Charles R. Vossbrinck

Host Feeding Patterns of Culex Mosquitoes and West Nile Virus Transmission, Northeastern United States

Culex salinarius is a bridge vector to humans, while Cx. pipiens and Cx. restuans are more efficient enzootic vectors.

Identification and characterization of three Encephalitozoon cuniculi strains

Microsporidia are increasingly recognized as causing opportunistic infections in immunocompromised individuals. Encephalitozoon cuniculi is probably the most studied mammalian microsporidian that infects insects and mammals, including man. In this study, 8 E. cuniculi isolates were compared and were found to fall into 3 strains. Strain type I includes the rabbit type isolate, as well as isolates from an additional rabbit, a dwarf rabbit, and a mouse. Strain type II includes 2 murine isolates and strain type III includes 2 isolates obtained from domestic dogs. By SDS-PAGE, the 3 strains differ primarily in the molecular weight range of 54-59 kDa where strain type I displays an apparent broad singlet at 57 kDa, strain type II displays an apparent doublet at 54 and 58 kDa, and strain type III displays an apparent broad band at 59 kDa. Antigenic differences were detected in the molecular weight regions of 54-58 kDa as well as 28-40 kDa by Western blot immunodetection using murine antisera raised against E. cuniculi, Encephalitozoon hellem, and the Encephalitozoon-like Septata intestinalis. Polymerase chain reaction (PCR) products containing only small subunit rDNA sequences from the different E. cuniculi isolates formed homoduplexes whereas PCR products containing intergenic rRNA gene sequences formed heteroduplexes in mobility shift analyses. Fok I digestion of the PCR products containing the intergenic rRNA gene region resulted in unique restriction fragment length polymorphism patterns, and DNA sequencing demonstrated that in the intergenic spacer region, the sequence 5-GTTT-3 was repeated 3 times in strain type I, twice in strain type II, and 4 times in strain type III. This study indicates that there exist at least 3 E. cuniculi strains which may become important in the epidemiology of human E. cuniculi infections. Furthermore, as additional E. cuniculi isolates are characterized, these strains will be named or reclassified once the criteria for taxonomy and phylogenetic tree construction for microsporidia become better defined.

Small subunit ribosomal DNA phylogeny of various microsporidia with emphasis on AIDS related forms.

ABSTRACT. Phylogenetic analysis of the small subunit ribosomal DNA of a broad range of representative microsporidia including five species from humans (Enterocytozoon bieneusi, Nosema corneum, Septata intestinalis, Encephalitozoon hellem and Encephalitozoon cuniculi), reveals that human microsporidia are polyphyletic in origin. Septata intestinalis and E. hellem are very similar to the mammalian parasite E. cuniculi. Based on the results of our phylogenetic analysis, we suggest that S. intestinalis be designated Encephalitozoon intestinalis. Furthermore, analysis of our data indicates that N. corneum is much more closely related to the insect parasite Endoreticulatus schubergi than it is to other Nosema species. This finding is supported by recent studies which have shown a similarity between E. schubergi and N. corneum based on the origin and development of the parasitophorous vacuole. Thus these opportunistic microsporidian parasites can originate from hosts closely or distantly related to humans. Finally, the phylogeny based on small subunit ribosomal DNA sequences is highly inconsistent with traditional classifications based on morphological characters. Many of the important morphological characters (diplokaryon, sporophorous vesicle, and meiosis) appear to have multiple origins.

Ribosomal Dna Sequences of Encephalitozoon Hellem and Encephalitozoon Cuniculi: Species Identification and Phylogenetic Construction

ABSTRACT. A segment of ribosomal DNA, about 1,350 base pairs long, was amplified from the microsporidian species Encephalitozoon hellem, isolated from AIDS patients, and Encephalitozoon cuniculi. the amplified DNA segment extends from position 530 in the small ribosomal RNA subunit to position 580 in the large ribosomal RNA subunit. A comparison of sequence data from this region for Encephalitozoon hellem and Encephalitozoon cuniculi shows relatively high sequence similarity, supporting the placement of these two organisms in the same genus. At the same time, sequence differences between the two organisms confirm that they are not the same species. Three separate isolates of E. hellem were sequenced in the highly variable intervening spacer region. the sequence was identical for all three isolates. Within the amplified DNA segment, regions were sequenced which yield highly variable, moderately variable and highly conserved sequence information, appropriate for comparison with other species in the phylum Microspora at all taxonomic levels. We suggest that sequence data from these regions be included in future species descriptions for the purposes of species identification and phylogenetic analysis. Restriction digests of the amplified region are presented and give a rapid method for distinguishing between the two Encephalitozoon species.

