Presence of Double-Stranded RNAs in Human and Calf Isolates of Cryptosporidium parvum

Abstract

We examined the occurrence of 2 virus-like double-stranded (ds)RNAs in human and calf isolates of Cryptosporidium parvum senso latu and other microorganisms, including 7 other members of the genus. A total of 32 isolates of C. parvum, 16 from humans (5 from acquired immune deficiency syndrome patients) and 16 from calves, were analyzed. Ethidium bromide staining, or Northern blot analysis, or reverse transcription/polymerase chain reaction, or all 3 methods, revealed that both genotype 1 and genotype 2 isolates of C. parvum possessed these dsRNAs. No other Cryptosporidium spp. or other organisms examined possessed these dsRNAs. Comparison analysis of partial cDNA sequences of dsRNAs from human and calf isolates revealed a high degree of similarity (Ͼ92% and Ͼ93% identical nucleotides for large and small dsRNAs, respectively). Slight, consistent differences in nucleotide sequences could be seen at select sites and were associated with an isolate being either genotype 1 or 2. Because of the widespread distribution of the dsRNAs, the similarity of these molecules between isolates, and high host specificity, these nucleic acids may prove to represent species-specific molecular markers for C. parvum. Evidence also suggests that the dsRNA can be utilized for molecular genotyping of C. parvum.

Association of RNA Polymerase Complexes of the Parasitic Protozoan Cryptosporidium parvum with Virus-Like Particles: Heterogeneous System

“…Additional indirect evidence of protein coding capacity of the dsRNAs exists as a result of a comparative analysis of the sequences of dsRNAs from a number of isolates of C. parvum with two distinct genotypes. The majority of nucleotide substitutions occur in the third positions of the codons in putative ORFs and fail to change the protein sequences (19). However, to date, there is no direct evidence for the presence of proteins encoded by the dsRNAs, nor have virus particles been identified in the previous studies (17)(18)(19).…”

“…All isolates of C. parvum tested thus far are known to harbor both large (L) and small (S) extrachromosomal viral doublestranded RNA (dsRNA) segments (17,19). The cDNAs of both dsRNAs have been cloned and sequenced, and only a single long open reading frame (ORF) has been identified in each dsRNA.…”

“…Because of the lack of adequate treatment, other types of therapeutic targets are being sought, and extrachromosomal genetic elements are considered potential targets. For example, the extranuclear plastid genomes of Plasmodium and Toxoplasma spp., both parasites distantly related to C. parvum, are essential for parasite survival, and inhibition of plastid replication blocks the multiplication of parasites (9).All isolates of C. parvum tested thus far are known to harbor both large (L) and small (S) extrachromosomal viral doublestranded RNA (dsRNA) segments (17,19). The cDNAs of both dsRNAs have been cloned and sequenced, and only a single long open reading frame (ORF) has been identified in each dsRNA.…”

Transient transfection of Cryptosporidium parvum using green fluorescent protein (GFP) as a marker

“…Cryptosporidium parvum has been shown to harbor a dsRNA virus (CPV) with two diverse constituents: large (L) and small (S) extra-chromosomal viral double-stranded RNA (dsRNA) segments (CPV-L and CPV-S), which are 1,786 nt and 1,374 nt respectively in length with only a single long open reading frame (ORF) each [7,8].…”

“…The development of effective transfection methods, will result in a better understanding of this parasite's biology and its relationship with the host and will also lead to more efficient molecule-based vaccines and drugs [5,6]. Double-stranded RNA (dsRNA) viruses, belonging to the family Totiviridae, have been identified in several protozoan parasites, including Cryptosporidium [7,8].…”