The comparative analysis of complete genome sequences from two South African betabaculoviruses: Phthorimaea operculella granulovirus and Plutella xylostella granulovirus.

The complete genomes of two novel South African betabaculovirus isolates, namely Phthorimaea operculella granulovirus (PhopGV-SA) and Plutella xylostella granulovirus (PlxyGV-SA), were sequenced and compared to the respective reference isolates PhopGV-1346 and PlxyGV-K1. For both isolates, the genome size and guanine-cytosine (GC) content were similar to those of the respective reference genomes. However, numerous-single nucleotide polymorphisms (SNPs) and several insertions/deletions were observed, revealing the novelty of the isolates. Focus was placed on analysing the observed insertion/deletion events by conducting amino acid sequence alignments for all ORFs of each isolate against all respective ORFs in the corresponding reference isolate. Certain ORFs in each granulovirus genome contained significant insertion/deletion events. In addition, the PlxyGV-SA genome had single-nucleotide insertions/deletions in ORFs 38 and 49 that resulted in the extension and complete overlap of these two ORFs with the neighbouring ORFs 39 and 48, respectively. These novel isolates have significant potential for development and application as biopesticides in South Africa, and the genetic variations observed may have important implications for the biological activity and management of host resistance in the field.

Human defined antigenic region on the nucleoprotein of Crimean-Congo hemorrhagic fever virus identified using truncated proteins and a bioinformatics approach.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne viral zoonosis widely distributed in Africa, Asia and eastern Europe. In this study, amino acid sequence data for the CCHFV nucleoprotein (NP) was used to identify potential linear epitopic regions which were subsequently included in the design of large and small truncated recombinant NP antigens and peptide libraries. Two truncated recombinant CCHFV NP antigens were prepared based on results of prediction studies to include epitopic regions and exclude hydrophobic regions that could influence protein expression and solubility. Serum samples were collected from acute and convalescent patients. An IgG antibody response was detected in 16/16 samples tested using the large recombinant NP-based ELISA and in 2/16 using the small recombinant NP-based ELISA. A total of 60 peptides covering predicted epitopic regions of the NP were synthesized and peptide NRGGDENPRGPVSR at amino acid position 182-195, reacted with 13/16 human serum samples. In summary, functional assays are required to determine the biological activity of predicted epitopes for development of peptide based assays for antibody detection. Bacterially expressed complete NP antigens have previously been shown to be useful tools for antibody detection. Truncation of the antigen to remove the hydrophobic C terminus had no impact on the ability of the antigen to detect IgG antibody in human sera. The results indicate that the region from amino acids 123 to 396 includes a highly antigenic region of the NP with application in development of antibody detection assays.

Identification of bacterial pathogens in patients with endophthalmitis by 16S ribosomal DNA typing.

PURPOSE: To determine whether sequence analysis of 16S ribosomal DNA (rDNA) can be used to detect bacterial pathogens in patients with postoperative endophthalmitis. METHODS: In 10 eyes of 10 patients, vitreous specimens were collected for culture and rDNA typing. Variable segments of each ribosomal DNA specimen were amplified by polymerase chain reaction (PCR), sequenced, and aligned by BLAST, a computer alignment program, against sequences in GenBank at the National Institutes of Health. RESULTS: Specimens were available from five eyes with bacterial endophthalmitis diagnosed by Gram stain or culture. Amplified 16s rDNA sequences from the eyes of three patients were identical to microbiologic results. Polymerase chain reaction results were negative in two cases in which unusual organisms were detected. All five control specimens from patients with nonbacterial endophthalmitis or uveitis were PCR negative. Approximately 48 to 72 hours are required under ideal conditions for final species identification with this ribosomal typing technique. CONCLUSIONS: 16S rDNA typing shows potential as a relatively rapid technique for identifying bacteria in vitreous samples.

16S ribosomal DNA typing for identification of pathogens in patients with bacterial keratitis.

