Molecular detection of parvovirus in Manchurian chipmunks (Tamias sibiricus asiaticus) captured in Korea

Cross-species transmission of viral diseases alarms our global community for its potential of novel pandemic events. Of various viral pathogens noted recently, parvoviruses have posed public health threats not only to humans but to wild animals. To investigate the prevalence of parvoviruses in wild Manchurian chipmunks, here we detected genetic fragments of the nonstructural protein of parvovirus by polymerase chain reaction in wild Manchurian chipmunk specimens captured in the central and southern regions of South Korea and compared their sequence homology with references. Of a total of 348 specimens examined, chipmunk parvovirus (ChpPV)-specific gene fragments were detected with a 31.32 % rate (109 chipmunks of 348) in their kidney, liver, lung, and spleen samples, and the chipmunks captured in Gangwon Province exhibited the highest positive rate (45.37%), followed by Gyeongsang (35.29%), Gyeonggi (31.03%), Chungcheong (20.00%), and Jeolla (19.70%). When compared with the reference sequences registered in GenBank, a partial ChpPV sequence showed 97.70% identity to the previously reported Korean strain at the nucleic acid level. In the phylogenetic analysis, ChpPV exhibited closer relationship to primate parvoviruses, erythroviruses, and bovine parvovirus than to adeno-associated viruses. Despite limited sample size and genetic sequences examined in this study, our results underline the prevalence of ChpPV in Korea and emphasize the need of close surveillance of parvoviruses in wild animals.

Identification and genomic characterization of bovine parvovirus 1 in yaks

Bovine parvovirus 1 (BPV1) is a causative agent of respiratory, gastrointestinal, and reproductive cattle diseases. We collected 149 yak diarrhea fecal samples from 9 farms in the Qinghai-Tibet Plateau. The samples were screened for BPV1 by PCR, and 2 samples were positive for BPV1. The complete genomes of these BPV1 isolates were sequenced successfully. The sequences of these 2 variants were both 5,515 bp in length and shared 96.5–96.8% identity with 2 previously reported BPV1 genomes (GenBank DQ335247, NC_001540). Twenty-six identical amino acid mutations were found in the 2 yak variants, including 7 amino acid substitutions in receptor-binding regions of the VP2 protein, and 5 amino acid substitutions in the NS1 protein C-terminal region that functions to activate transcription. The new genome sequences contribute to better understanding of the evolution and molecular characteristics of BPV1.

Analysis of irradiated Argentinean fetal bovine serum for adventitious agents

Fetal bovine serum (FBS) used in cell culture may be contaminated with adventitious agents, which can affect the production of biologicals and the results of clinical laboratory tests. We carried out a retrospective study to determine the incidence of adventitious agent contamination of Argentinean irradiated FBS dating from 2015 to 2019. We analyzed FBS batches for mycoplasma and adventitious viruses (bovine pestiviruses, bovine adenovirus, bluetongue virus, bovine parainfluenza virus 3, rabies virus, bovine parvovirus, bovine herpesvirus 1, bovine respiratory syncytial virus, and reovirus). Cell passages followed by direct immunofluorescence were carried out to check viability of the mentioned adventitious agents. Also, molecular detection of mycoplasma and pestiviruses was performed on the FBS samples. The presence of neutralizing antibodies against pestiviruses was determined. Molecular analyses indicated that frequencies of mycoplasma and pestiviruses in FBS were 14% and 84%, respectively. All of the batches were seronegative for pestiviral antibodies. After cell passages, all FBS samples were negative for hemadsorbent agents and by immunofluorescence for all of the viral species analyzed; PCR assays were negative for mycoplasma and pestiviruses. Our results demonstrate that, of all adventitious agents tested, local FBS batches only had traces of mycoplasma and pestiviruses; gamma irradiation was effective in inactivating them.

