were in the same phylogenetic cluster as strain 27a. The changes in the cluster, specifically marker amino acids, and their potential role in enhancing pathogenicity are discussed in this study. Furthermore, the evolutionary tree map results showed that the strains in China were evolving in two directions: one was becoming increasingly similar to early NADL-2 strains, while the other was evolving toward 27a-like strains. We also compared the proliferation ability of the isolated strains in susceptible cells by analyzing the multistep growth curves. The results showed that the virulence titer of the mutant strain was high. In summary, this study introduced the latest changes in PPV and discussed the virus characteristics that were considered to affect virulence.
First investigation on the presence of porcine parvovirus type 3 in domestic pig farms without reproductive failure in the Democratic Republic of Congo
2021, Veterinary and Animal SciencePorcine Parvovirus (PPV) is one of the major pathogens responsible for reproductive failure in sows. However, the information on its frequency in the Democratic Republic of Congo (DRC) is largely unknown. Thus, the present study was carried out to detect and genetically characterize some of known Parvovirus namely porcine parvovirus 1, 2, 3, 4, porcine bocavirus (PBoV) 1, and porcine bocavirus-like virus (PBolikeV) in 80 randomly selected archive pig farm samples during an African swine fever (ASF) survey in South Kivu, eastern DRC by polymerase chain reaction (PCR). The majority of animals analyzed (82.5%) were local breeds, and most of them (87.5%) were adults (above one year old). The majority of the animals (65%) were from the free range farms. The PCR result indicated that only PPV3 was detected in 14/80 pigs. Seven swine herds (8.7%) were co-infected with PPV3 and ASFV. Morever, a significantly high PPV3 infection rate was observed in the spleen (66.7%, P<0.0001) compared to the others type of samples. Further, the phylogenetic analysis of partial PPV3 sequences revealed one clade of PPV3 clustered with PPV3 isolates reported in a previous study in Cameroun, China, Slovakia, Germany, and China. This study is the first to report the detection of PPV in DRC. Further studies are needed to assess the levels of PPV3 viremia and the impact in co-infections with other endemic pig viruses, including ASFV.
Isolation and phylogenetic analysis of a new Porcine parvovirus strain GD2013 in China
2020, Journal of Virological MethodsPorcine parvovirus (PPV), a causative agent of an infectious reproductive disorder causing stillbirth, mummification, embryonic death and infertility (SMEDI) syndrome in swine, is a threat to both domestic pigs and wild boars regardless of age and gender. Recent studies found that the observed average substitution rate in the PPV genome was close to those of the RNA viruses and new strains showing serological neutralization activities different from that of the vaccine strain NADL-2 have been reported. These observations have increased the need for the development of new commercial vaccine strains.
In this study, a new PPV strain, GD2013, was isolated from Guangdong, China, and its entire genome sequenced. A phylogenetic tree based on the complete coding region of the genomes of 32 PPV strains was constructed using the Bayesian Markov Chain Monte Carlo (MCMC) method. The results showed that strain GD2013 fell into the same phylogenetic cluster as the classical vaccine strains NADL-2 and POVCAP, suggesting a close relationship to the vaccine strains. Multiple sequence alignments and amino acid mutation analyses of the PPV VP2 gene revealed a new amino acid polymorphism site at Thr45 on VP2 that could be used to identify low virulence strains as vaccine candidates. Selective pressure analysis of the NS1 and VP2 genes by calculating the mean rates of non-synonymous substitutions (dN) over synonymous substitutions (dS) implied that both of these genes were under negative selection. Therefore, by using phylogenetic and amino acid mutation analyses, a likely candidate strain suitable for evaluation as an attenuated vaccine strain was identified.
Increasing diversity of swine parvoviruses and their epidemiology in African pigs
2019, Infection, Genetics and EvolutionCitation Excerpt :PPV1 has a small, single-stranded, negative-sense DNA genome of approximately 5 kb which is packaged in a non-enveloped viral capsid (Molitor et al., 1984). The genome has a unique feature of distinct palindromic hairpin termini and contains two major open reading frames (ORFs) (Bergeron et al., 1993; Bergeron et al., 1996). The ORF1 found at the 5′ end of the viral genome codes for non-structural proteins 1 (NS1) which could be spliced alternatively to obtain two additional non-structural proteins (NS2 and NS3).
Detection of infectious viral agents has been on the increase globally with the advent and usage of more sensitive and selective novel molecular techniques in the epidemiological study of viral diseases of economic importance to the swine industry. The observation is not different for the pig-infecting member of the subfamily Parvovirinae in the family Parvoviridae as the application of novel molecular methods like metagenomics has brought about the detection of many other novel members of the group. Surprisingly, the list keeps increasing day by day with some of them possessing zoonotic potentials. In the last one decade, not less than ten novel swine-infecting viruses have been added to the subfamily, and ceaseless efforts have been in top gear to determine the occurrence and prevalence of the old and new swine parvoviruses in herds of pig-producing countries worldwide. The story, however, is on the contrary on the African continent as there is presently a dearth of information on surveillance initiatives of the viruses among swine herds of pig-producing countries in the region. Timely detection and characterization of the viral pathogens is highly imperative for the implementation of effective control and prevention of its spread. This review therefore presents a concise overview on the epidemiology of novel porcine parvoviruses globally and also provides up-to-date highlights on the reported cases of the viral agents in the African sub-region.
Rapid and specific detection of porcine parvovirus by isothermal recombinase polymerase amplification assays
2016, Molecular and Cellular ProbesPorcine parvovirus (PPV) is a major cause of swine reproductive failure and reported in many countries worldwide. Recombinase polymerase amplification (RPA) assays using a real-time fluorescent detection (PPV real-time RPA assay) and a lateral flow dipstick (PPV RPA LFD assay) were developed targeting PPV NS1 gene. The detection limit of PPV real-time RPA assay was 300 copies per reaction within 9 min at 38 °C, while the RPA LFD assay has a detection limit of 400 copies per reaction in less than 20 min at 38 °C. In both assays, there were no cross-reactions with porcine circovirus type 2, pseudorabies virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus, and foot-and-mouth disease virus. Based on a total of 128 clinical samples examined, the sensitivity and the specificity of the developed RPA assays for identification of PPV was 94.4% and 100%, respectively, when compared to real-time (qPCR) assay. Therefore, the RPA assay provides a rapid, sensitive and specific alternative for PPV detection.
Although PPV has been described as a cellular contaminant, few recent studies about the presence of this virus in cell cultures, serum, and trypsin were found in the literature. The purpose of this study was to detect the presence of porcine parvovirus (PPV) by polymerase chain reaction (PCR) in cell cultures, serum, and trypsin used in official public laboratories of educational institutes and research centers. We tested samples of cell cultures (88), batches of trypsin (10), and fetal bovine serum (13) from different manufacturers. The PCR for beta-actin and GAPDH was used to evaluate the efficiency of DNA extraction from samples. The PPV DNA was detected in 52 of 88 (59.1%) cell culture samples. One in ten batches of trypsin tested for PPV DNA was positive. In no sample of fetal bovine serum, amplification of PPV DNA was observed. Positive samples were tested and confirmed by another analyst. In addition, all positive samples were sequenced. Our results indicate that regular PCR testing for PPV in cell cultures and their supplies is important.