Natural polymorphisms and resistance-associated mutations in the fusion protein of respiratory syncytial virus (RSV): effects on RSV susceptibility to palivizumab.

Specific mutations in respiratory syncytial virus (RSV) fusion protein can cause palivizumab resistance. We assessed the incidence of sequence polymorphisms and palivizumab resistance in clinical RSV isolates collected from immunoprophylaxis-naive subjects. Polymorphisms were identified at low frequency, and only polymorphic mutations in antigenic site A (<1% of all polymorphisms) conferred palivizumab resistance.

Analysis of respiratory syncytial virus preclinical and clinical variants resistant to neutralization by monoclonal antibodies palivizumab and/or motavizumab.

BACKGROUND: Palivizumab is a US Food and Drug Administration-approved monoclonal antibody for the prevention of respiratory syncytial virus (RSV) lower respiratory disease in high-risk infants. Motavizumab, derived from palivizumab with enhanced antiviral activity, has recently been tested in humans. Although palivizumab escape mutants have been generated in the laboratory, the development of resistant RSV in patients receiving palivizumab has not been reported previously. METHODS: We generated palivizumab and motavizumab escape mutants in vitro and examined the development of resistant mutants in RSV-breakthrough patients receiving immunoprophylaxis. The effect of these mutations on neutralization by palivizumab and motavizumab and in vitro fitness was studied. RESULTS: Antibody-resistant RSV variants selected in vitro had mutations at position 272 of the fusion protein, from lysine to asparagine, methionine, threonine, glutamine, or glutamate. Variants containing mutations at positions 272 and 275 were detected in breakthrough patients. All these variants were resistant to palivizumab, but only the glutamate variant at position 272 demonstrated resistance to motavizumab. Mixtures of wild-type and variant RSV soon lost the resistant phenotype in the absence of selection. CONCLUSIONS: Resistant RSV variants were detected in a small subset (∼ 5%) of RSV breakthrough cases. The fitness of these variants was impaired, compared to wild-type RSV.

An STR forensic typing system for genetic individualization of domestic cat (Felis catus) samples.

A forensic genotyping panel of 11 tetranucleotide STR loci from the domestic cat was characterized and evaluated for genetic individualization of cat tissues. We first examined 49 candidate STR loci and their frequency assessment in domestic cat populations. The STR loci (3-4 base pair repeat motifs), mapped in the cat genome relative to 579 coding loci and 255 STR loci, are well distributed across the 18 feline autosomes. All loci exhibit Mendelian inheritance in a multi-generation pedigree. Eleven loci that were unlinked and were highly heterozygous in cat breeds were selected for a forensic panel. Heterozygosity values obtained for the independent loci, ranged from 0.60-0.82, while the average cat breed heterozygosity obtained for the 11 locus panel was 0.71 (range of 0.57-0.83). A small sample set of outbred domestic cats displayed a heterozygosity of 0.86 for the 11 locus panel. The power of discrimination of the panel is moderate to high in the cat breeds examined, with an average P(m) of 3.7E-06. The panel shows good potential for genetic individualization within outbred domestic cats with a P(m) of 5.31E-08. A multiplex protocol, designed for the co-amplification of the 11 loci and a gender-identifying locus, is species specific and robust, generating a product profile with as little as 0.125 nanograms of genomic DNA.

Comprehensive panel of real-time TaqMan polymerase chain reaction assays for detection and absolute quantification of filoviruses, arenaviruses, and New World hantaviruses.

Viral hemorrhagic fever is caused by a diverse group of single-stranded, negative-sense or positive-sense RNA viruses belonging to the families Filoviridae (Ebola and Marburg), Arenaviridae (Lassa, Junin, Machupo, Sabia, and Guanarito), and Bunyaviridae (hantavirus). Disease characteristics in these families mark each with the potential to be used as a biological threat agent. Because other diseases have similar clinical symptoms, specific laboratory diagnostic tests are necessary to provide the differential diagnosis during outbreaks and for instituting acceptable quarantine procedures. We designed 48 TaqMan-based polymerase chain reaction (PCR) assays for specific and absolute quantitative detection of multiple hemorrhagic fever viruses. Forty-six assays were determined to be virus-specific, and two were designated as pan assays for Marburg virus. The limit of detection for the assays ranged from 10 to 0.001 plaque-forming units (PFU)/PCR. Although these real-time hemorrhagic fever virus assays are qualitative (presence of target), they are also quantitative (measure a single DNA/RNA target sequence in an unknown sample and express the final results as an absolute value (e.g., viral load, PFUs, or copies/mL) on the basis of concentration of standard samples and can be used in viral load, vaccine, and antiviral drug studies.

Quantitative polymerase chain reaction-based assay for estimating DNA yield extracted from domestic cat specimens.

A quantitative polymerase chain reaction (PCR) assay has been developed for the quantification of genomic DNA extracted from domestic cat samples. The assay, which targets highly repetitive genomic short interspersed nuclear elements (SINE), can be performed rapidly and is highly sensitive, detecting as little as 10 fg of feline genomic DNA. The assay was linear over a 10(6) dilution range. We have recently developed a short tandem repeat (STR) multiplex panel for forensic analysis of feline specimens. The SINE assay is an integral part of the forensic typing system. The sensitivity of the assay will enable forensic examiners to determine the likelihood of success of genotyping sample extracts with the STR panel without sacrificing valuable DNA necessary to perform genotyping of samples.