Imagine a virus that can infect Protists! Although it is common knowledge that viruses are as ubiquitous as other microorganisms, and that each living species probably has a set of viruses that they specifically play host to, protozoan viruses are not exactly virology textbook material. Without exception though, these strange entities can still be classified under the Baltimore system of classification. Trichomonas, Babesia, Giardia, Eimeria, Leishmania and Cryptosporidium, among others, host some of the recently discovered ones. For this blog, I am going to focus on the virus that infects the protozoan Cryptosporidium, for no reason other than that it affects my favorite Apicomplexan.
In 1995, Gallimore and his colleagues, published the first report of a picobirnavirus with an "atypical genome" that they had found in human fecal samples that were positive for Cryptosporidium parvum oocysts. They had detected two fragments of 1.75 and 1.55 Kbp size with Polyacrylamide Gel Electrophoresis. They found the genome "atypical" because it was considered far too small in comparison with previously described "typical" picobirnaviruses.
If you know how naming in virology works, you probably have deduced that the virus under scrutiny is small and has two segments in its RNA genome.
(Image from EMDB - 1459)
The initial description was followed by others. A publication by Khramtsov et al. in 1997, shows how researchers attempted to characterize the virus. They found that the "novel" extrachromosomal dsRNA that they had isolated from the oocysts of Cryptosporidium parvum was found only in that species (viz. C. parvum), but was absent in other "non-parvum" species that they tested. I found the "non-parvum" species very interesting indeed! C. serpentis of snakes, C. andersoni of cattle, C. baileyi of birds, C. muris of mice and an ostrich isolate were the ones they had tested. Since the ostrich fecal isolate used cannot be identified with any degree of certainty, I offer no comments on it. Other than the odd-ball C. baileyi however, the other three species belong to the gastric group of Cryptosporidium, whereas C.parvum belongs to the intestinal group. So, it would be fairly interesting to see if both groups harbour the virus and if the other intestinal species actually lack it entirely.
Act III: Climax
Khramtsov et al., also found that the larger of the two segments after cloning proved to be a single open reading frame that encoded a protein that was homologous to a virally coded RNA dependent RNA polymerase and that the smaller one was homologous to a MAP kinase. The RNAs were also susceptible to degradation by RNAse A, possibly because they were unencapsidated.
In 2000, Khramtsov et al., isolated the ds RNA from both the calf and human isolates of Cryptosporidium parvum sensu lato, which were then known as C.parvum genotype II and C.parvum genotype I respectively. We know now that the human isolates were in fact C. hominis. Thus, our question must be modified. Can intestinal species other than the ones we now know as C.hominis and C.parvum be infected by the virus?
Subsequent research has found double stranded RNA molecules with 86% similarity in nucleic acid extracts from C.felis and C.meleagridis, both of which most definitely belong to the intestinal group.
Act IV: Falling action
In 2004, Kniel et al., had the absolutely brilliant idea of utilizing the Cryptosporidium parvum virus (CPV; not to be confused with Canine Parvovirus or Cowpox virus) antigen as a target for the detection of C. parvum oocysts. SDS-PAGE analysis of the CPV antigen showed that it was a 40kDa protein. However, for reasons unexplored, the anti- recombinant CPV antibodies raised in rabbits cross-reacted with another 30 kDa protein as well. Immunofluorescence indicated that the antigen localized at the apical end of the parasite and once again no one knows why. The claim of the principle to remarkable sensitivity is well supported by the fact that one single oocyst suspended in water could be identified using an immunoblot assay. However, the long-lasting CPV antigen, which is not degraded even after 3 months, cannot be used to differentiate infectious oocysts from non-infectious ones, which severely dampened the enthusiasm of diagnosticians.
