Application of Enzyme-Linked Immunosorbent Assay for the Seroepizootiological Survey of Antibodies against Porcine Cytomegalovirus.

Tajima, Tomoko; Hironao, Takeshi; Kajikawa, Taketsugu; Kawamura, Hikaru

doi:10.1292/jvms.55.421

Cited by 20 publications

(21 citation statements)

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“…Human cytomegalovirus (HCMV) has a variety of direct and indirect effects in the allograft recipient (6). Given the high frequency of seropositivity of swine for PCMV and the efficiency of transmission of HCMV from donor to transplant recipient (6), PCMV may represent another potential risk to humans.At present the laboratory detection of PCMV is carried out by serology or qualitative PCR (8,13,14). We now describe the development of a quantitative-competitive PCR (QC-PCR) assay and its application to the measurement of viral load in porcine tissues.…”

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Quantitation of Porcine Cytomegalovirus in Pig Tissues by PCR

et al. 2001

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A quantitative-competitive PCR for the quantification of porcine cytomegalovirus (PCMV) was developed. The virus was detected in a variety of pig organs (including potential xenotransplant donations), with viral loads ranging from <10 to 97 genome copies/g of DNA. This assay will have significant utility for studying the activation and replication of PCMV and in swine models for allo-and xenotransplantation.Porcine cytomegalovirus (PCMV) is a betaherpesvirus which causes generalized infection in newborn piglets. It has a worldwide distribution and is present in 90% of herds in the United Kingdom, many of which are from well-managed, high-healthstatus farms (3). Epidemiological features of the virus include evidence of latency and crossplacental infection. Xenotransplantation has the potential to relieve the current donor organ shortage associated with human allotransplantation, and for both practical and ethical reasons, the pig has become the most likely source of such tissues. As clinical trials of xenotransplantation are beginning, concerns have been raised about the potential transmission of porcine organisms to immunocompromised human recipients of porcine tissues (1, 4, 5). As with allotransplantation, there are potential risks associated with the transmission of porcine viruses through xenograft tissues. Although transmission to humans has not been demonstrated in vivo, porcine endogenous retroviruses have been shown to replicate in human cells in vitro (11,15). Human cytomegalovirus (HCMV) has a variety of direct and indirect effects in the allograft recipient (6). Given the high frequency of seropositivity of swine for PCMV and the efficiency of transmission of HCMV from donor to transplant recipient (6), PCMV may represent another potential risk to humans.At present the laboratory detection of PCMV is carried out by serology or qualitative PCR (8,13,14). We now describe the development of a quantitative-competitive PCR (QC-PCR) assay and its application to the measurement of viral load in porcine tissues. The QC-PCR is based on the coamplification of the test sample with an internal competitor, which differs from the wild-type sequence by the presence of a restriction endonuclease site in the middle of the amplicon. To our knowledge this is the first quantitative PCR assay for the quantification of PCMV to be reported.The qualitative and QC-PCR assays amplify a region of the polymerase gene (14). Primers PCMVF1 (5Ј CCTATGTTGG CACTGATACTTGAC 3Ј) and PCMVR1 (5Ј CCCTGAAAA TCACCGTCTGAGAGA 3Ј) were initially used to amplify a 236-bp region of the gene from tissue culture supernatant of porcine alveolar macrophages infected with PCMV strain B6. The amplicon was then cloned into the pGEM-T Easy vector (Promega). PCR-mediated site-directed mutagenesis, similar to the method described by Fox et al. (7), was used to create a SmaI restriction endonuclease site in the middle of the PCMV amplicon sequence by altering two nucleotides. Two complementary mutagenic primers were used in two separate PCRs; primers PCMVF1...

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“…At present the laboratory detection of PCMV is carried out by serology or qualitative PCR (8,13,14). We now describe the development of a quantitative-competitive PCR (QC-PCR) assay and its application to the measurement of viral load in porcine tissues.…”

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Quantitation of Porcine Cytomegalovirus in Pig Tissues by PCR

et al. 2001

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“…In the United Kingdom, 50% of the herds were reported as infected, while the seroprevalence in Japan and the Netherlands was extremely high, with more than 99% and 93% of tested pigs positive, respectively. Prevalence of PCMV among Canadian pigs was reported to be 59% by PCR [8,[10][11][12][13].…”

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Molecular survey of Cytomegalovirus shedding profile in commercial pig herds in Brazil

Dutra

Moreno

Amigo

et al. 2016

J Infect Dev Ctries

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Introduction: Porcine cytomegalovirus (PCMV) causes rhinitis in both young and older pigs. The present study describes the detection and characterization of shedding profiles of PCMV in nine farrow-to-finish Brazilian swine herds. Methodology: Tonsil swabs from sows, nursery and grow-finish pigs of nine farrow-to-finish commercial herds (n = 756) were evaluated for the presence of PCMV by PCR. Results: The virus was detected in all herds. Positive samples were concentrated in piglets of ages varying from 40 to 60 days (nursery phase), while none of the sows were positive for PCMV detection. Conclusions: These findings corroborate the literature regarding PCMV worldwide distribution, and introduce the first report of PCMV shedding profile in Brazilian pig farms.

