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Kay S Faaberg

Publications and source records attributed to Kay S Faaberg.

11 recordsLinked to original sources
PubMed2006

Evaluation of a rapid fecal PCR test for detection of Mycobacterium avium subsp. paratuberculosis in dairy cattle.

A high-throughput TaqMan PCR assay for detection of bovine paratuberculosis was evaluated by using fecal samples from 1,808 dairy cattle in seven naturally infected herds and 347 dairy cattle in seven herds considered free of paratuberculosis. Fecal, blood, and milk samples were submitted to laboratories where the PCR-based assay, three different fecal culture procedures for Mycobacterium avium subsp. paratuberculosis (centrifugation, sedimentation, and the BACTEC filter concentration method), two serologic enzyme-linked immunosorbent assays (ELISAs), and one milk ELISA were performed. Results from testing of dairy cattle in herds free of M. avium subsp. paratuberculosis showed that the PCR assay's specificity was 99.7%. Twenty-three percent of the dairy cows that were fecal culture positive by at least one of the three methods were positive by the PCR assay. By Bayesian non-"gold standard" analysis methods, the TaqMan PCR assay had a higher specificity than the serum ELISAs (99.3%; 95% confidence interval [CI]=98.6 to 99.7%) and a test sensitivity similar to that of the serum ELISAs (29%; 95% CI=24 to 35%). By classical methods, the estimated relative sensitivity of the fecal PCR assay was 4% for light and moderate fecal shedders (compared to 12 to 13% for the ELISAs) and 76% for heavy fecal shedders (compared to 67% for the milk ELISA). The PCR assay has higher sensitivity for detection of heavy fecal shedders than the evaluated milk ELISA but lower sensitivity than a serum or milk ELISA for detection of light and moderate fecal shedders. This assay can be used as a quick test for detection of cattle with heavy fecal shedding, those cattle with the highest risk of transmitting infection to susceptible cattle.

Animals↗
PubMed2006

Complete genome analysis of RFLP 184 isolates of porcine reproductive and respiratory syndrome virus.

Two full-length genomes of recently emerged virulent isolates of porcine reproductive and respiratory syndrome virus (PRRSV) were sequenced and compared to other PRRSV strains. The results revealed that these two isolates (named MN184), of North American lineage, represented the shortest PRRSV genomes sequenced to date with a nucleotide length of 15019 bases. Genetic analysis demonstrated that the two isolates were not identical and shared approximately 87 and 59% nucleotide identity with prototype North American strain VR-2332 and European strain Lelystad, respectively. Three quite variable regions were identified, corresponding to putative nsp1beta, putative nsp2 and ORF5. Nsp2, the most variable region, shared only 66-70% amino acid similarity to other sequenced North American-like PRRSV nsp2 proteins. Further study revealed that the nsp2 protein of the MN184 isolates contained three discontinuous deletions when compared to strain VR-2332 nsp2 protein, with the sizes of 111, 1, and 19 amino acids corresponding to strain VR-2332 positions 324-434, 486 and 505-523, respectively. The results suggest that targeted manipulation of PRRSV through nsp2 modification by reverse genetics may yield promising vectors for vaccine development, as has been recently demonstrated [Han, J., Faaberg, K.S., Wang, Y., Liu, H., 2005. Non-structural protein 2 mutants of PRRSV strain VR-2332 infectious clone based on deletions seen in RFLP184 isolates are viable. In: PRRS International Symposium Proceedings, vol. 8, Saint Louis, MO].

Amino Acid Sequence↗
PubMed2006

Neutralizing antibody responses of pigs infected with natural GP5 N-glycan mutants of porcine reproductive and respiratory syndrome virus.