Further research along the same lines was carried out in 2008 by Jenkins et al. The near flawless principle quickly deteriorated because of the sheer complexity of protein folding. The researchers made monoclonal Abs against the 40 kDa protein, because they thought a hybridoma would provide "an unlimited source of detection reagent" and increase assay sensitivity. But, despite using an indirect IFA, the sensitivity was not much higher than the conventional IFA that targets the outer wall protein. Nevertheless, on a dot blot assay, the sensitivity was seemingly incredible. As low as 100 non-bleach treated oocysts could be detected. The authors however agree that this jaw dropping oddity was probably due to contamination and the binding of MAbs by Fc receptors on contaminant bacteria, because bleach treated oocysts (which are standards for research), could not be detected well. Another wall that the researchers ran up against was the native confirmation of CPV40, which did not allow the binding of the antibody made against the recombinant antigen. It is possible that the recombinant protein made in E.coli did not fold the same way due to lack of eukaryotic chaperone proteins. To circumvent the binding issue, the proteins had to be extracted under denaturing conditions. Under non-denaturing conditions, yet another eccentricity was observed. The MAb could bind to proteins of size 77 and 125 kDa respectively, which were presumed to be viral capsid dimers and tetramers. This presumption begs to be challenged. If the epitope is not exposed in the monomeric capsid, then it surely must not be exposed in the dimeric or higher forms given the regularity and symmetry of the virus. In the end, the detection system is still not fully ready for use.
Act V: Denoument
That same year , the executive committee of International Committee on the Taxonomy of Viruses agreed to formally recognize the virus, which became known as 'Cryptosporidium parvum virus 1' (CSpV1), and establish a new genus called Cryspovirus in the family Partitiviridae, in which it is currently ensconced.
A short paper by Jenkins et al., published in 2008 (Was 2008 the year of the Cryspovirus?) sought to give the virus a role. "Fecundity" , that is , oocyst producing ability of two strains of C. parvum were compared, one of which had the virus and one of which did not. Calves infected with C. parvum with virus had a 5 fold higher oocyst output in their feces compared to the calves infected with C.parvum without the virus. Calf immunity and other variables cast doubts on the report. Does the virus affect the replication of the parasite (given that it is only cytoplasmic) or is the proposal just a fanciful conjecture?
A description paper was later published, cementing the name and the taxonomy. CSpV1 shows vertical transmission only. The virions are isometric, have a diameter of 31nm with short protrusions on their surface.
The story of the Cryspovirus continues to unfold. We are yet to come to a full understanding of its significance in the life history of Cryptosproridium and hence its importance to us. I'm looking forward to future publications!
References :
Gallimore CI, Green J, Casemore DP, Brown DW. Detection of a picobirnavirus associated with Cryptosporidium positive stools from humans. Arch Virol. 1995;140(7):1275-8.
Khramtsov NV, Woods KM, Nesterenko MV, Dykstra CC, Upton SJ. Virus-like, double-stranded RNAs in the parasitic protozoan Cryptosporidium parvum. Mol Microbiol. 1997 Oct;26(2):289-300.
Khramtsov NV, Chung PA, Dykstra CC, Griffiths JK, Morgan UM, Arrowood MJ, Upton SJ. Presence of double-stranded RNAs in human and calf isolates of Cryptosporidium parvum. J Parasitol. 2000 Apr;86(2):275-82.
Leoni F, Gallimore CI, Green J, McLauchlin J. Characterisation of small double stranded RNA molecule in Cryptosporidium hominis, Cryptosporidium felis and Cryptosporidium meleagridis. Parasitol Int. 2006 Dec;55(4):299-306.
Kniel KE, Higgins JA, Trout JM, Fayer R, Jenkins MC. Characterization and potential use of a Cryptosporidium parvum virus (CPV) antigen for detecting C. parvum oocysts. J Microbiol Methods. 2004 Aug;58(2):189-95.
Jenkins MC, O'Brien CN, Trout JM. Detection of Cryptosporidium parvum oocysts by dot-blotting using monoclonal antibodies to Cryptosporidium parvum virus 40-kDa capsid protein. J Parasitol. 2008 Feb;94(1):94-8.
Nibert ML, Woods KM, Upton SJ, Ghabrial SA. Cryspovirus: a new genus of protozoan viruses in the family Partitiviridae. Arch Virol. 2009;154(12):1959-65.
Jenkins MC, Higgins J, Abrahante JE, Kniel KE, O'Brien C, Trout J, Lancto CA, Abrahamsen MS, Fayer R. Fecundity of Cryptosporidium parvum is correlated with intracellular levels of the viral symbiont CPV. Int J Parasitol. 2008 Jul;38(8-9):1051-5.
