Andrew K. Cheung

Detection of two porcine circovirus type 2 genotypic groups in United States swine herds

Summary.In late 2005, sporadic cases of an acute onset disease of high mortality were observed in 10- to 16-week-old growing pigs among several swine herds of the United States. Tissues from the affected pigs in Kansas, Iowa, and North Carolina were examined, and porcine circovirus type 2 (PCV2) was detected consistently among these tissues. Phylogenetically, PCV2 can be divided into two major genotypic groups, PCV2-group 1 and PCV2-group 2. Whereas PCV2-group 1 isolates were detected in all the diseased animals, only two of the diseased animals harbored PCV2-group 2 isolates. This observation is important because PCV2-group 1 isolates had never been reported in the United States before (GenBank as of May 16, 2006), and they are closely related to the PCV2-group 1 isolates that have been described in Europe and Asia, previously. Our analysis revealed that each genotypic group contains a distinct stretch of nucleotide or amino acid sequence that may serve as a signature motif for PCV2-group 1 or PCV2-group 2 isolates.

Kinetics of porcine circovirus type 2 replication

Summary. The kinetics of porcine circovirus type 2 (PCV2) replication in PK15 cells was examined. During productive infection, viral antigens, RNA transcripts and progeny viruses all increased in a time dependent manner. Viral antigens were observed in a few cells at 18 hour postinfection (h p.i.) and cell-free progeny viruses began to appear at about 30 h p.i. Viral transcripts were detected by 18 h p.i. and the capsid protein RNA of 950 nucleotides (nt) was the most abundant RNA species. Two other RNAs of sizes 750 and 450 nt, derived from the predicted replication associated protein (Rep) gene region, were also detected. These two RNAs share 3′ common nucleotide sequences and they are transcribed in the same orientation as the proposed unspliced Rep RNA or the recently described Rep’ RNA. The 35 kD capsid protein was observed at 30 h p.i. by Western blot analysis and it appeared to be the most immunodominant protein in swine exposed to PCV2. The capsid proteins of PCV type 1 and PCV2 each contain a nuclear localization signal sequence capable of targeting a reporter protein to the nucleoli of transfected cells when the capsid proteins were expressed as 3′ fusion polypeptides. Although previous reports indicated that PCV2 capsid proteins localized predominantly in the nuclei of infected cells, we observed an abundant amount of PCV2 capsid proteins in the cytoplasm of many cells of the infected cultures. The cells that exhibited cytoplasmic capsid proteins also contained virus nucleic acids, indicating that these proteins were synthesized by the infected cells and not through uptake from the culture medium. Elucidation of the changes that affect the localization pattern of PCV2 capsid proteins, nuclear versus cytoplasmic, requires further investigation.

Identification and molecular cloning of a novel porcine parvovirus

A novel porcine parvovirus, PPV4, was identified in the lung lavage of a diseased pig coinfected with porcine circovirus type 2. This virus exhibits limited similarity to its closest relative, bovine parvovirus 2, but resembles viruses of the genus Bocavirus (bovine parvovirus, canine minute virus and human bocavirus) that encode an additional ORF3. The ORF3 of PPV4 is predicted to encode a protein of 204 amino acid residues, which is similar in size to the ORF3-encoded proteins of the bocaviruses. Whereas the ORF3-encoded proteins of bocaviruses share significant similarity with each other, the PPV4 ORF3 encoded protein does not exhibit homology with any protein in the GenBank non-redundant database.

Homologous recombination within the capsid gene of porcine circovirus type 2 subgroup viruses via natural co-infection.

Several studies had reported homologous recombination between two porcine circovirus (PCV) type 2 subgroup viruses, PCV2a and PCV2b. The recombination events described thus far mapped either within the Rep gene sequences or the sequences flanking the Rep gene region. Previously, the presence of both PCV2a and PCV2b DNA sequences in tissues of the same infected swine from the 2005 United States PCV-associated disease outbreak was reported, which indicates that the animal was co-infected with both PCV2 subgroup viruses. Here, two naturally occurring chimeric genomes were identified that exhibited homologous recombination within the capsid gene sequences, and infectious viruses were recovered from both chimeric genomes after transfection into tissue culture cells.

Detection of a novel porcine parvovirus, PPV4, in chinese swine herds

To determine whether the novel porcine parvovirus type 4 (PPV4) recently reported in America is prevalent in China, a set of specific primers was designed and used for molecular survey of PPV4 among the clinical samples collected from various provinces of China between 2006 and 2010. The results showed that PPV4 is present in Chinese swine herds at a rate of 2.09% (12/573) among the clinical samples examined and 0.76% (1/132) among the samples taken from healthy animals. We also noted that PPV4 was not detected in samples taken prior to 2009. Analysis of the coding sequences showed that the Chinese and American PPV4 genome sequences are closely related with greater than 99% nucleotide sequence identity. Similar to a previous study, viral genomes in head-to-tail configuration of various lengths of the non-coding region were detected. Our findings confirmed that PPV4 is a unique recently discovered virus in pigs. Phylogenetically, PPV4 is most closely related to bovine parvovirus 2 (BPV2, which is not a Bocavirus and is not assigned to any Parvovirinae genus) and shares limited ORF1 (33.6%) and ORF2 (24.5%) amino acid identity. With respect to genome structure and organization, PPV4 encodes an ORF3 in the middle of the viral genome that resembles the Bocavirus genus. However, the PPV4 ORF3 encoded protein shares minimal amino acid identity with the ORF3 encoded proteins of the Bocavirus genus.