Parvovirus B19 and Human Parvovirus 4 Encode a Homologous "X Protein" in a Reading Frame Overlapping the VP1 Capsid Gene

30 years ago, researchers noticed that the capsid (VP1) gene of B19 parvovirus might encode a second protein, called "X", in an overlapping reading frame. Since then, experimental approaches failed to detect it. In contrast, sequence analyses can reliably predict whether a protein is expressed from an overlapping frame, provided that it is beneficial to the virus and thus under selection pressure. We used a dedicated software, Synplot2, to identify regions of VP1 likely to encode functional proteins in overlapping frames. Synplot2 detected the X open reading frame and confirmed it is under highly significant selection pressure. We discovered that the X protein is homologous to the ARF1 protein of human parvovirus 4, another suspected protein encoded in a frame overlapping VP1. These findings provide compelling evidence that the X protein must be expressed and functional. We predict that it contains a predicted transmembrane region. We found that the X frame contains a potential AUG start codon in parvovirus B19 and in all related species. Yet no currently known viral transcript has the potential to encode the X protein in a monocistronic fashion. Therefore, the X protein is probably expressed either from an unmapped monocistronic mRNA, or translated by a non-canonical mechanism from the VP1 mRNA or from a short transcript, R3, which has no currently known function. Finally, Synplot2 also detected proteins likely to be expressed from a frame overlapping VP1 in species distantly related to parvovirus B19: porcine parvovirus 2 and bovine parvovirus 3.

Structure of an Enteric Pathogen, Bovine Parvovirus

ABSTRACTBovine parvovirus (BPV), the causative agent of respiratory and gastrointestinal disease in cows, is the type member of theBocaparvovirusgenus of theParvoviridaefamily. Toward efforts to obtain a template for the development of vaccines and small-molecule inhibitors for this pathogen, the structure of the BPV capsid, assembled from the major capsid viral protein 2 (VP2), was determined using X-ray crystallography as well as cryo-electron microscopy and three-dimensional image reconstruction (cryo-reconstruction) to 3.2- and 8.8-Å resolutions, respectively. The VP2 region ordered in the crystal structure, from residues 39 to 536, conserves the parvoviral eight-stranded jellyroll motif and an αA helix. The BPV capsid displays common parvovirus features: a channel at and depressions surrounding the 5-fold axes and protrusions surrounding the 3-fold axes. However, rather than a depression centered at the 2-fold axes, a raised surface loop divides this feature in BPV. Additional observed density in the capsid interior in the cryo-reconstructed map, compared to the crystal structure, is interpreted as 10 additional N-terminal residues, residues 29 to 38, that radially extend the channel under the 5-fold axis, as observed for human bocavirus 1 (HBoV1). Surface loops of various lengths and conformations extend from the core jellyroll motif of VP2. These loops confer the unique surface topology of the BPV capsid, making it strikingly different from HBoV1 as well as the type members of otherParvovirinaegenera for which structures have been determined. For the type members, regions structurally analogous to those decorating the BPV capsid surface serve as determinants of receptor recognition, tissue and host tropism, pathogenicity, and antigenicity.IMPORTANCEBovine parvovirus (BPV), identified in the 1960s in diarrheic calves, is the type member of theBocaparvovirusgenus of the nonenveloped, single-stranded DNA (ssDNA)Parvoviridaefamily. The recent isolation of human bocaparvoviruses from children with severe respiratory and gastrointestinal infections has generated interest in understanding the life cycle and pathogenesis of these emerging viruses. We have determined the high-resolution structure of the BPV capsid assembled from its predominant capsid protein VP2, known to be involved in a myriad of functions during host cell entry, pathogenesis, and antigenicity for other members of theParvovirinae. Our results show the conservation of the core secondary structural elements and the location of the N-terminal residues for the known bocaparvovirus capsid structures. However, surface loops with high variability in sequence and conformation give BPV a unique capsid surface topology. Similar analogous regions in otherParvovirinaetype members are important as determinants of receptor recognition, tissue and host tropism, pathogenicity, and antigenicity.