Infection studies on human cell lines with porcine circovirus type 1 and porcine circovirus type 2

Circoviruses lack an autonomous DNA polymerase and are dependent on the replication machinery of the host cell for de novo DNA synthesis. Accordingly, the viral DNA needs to cross both the plasma membrane and the nuclear envelope before replication can occur. Here we report on the subcellular distribution of the beak and feather disease virus (BFDV) capsid protein (CP) and replication-associated protein (Rep) expressed via recombinant baculoviruses in an insect cell system and test the hypothesis that the CP is responsible for transporting the viral genome, as well as Rep, across the nuclear envelope. The intracellular localization of the BFDV CP was found to be directed by three partially overlapping bipartite nuclear localization signals (NLSs) situated between residues 16 and 56 at the N terminus of the protein. Moreover, a DNA binding region was also mapped to the N terminus of the protein and falls within the region containing the three putative NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome. Interestingly, whereas Rep expressed on its own in insect cells is restricted to the cytoplasm, coexpression with CP alters the subcellular localization of Rep to the nucleus, strongly suggesting that an interaction with CP facilitates movement of Rep into the nucleus.Circoviruses are animal viruses that have small (ϳ2-kb), covalently closed, circular, single-stranded DNA (ssDNA) genomes encapsidated within nonenveloped icosahedral virions (8). Members of the family Circoviridae are divided into genera based on their specific genome organization and host range. Porcine circovirus type 1 (PCV1), Porcine circovirus type 2 (PCV2) (12), and Beak and feather disease virus (BFDV) are the only formally recognized members of the genus Circovirus. In recent years, a number of novel circovirus-like viruses have been identified in nonpsittacine avian species. These include Columbid circovirus (34), Goose circovirus (34), Canary circovirus (29), and Duck circovirus (14), which have all been tentatively classified as members of the genus. All of these viruses possess ambisense genomes with two major open reading frames (ORFs) carried on opposite strands of the replicative double-stranded DNA (dsDNA) intermediate (33). These encode the replication-associated protein (Rep) and capsid protein (CP) from the virion and complementary strands, respectively (25, 27) (23, 26).Circoviruses are dependent on the replication machinery of the host cell for de novo DNA synthesis (26). Although Rep is required to initialize viral replication, continuation of the process is dependent upon cellular enzymes expressed during S phase and commences only after the host cell has passed through mitosis (32). Since DNA synthesis occurs exclusively in the nucleus, the viral DNA needs to cross both the plasma membrane and the nuclear envelope before a productive infection can be established. The strict size limitations associated w...

Section: Discussionmentioning

confidence: 74%

The Capsid Protein of Beak and Feather Disease Virus Binds to the Viral DNA and Is Responsible for Transporting the Replication-Associated Protein into the Nucleus

Heath

Williamson

Rybicki

2006

“…In this study, the results of screening plasma samples from 96 U.S. blood donors and 113 Central African bush hunters via pan-Rep PCR were also negative (Table 1). A study showed that viral protein expression, cytopathic effect, and DNA persistence occurs in human cell lines infected with PCV2, but the virus could not be passed to new cultures (14). One study reported the presence of PCV-reactive antibodies, although with somewhat distinctive properties in sera of humans, cows, and mice (36), while another reported the lack of PCV antibodies in cows and horses (8).…”

Section: Discussionmentioning

confidence: 99%

Multiple Diverse Circoviruses Infect Farm Animals and Are Commonly Found in Human and Chimpanzee Feces

Kapoor

Slikas

et al. 2010

335

399

Circoviruses are known to infect birds and pigs and can cause a wide range of severe symptoms with significant economic impact. Using viral metagenomics, we identified circovirus-like DNA sequences and characterized 15 circular viral DNA genomes in stool samples from humans in Pakistan, Nigeria, Tunisia, and the United States and from wild chimpanzees. Distinct genomic features and phylogenetic analysis indicate that some viral genomes were part of a previously unrecognized genus in the Circoviridae family we tentatively named "Cyclovirus" whose genetic diversity is comparable to that of all the known species in the Circovirus genus. Circoviridae detection in the stools of U.S. adults was limited to porcine circoviruses which were also found in most U.S. pork products. To determine whether the divergent cycloviruses found in non-U.S. human stools were of dietary origin, we genetically compared them to the cycloviruses in muscle tissue samples of commonly eaten farm animals in Pakistan and Nigeria. Limited genetic overlap between cycloviruses in human stool samples and local cow, goat, sheep, camel, and chicken meat samples indicated that the majority of the 25 Cyclovirus species identified might be human viruses. We show that the genetic diversity of small circular DNA viral genomes in various mammals, including humans, is significantly larger than previously recognized, and frequent exposure through meat consumption and contact with animal or human feces provides ample opportunities for cyclovirus transmission. Determining the role of cycloviruses, found in 7 to 17% of non-U.S. human stools and 3 to 55% of non-U.S. meat samples tested, in both human and animal diseases is now facilitated by knowledge of their genomes.

“…A single human gut biopsy specimen has been reported to contain PCV2 DNA, but contamination from human feces containing PCV2 from consumed pork cannot be excluded (6). A large PCR screen of human plasma and tissues did not detect any PCV2 DNA, and inoculations of various human cell lines with PCV2 were nonproductive (12,13). Serological testing for PCV exposure has yielded ambivalent results, with one group reporting a high rate of human seroreactivity to PCV1, although with an altered binding profile relative to pig serum samples, indicating the possible presence of a related but distinct virus (33).…”

Section: Vol 84 2010 Adventitious Virus In Live-attenuated Vaccine mentioning

confidence: 99%

Viral Nucleic Acids in Live-Attenuated Vaccines: Detection of Minority Variants and an Adventitious Virus

Victoria

Wang²,

Jones

et al. 2010

303

218

Metagenomics and a panmicrobial microarray were used to examine eight live-attenuated viral vaccines. Viral nucleic acids in trivalent oral poliovirus (OPV), rubella, measles, yellow fever, varicella-zoster, multivalent measles/mumps/rubella, and two rotavirus live vaccines were partially purified, randomly amplified, and pyrosequenced. Over half a million sequence reads were generated covering from 20 to 99% of the attenuated viral genomes at depths reaching up to 8,000 reads per nucleotides. Mutations and minority variants, relative to vaccine strains, not known to affect attenuation were detected in OPV, mumps virus, and varicella-zoster virus. The anticipated detection of endogenous retroviral sequences from the producer avian and primate cells was confirmed. Avian leukosis virus (ALV), previously shown to be noninfectious for humans, was present as RNA in viral particles, while simian retrovirus (SRV) was present as genetically defective DNA. Rotarix, an orally administered rotavirus vaccine, contained porcine circovirus-1 (PCV1), a highly prevalent nonpathogenic pig virus, which has not been shown to be infectious in humans. Hybridization of vaccine nucleic acids to a panmicrobial microarray confirmed the presence of endogenous retroviral and PCV1 nucleic acids. Deep sequencing and microarrays can therefore detect attenuated virus sequence changes, minority variants, and adventitious viruses and help maintain the current safety record of live-attenuated viral vaccines.