In order to assess the effect of the N-glycans associated with the GP5 neutralization epitope of porcine reproductive and respiratory syndrome virus (PRRSV) on the neutralizing antibody (Ab) response of swine, groups of young pigs were infected with PRRSV strains differing in N-glycosylation pattern. The humoral immune response to strain VR-2332, harboring four potential N-glycan sites, was compared to that of two natural field isolates carrying mutations either abolishing the N-glycosylation site at position 44 (N44) or the two N-glycosylation sites in the hypervariable region upstream of the neutralization epitope (HV-1). The pigs were bled at intervals and their sera were assayed for neutralizing Abs by indirect and competition ELISAs using peptides containing the GP5 neutralization epitope, and selectively for infectivity neutralization of a number of PRRSV strains. In addition, viremia was monitored by quantitative RT-PCR, and anti-N-protein Ab formation was measured by HerdChek ELISA. The neutralizing Ab responses as measured by peptide ELISA varied greatly between individual pigs infected with each PRRSV strain. Some pigs generated high titers of peptide binding Abs between 7 and 28 days post infection (p.i.), whereas other pigs had not generated a response by 90 days p.i. However, the HV-1-infected pigs generated Abs to the neutralization epitope more rapidly and to a 5-10 times higher level than VR-2332-infected pigs, and the Abs neutralized the homologous HV-1 virus 10-20 times more efficiently than PRRSV strains VR-2332, N44, MN184, or SDSU73. In contrast, most N44-infected pigs generated neutralizing Abs only after 42 days p.i. and only to low levels. The results suggest that the deletions of the N-glycans or other amino acid substitutions in the GP5 ectodomains of the mutants affect the immunogenicity of the neutralization epitope and the specificity of the Abs raised to it but not the sensitivity of the virions to Ab neutralization.

Amino Acid Sequence↗
PubMed2004

Characterization of heteroclite subgenomic RNAs associated with PRRSV infection.

In this study, porcine reproductive and respiratory syndrome virus (PRRSV) heteroclite (uncommon forms) RNAs were characterized. Nucleotide sequencing of 11 additional defective RNA species verified that heteroclites are formed between the 5' and 3' termini of PRRSV at short stretches of identity, with variability seen between the junction sites utilized. Northern blot and RT-PCR analyses indicated that heteroclite RNA species were likely to be packaged into purified virions. To study whether heteroclite RNAs and viral genomic RNAs could be packaged into the same virions, PRRSV strain VR-2332 was purified by sucrose density gradient centrifugation. RT-PCR amplification of the viral RNAs isolated from three distinct gradient bands, using genomic- and heteroclite-specific primer pairs, demonstrated that heteroclite RNAs could not be readily dissociated from genomic RNA. Partial segregation of full-length and larger heteroclite genomes to the upper two gradient bands was seen, but smaller species could be found in all three fractions. These results strongly suggest that heteroclite RNAs retain the PRRSV RNA packaging signal. In vitro transcription and translation of one heteroclite cDNA clone verified that the RNA could express a predicted 32.6 kDa protein, indicating that these RNA species have the potential to produce abnormal proteins in infected cells.

Amino Acid Sequence↗
PubMed2004

Splice junction map of simian parvovirus transcripts.

The transcription map of simian parvovirus (SPV), an Erythrovirus similar to Parvovirus B19, was investigated. RNA was extracted from tissues of experimentally infected cynomolgus macaques and subjected to reverse transcription-PCR with SPV-specific primers. The PCR products were cloned and sequenced to identify splice junctions. A total of 14 distinct sequences were identified as putative partial transcripts. Of these, 13 were spliced; a single unspliced transcript putatively encoded NS1. Sequence analysis revealed that spliced partial transcripts may encode portions of open reading frames for the major capsid proteins VP1 and VP2 and smaller, unknown proteins. These unspliced and spliced transcripts and putative proteins encoded by SPV were similar to those of B19. Initial splice junctions at nucleotides 279 and 333 were analogous to those at nucleotides 406 and 441, respectively, in B19. Seven of the 10 splices identified had typical GT/AG donor/acceptor junctions. The splice sites were confirmed by Northern blotting and autoradiography. In contrast to B19, which has a maximum of two splices per transcript, up to three splices were observed in SPV transcripts. A spliced transcript putatively encoding a truncated version of NS1, as seen with minute virus of mice and adeno-associated virus 2, was also observed. The findings indicate that that the splicing pattern of transcripts of SPV and B19 is similar, but SPV also has coding strategies in common with other parvoviruses.