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“…The traditional method for diagnosing PCMV infection involves virus-isolation and detection of virus-antigen or specific antibodies by indirect immunofluorescence test or enzyme-linked immunosorbent assay [1,22,23]. PCMV can be isolated from affected animals by sampling the nasal mucosa, lung and kidney, and by making direct cultures of lung macrophages if possible.…”

Section: Discussionmentioning

confidence: 99%

Identification of the Porcine Cytomegalovirus Major Capsid Protein Gene.

Rupasinghe

Iwatsuki‐Horimoto²,

Sugii

et al. 2001

The Journal of Veterinary Medical Science

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ABSTRACT. A major capsid protein (MCP) gene homologue of porcine cytomegalovirus (PCMV) was identified. Sequence analysis indicated that the PCMV MCP gene is 4,026 nucleotides in length encoding a protein of 1,341 amino acid residues. The predicted molecular weight of the PCMV MCP is 151,456 Da, equivalent to those of other herpesvirus MCP counterparts. Phylogenetic analysis using herpesviral MCP gene sequences confirmed that PCMV is a betaherpesvirus with higher homology with human herpesvirus-6 and -7 than human and mouse cytomegaloviruses. The serum of pig experimentally infected with PCMV did not react with bacterially expressed MCP, suggesting that the PCMV MCP may not be related to the humoral immune response in the course of PCMV infection. Also, we established polymerase chain reaction (PCR) protocols using primers corresponding to MCP gene sequences for detection of PCMV infection. The PCR protocol would be effective for the diagnosis of slow-growing PCMV infection, for which traditional methods involving virus-isolation are not useful. KEY WORDS: betaherpesvirus, major capsid protein, polymerase chain reaction, porcine cytomegalovirus.J. Vet. Med. Sci. 63(6): 609-618, 2001 Porcine cyomegalovirus (PCMV), first described in 1955 [6], usually induces a silent infection in adult pigs but often a fatal, generalized infection in piglets. In utero infection in sows can cross the placenta and infect the fetuses leading to fetal death or birth of weak piglets [3,17]. As PCMV exhibits a relatively protracted replication cycle, a slowly developing cytopathology characterized by cytomegaly, and a restricted host range, it is grouped into the subfamily Betaherpesvirinae [18]. Recently, we and other research groups confirmed that PCMV is a betaherpesvirus based on sequence analyses of DNA polymerase gene fragments [19,29].The major capsid protein (MCP) gene appears to be conserved among the herpesviruses [5,15], suggesting that MCP should play essential roles in vital processes in the viral replication cycle such as assembly and maturation. Herpes simplex virus-1 (HSV-1) has been shown to encode seven capsid proteins. Of these, MCP is the structural subunit of the hexons and pentons, and is the principal structural element making up the matrix of the icosahedral capsid [16]. In addition, in some herpesviruses such as HSV-1 and varicella-zoster virus (VZV), MCPs serve as an antigen for the humoral immune response during the course of infection, being highly reactive with human antisera as well as being responsible for cell-mediated immunity [7,20,27].The MCPs of many herpesviruses have been shown to have similar molecular masses, e.g., between 135 kDa and 160 kDa for human herpesviruses [2,4,10,14,15]. In this study, we identified the MCP gene of PCMV and expressed the protein in a bacterial expression system. In addition, we also analyzed the evolutionary position of PCMV in the herpesvirus group using MCP sequence information. We also established a polymerase chain reaction (PCR) protocol for specific detection ...

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Application of Enzyme-Linked Immunosorbent Assay for the Seroepizootiological Survey of Antibodies against Porcine Cytomegalovirus.

Cited by 20 publications

References 10 publications

Quantitation of Porcine Cytomegalovirus in Pig Tissues by PCR

Quantitation of Porcine Cytomegalovirus in Pig Tissues by PCR

Molecular survey of Cytomegalovirus shedding profile in commercial pig herds in Brazil

Identification of the Porcine Cytomegalovirus Major Capsid Protein Gene.

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