Identification of the essential and non-essential transcription units for protein synthesis, DNA replication and infectious virus production of Porcine circovirus type 1

Summary.A plasmid-based transfection system capable of yielding infectious Porcine circovirus type 1 (PCV1) was established and mutational analysis was conducted to investigate the involvement of each viral transcription unit in protein synthesis, DNA replication and progeny virus production. During PCV1 replication in PK15 cells, twelve viral-specific RNAs are synthesized. They include the capsid protein RNA (CR), eight Rep-associated RNAs (Rep, Rep’, Rep3a, Rep3b, Rep3c-1, Rep3c-2, Rep3c-3 and Rep3c-4), and three NS-associated RNAs (NS462, NS642 and NS0). A stop codon introduced at the 5’-end of CR did not affect Rep-associated antigens or viral DNA synthesis. Altering the consensus dinucleotide at the splice junctions of the Rep3 RNAs and NS462 or introducing an early termination codon in Rep3c-4 and NS0 also did not have any affect on virus replication. However, mutations in Rep and Rep’ caused greater than 99% reduction of protein synthesis and complete shut down of viral DNA replication. NS642 could not be assayed in this study because silent mutation at the splice junction was not possible. However, it is probably equivalent to the non-essential RNA (NS672) of PCV type 2. Thus, only two proteins, Rep and Rep’, are essential for PCV1 protein, DNA and infectious virus biosynthesis.

A divergent clade of circular single-stranded DNA viruses from pig feces.

Using metagenomics and molecular cloning methods, we characterized five novel small, circular viral genomes from pig feces that are distantly related to chimpanzee and porcine stool-associated circular viruses, (ChiSCV and PoSCV1). Phylogenetic analysis placed these viruses into a highly divergent clade of this rapidly growing new viral family. This new clade of viruses, provisionally named porcine stool-associated circular virus 2 and 3 (PoSCV2 and PoSCV3), encodes a stem–loop structure (presumably the origin of DNA replication) in the small intergenic region and a replication initiator protein commonly found in other biological systems that replicate their genomes via the rolling–circle mechanism. Furthermore, these viruses also exhibit three additional overlapping open reading frames in the large intergenic region between the capsid and replication initiator protein genes.

Nucleotide sequence of coronavirus TGEV genomic RNA: evidence for 3 mRNA species between the peplomer and matrix protein genes

Abstract The region of the TGEV genome between the E1-matrix protein gene and the E2-peplomer protein gene has been sequenced from a cDNA clone. The consensus recognition sequence, 5 ′AA TT CTAAAC was found upstream from 3 large open reading frames. In coronaviruses these homologous recognition sequences are involved in the initiation of transcription suggesting that there are 3 mRNA species in this region of the TGEV genome. Northern blot analysis and nuclease S1 mapping confirmed the presence of 3 mRNA species between mRNA 3 encoding the E2-peplomer protein and mRNA 6 encoding the E1-matrix protein. The 5′ regions of these 3 mRNAs encode potential polypeptides of predicted molecular weight; 7859, 27744 and 9287, respectively. The potential translation product of ORF B (27744 Da) is considerably larger than previously reported and could be difficult to distinguish by size from the E1-matrix protein.

Diversity of viruses detected by deep sequencing in pigs from a common background

Although advances in nucleic acid sequencing have enabled the discovery of many infectious agents, challenges remain for scientists and veterinary diagnosticians trying to design animal studies with a minimum of variables and to interpret laboratory results. To evaluate pyrosequencing technology as a potential screening method to estimate the virome in pigs, fecal samples were collected from 4 pigs out of a group of 175 that had been raised together since birth. A number of viruses were detected, demonstrating the application of this technology to determine the background “noise” in the pigs. However, pyrosequencing also demonstrated the diversity of viruses within a group of animals and how that can confound experimental design and obscure a definitive diagnosis.

Identification of an amino acid domain encoded by the capsid gene of porcine circovirus type 2 that modulates intracellular viral protein distribution during replication

Previous work showed that distinct amino acid motifs are encoded by the Rep, Cap and ORF3 genes of two subgroups of porcine circoviruses (PCV), PCV2a and PCV2b. At a specific location of the gene, a certain amino acid residue or sequence is preferred. Specifically, two amino acid domains located in the capsid protein, designated motif-1 (six residues located at positions 86-91) and motif-2 (four residues at positions 190-191-206-210), have been identified to associate with either PCV2a (motif-1a:T(N)KI(S)I and motif-2a:SRKD) or PCV2b (motif-1b:S(N)PR(S)V and motif-2b:AGIE) preferentially. In this study, the protein distribution pattern of a PCV2a isolate and a PCV2b isolate was examined. Each virus exhibited a different viral protein pattern during replication in porcine kidney cells and the viral protein distribution pattern was associated with amino acid motif-2 but not motif-1. The results also showed that a more robust accumulation of viral proteins in the nucleus was associated with motif-2b than with motif-2a. In addition, viruses containing motif-2b replicated better than viruses containing motif-2a in porcine kidney cells.