The identification of pathogens in patients with bacterial keratitis remains problematic because standard diagnostic tests are negative for 40 to 60% of patients. A cross-sectional study was undertaken to determine if PCR and sequence analysis of 16S ribosomal DNA (rDNA) could be used to detect bacterial pathogens in patients with keratitis. Corneal specimens were collected for culture and rDNA typing. Variable segments of each rDNA specimen were amplified by PCR, sequenced, and aligned with the sequences in GenBank. Eleven patients had microbiologically documented bacterial keratitis, while 17 patients had keratitis due to other causes. Nine (82%) of 11 bacterial keratitis patients were PCR positive; each sequencing result matched the culture results. Seventeen (100%) patients with nonbacterial keratitis were PCR negative. Our data suggest that 16S rDNA typing holds promise as a rapid alternative to culture for identifying pathogens in patients with bacterial keratitis.

Vitreitis and Waldenström's macroglobulinemia.

PURPOSE: To describe vitreitis in a patient with Waldenström's macroglobulinemia. METHODS: Case report and review of pertinent literature. RESULTS: A 90-year-old man developed vitreitis 10 years after a systemic diagnosis of a lymphoproliferative disorder. Numerous small, normal-appearing lymphocytes were seen on pathologic examination of the vitreous. He developed worsening lymphadenopathy and was diagnosed with Waldenström's macroglobulinemia after systemic review. CONCLUSION: Chronic lymphoproliferative diseases such as Waldenstrom's macroglobulinemia may cause vitreitis.

Polymerase chain reaction-based assays of vitreous samples for the diagnosis of viral retinitis. Use in diagnostic dilemmas.

OBJECTIVE: This study aimed to review the authors' results using polymerase chain reaction (PCR)-based assays for the diagnosis of viral retinitis. DESIGN: The study design was a retrospective case series. PARTICIPANTS: Thirty-seven patients (38 eyes) with active retinitis from whom vitreous biopsy specimens were received in the authors' laboratory for diagnostic evaluation. INTERVENTION: Vitreous biopsy specimens were evaluated with previously described PCR-based assays for cytomegalovirus (CMV), varicella zoster virus (VZV), and herpes simplex virus (HSV) DNA; clinical histories were reviewed. MAIN OUTCOME MEASURES: Laboratory findings and clinical course were measured. RESULTS: The results of the authors' assays were consistent with the long-term clinical course of each patient. Cytomegalovirus, VZV, or HSV DNA was detected in the vitreous from 24 patients. Cytomegalovirus DNA was detected in vitreous biopsy specimens from 10 patients (11 eyes). Nine patients (ten eyes) with acquired immune deficiency syndrome ultimately were diagnosed with CMV retinitis as they were followed clinically over time. Varicella zoster virus DNA was detected in vitreous biopsy specimens from eight patients; seven adult patients were ultimately diagnosed with acute retinal necrosis or progressive outer retinal necrosis. Herpes simplex virus DNA was detected in vitreous biopsy specimens from six patients; five patients had previous or subsequent herpes encephalitis. No viral DNA was detected in the vitreous from 13 patients; all were ultimately diagnosed with toxoplasmosis, syphilis, Behcet disease, fungal endophthalmitis, or idiopathic inflammation. CONCLUSIONS: These data further support the use of PCR-based assays of vitreous specimens in the diagnostic evaluation of patients with infectious retinitis.

Nocardia scleritis.

PURPOSE: To describe a case of Nocardia scleritis, an unusual ocular infection. METHODS: Case report and review of pertinent literature. RESULTS: An 83-year-old man with leukocytoclastic vasculitis was initially examined for infectious necrotizing scleritis after explantation of an extruded scleral buckle. The patient was successfully treated with sulfonamides. CONCLUSIONS: Nocardia asteroides may cause infectious scleritis in the absence of cataract surgery or trauma. Treatment with sulfonamides can result in a satisfactory outcome.