A phylogenetic approach to following West Nile virus in Connecticut.

The 1999 outbreak of West Nile (WN) virus in the northeastern United States was the first known natural occurrence of this flavivirus in the Western Hemisphere. In 1999 and 2000, 82 independent Connecticut WN virus isolates were cultured from nine species of birds, five species of mosquitoes, and one striped skunk. Nucleotide sequences obtained from these isolates identified 30 genetic changes, compared with WN-NY99, in a 921-nt region of the viral genome beginning at nucleotide position 205 and ending at 1125. This region encodes portions of the nucleocapsid and envelope proteins and includes the entire coding regions for the premembrane and membrane proteins. Amino acid changes occurred at seven loci in six isolates relative to the WN-NY99 strain. Although 34 of the isolates showed sequences identical to the WN-NY99 isolate, we were able to show geographical-based clusters of mutations. In particular, 26 isolates were characterized by mutation of C to T at position 858. This group apparently originated in Stamford, CT and disseminated to sites located as far as 54 miles from Stamford. Sequences of WN virus isolated from both brain and heart tissues from the same avian host were identical in all 14 tested individual birds, suggesting that the mutations we have documented are real and not caused by culture, RNA extraction, or PCR procedures. We conclude that this portion of the viral genome will enable us to follow the geographical and temporal movement of variant WN virus strains as they adapt to North America.

Comparative rDNA Analysis of Microsporidia including AIDS Related Species

Molecular phylogenetic analysis has the potential for being a powerful tool for determining the sources of microsporidia isolated from AIDS patients. We have constructed a database of small subunit rDNA sequences of both human and non-human microsporidia in order to determine the sources of microsporidial infections in humans through comparative analysis. MATERIALS AND METHODS. DNA was liberated from spores by bead beating, amplified and sequenced as previously described [ 9 ] . Ribosomal DNA sequences were obtained by dideoxy sequencing of amplified DNA or from Genbank entries [I]. RESULTS AND DISCUSSION. The cladogram below illustrates a number of relationships relevant to the sources of these AIDS microsporidia.

Spectrosopic studies of an insect hemoglobin from the backswimmer Buenoa margaritacea (hemiptera: notonectidae)

Hemoglobin (Hb) isolated from the backswimmer Buenoa margaritacea has been analyzed spectroscopically. The met form at pH less than 6 shows a 30nm red shift in the Qv and Qo bands and a 5nm red shift in the Soret band compared to mammalian Hb, while only minor differences are seen in the spectra of the CO and O2 adducts of Hb from Buenoa and mammals. EPR spectra of the metHb show a superposition of signals; at low pH they are mainly of axial high-spin character, while at high pH a low-spin signal predominates with an O-type g-tensor (2.54, 2.61, 1.85) comparable to that of hydroxy myoglobin. Infrared spectra of Hb12C-16O at pH 8.2 reveal two major absorption bands at 1934 cm-1 and 1967 cm-1, which shift to 1892 cm-1 and 1923 cm-1, respectively, for Hb12C-18O. As isolated the Buenoa Hb consists of several isozymes, all of which have a histidine as the proximal ligand of the heme iron.

Characterization of hemoglobin from the backswimmer Buenoa margaritacea (Hemiptera: Notonectidae) reveals high electrophoretic heterogeneity

Abstract Backswimmers (Notonectidae) are one of the three families of insects which contain species known to make hemoglobin. In backswimmers the hemoglobin allows the insect to maintain a prolonged neutral buoyancy during its dive. Sephadex G-200 column chromatography, SDS-polyacrylamide gel electrophoresis and HPLC demonstrate a molecular weight for the monomeric hemoglobin og Buenoa margaritacea of 15,000–19,000 Da. u.v.-visible spectra at eight hemoglobin derivatives (Fe2+:deoxy, O2, CO, NO; Fe3+:H2O, OH−, CN−, imidazole) are given. Native gel electrophoresis of the cyanomet form shows nine bands, while isoelectric focusing shows 12. Bands isolated from preparative IEF gels form multiple bands when rerun, indicative of a change in aggregation status after isolation. Interactions among various hemoglobin components are seen using 2-dimensional “titration” electrophoresis.