Animals↗
PubMed2004

Characterization of emerging European-like porcine reproductive and respiratory syndrome virus isolates in the United States.

European-like field isolates of porcine reproductive and respiratory syndrome virus (PRRSV) have recently emerged in North America. The full-length genomic sequence of an index isolate characterized in 1999, strain EuroPRRSV, served as the reference strain for further studies of the evolution and epidemiology of European-like isolates (type 1) in the United States. Strain EuroPRRSV shared 90.1 to 100% amino acid identity with the prototype European strain, Lelystad, within the structural and nonstructural open reading frames (ORFs) and 95.3% overall nucleotide identity. The 5' untranslated region and two nonstructural regions within ORF 1 were closely examined due to significant divergence from strain Lelystad. A 51-bp deletion in a region within ORF 1a, coding for nonstructural protein 2 (NSP2), was observed. Sequence analysis of the structural ORFs 2 to 7 of additional European-like isolates indicated that these isolates share 93% nucleotide identity with one another and 95 to 96% identity with the Lelystad strain but only 70% identity with the North American reference strain VR-2332. Phylogenetic analysis with published PRRSV ORF 3, 5, and 7 nucleotide sequences indicated that these newly emerging isolates form a clade with the Lelystad and United Kingdom PRRSV isolates. Detailed analysis of four of these isolates with a panel of 60 monoclonal antibodies directed against the structural proteins confirmed a recognition pattern that was more consistent with strain Lelystad than with other North American isolates.

Amino Acid Sequence↗
PubMed2002

Functional properties of the predicted helicase of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the positive-strand RNA virus family Arteriviridae. Although considerable research has focused on this important pathogen, little is known about the function of most PRRSV proteins. To examine characteristics of putative nonstructural proteins (nsp) encoded in ORF1b, which have been identified by nucleotide similarity to domains of equine arteritis virus, defined genomic regions were cloned and expressed in the pRSET expression system. One region, nsp10, encoded a protein with a putative helicase domain and was further examined for functional helicase-like activities. PRRSV nsp10 was found to possess a thermolabile and pH-sensitive NTPase activity that was modulated by polynucleotides and to unwind dsRNA in a 5' to 3' polarity. These results provide the first evidence of the functional properties of PRRSV helicase and further support the finding that nidovirus helicases possess properties that distinguish them from other viral helicases.

3' Untranslated Regions↗
PubMed2002

Detection of bovine viral diarrhea virus by TaqMan reverse transcription polymerase chain reaction.

Detection and elimination of calves and cows persistently infected with bovine viral diarrhea virus (BVDV) is important for the control of this pathogen. Historically, BVDV detection involved cell culture isolation followed by virus detection through immunofluorescence or immunoperoxidase monolayer assay (IPMA) methods. More recently, immunohistochemistry (IHC) has been added as a routine test for BVDV detection. The detection of BVDV by gel-based reverse transcription polymerase chain reaction (RT-PCR) is more sensitive and rapid than by cell culture isolation, but test results can be compromised by sample contamination during nucleic acid amplification. This study was designed to develop a closed-tube format of BVDV nucleic acid amplification and detection, TaqMan RT-PCR. The results of this new technique were compared with those obtained with virus isolation, IPMA, and IHC. With TaqMan RT-PCR, BVDV was detected in many samples negative by IPMA, IHC, and virus isolation with the exception of 1 sample that was positive by IHC. TaqMan RT-PCR in a closed-tube format offers a rapid, economical, high volume, and sensitive method for BVDV detection without the concerns of amplified cDNA product contamination associated with open-tube gel-based PCR tests.

Animals↗