Michael W. Gaunt

Phylogenetic relationships of flaviviruses correlate with their epidemiology, disease association and biogeography.

Phylogenetic analysis of the Flavivirus genus, using either partial sequences of the non-structural 5 gene or the structural envelope gene, revealed an extensive series of clades defined by their epidemiology and disease associations. These phylogenies identified mosquito-borne, tick-borne and no-known-vector (NKV) virus clades, which could be further subdivided into clades defined by their principal vertebrate host. The mosquito-borne flaviviruses revealed two distinct epidemiological groups: (i) the neurotropic viruses, often associated with encephalitic disease in humans or livestock, correlated with the Culex species vector and bird reservoirs and (ii) the non-neurotropic viruses, associated with haemorrhagic disease in humans, correlated with the Aedes species vector and primate hosts. Thus, the tree topology describing the virus-host association may reflect differences in the feeding behaviour between Aedes and Culex mosquitoes. The tick-borne viruses also formed two distinct groups: one group associated with seabirds and the other, the tick-borne encephalitis complex viruses, associated primarily with rodents. The NKV flaviviruses formed three distinct groups: one group, which was closely related to the mosquito-borne viruses, associated with bats; a second group, which was more genetically distant, also associated with bats; and a third group associated with rodents. Each epidemiological group within the phylogenies revealed distinct geographical clusters in either the Old World or the New World, which for mosquito-borne viruses may reflect an Old World origin. The correlation between epidemiology, disease correlation and biogeography begins to define the complex evolutionary relationships between the virus, vector, vertebrate host and ecological niche.

The ecotopes and evolution of triatomine bugs (triatominae) and their associated trypanosomes

Triatomine bug species such as Microtriatoma trinidadensis, Eratyrus mucronatus, Belminus herreri, Panstrongylus lignarius, and Triatoma tibiamaculata are exquisitely adapted to specialist niches. This suggests a long evolutionary history, as well as the recent dramatic spread a few eclectic, domiciliated triatomine species. Virtually all species of the genus Rhodnius are primarily associated with palms. The genus Panstrongylus is predominantly associated with burrows and tree cavities and the genus Triatoma with terrestrial rocky habitats or rodent burrows. Two major sub-divisions have been defined within the species Trypanosoma cruzi, as T. cruzi 1 (Z1) and T. cruzi 2 (Z2). The affinities of a third group (Z3) are uncertain. Host and habitat associations lead us to propose that T. cruzi 1 (Z1) has evolved in an arboreal, palm tree habitat with the triatomine tribe Rhodniini, in association with the opossum Didelphis. Similarly we propose that T. cruzi (Z2) and Z3 evolved in a terrestrial habitat in burrows and in rocky locations with the triatomine tribe Triatomini, in association with edentates, and/or possibly ground dwelling marsupials. Both sub-divisions of T. cruzi may have been contemporary in South America up to 65 million years ago. Alternatively, T. cruzi 2 (Z2) may have evolved more recently from T. cruzi 1 (Z1) by host transfers into rodents, edentates, and primates. We have constructed a molecular phylogeny of haematophagous vectors, including triatomine bugs, which suggests that faecal transmission of trypanosomes may be the ancestral route. A molecular clock phylogeny suggests that Rhodnius and Triatoma diverged before the arrival, about 40 million years ago, of bats and rodents into South America.

The molecular epidemiology and phylogeography of Trypanosoma cruzi and parallel research on Leishmania: looking back and to the future

Trypanosoma cruzi is the protozoan agent of Chagas disease, and the most important parasitic disease in Latin America. Protozoa of the genus Leishmania are global agents of visceral and cutaneous leishmaniasis, fatal and disfiguring diseases. In the 1970s multilocus enzyme electrophoresis demonstrated that T. cruzi is a heterogeneous complex. Six zymodemes were described, corresponding with currently recognized lineages, TcI and TcIIa-e--now defined by multiple genetic markers. Molecular epidemiology has substantially resolved the phylogeography and ecological niches of the T. cruzi lineages. Genetic hybridization has fundamentally influenced T. cruzi evolution and epidemiology of Chagas disease. Genetic exchange of T. cruzi in vitro involves fusion of diploids and genome erosion, producing aneuploid hybrids. Transgenic fluorescent clones are new tools to elucidate molecular genetics and phenotypic variation. We speculate that pericardial sequestration plays a role in pathogenesis. Multilocus sequence typing, microsatellites and, ultimately, comparative genomics are improving understanding of T. cruzi population genetics. Similarly, in Leishmania, genetic groups have been defined, including epidemiologically important hybrids; genetic exchange can occur in the sand fly vector. We describe the profound impact of this parallel research on genetic diversity of T. cruzi and Leishmania, in the context of epidemiology, taxonomy and disease control.

Genome-Scale Multilocus Microsatellite Typing of Trypanosoma cruzi Discrete Typing Unit I Reveals Phylogeographic Structure and Specific Genotypes Linked to Human Infection

Trypanosoma cruzi is the most important parasitic infection in Latin America and is also genetically highly diverse, with at least six discrete typing units (DTUs) reported: Tc I, IIa, IIb, IIc, IId, and IIe. However, the current six-genotype classification is likely to be a poor reflection of the total genetic diversity present in this undeniably ancient parasite. To determine whether epidemiologically important information is “hidden” at the sub-DTU level, we developed a 48-marker panel of polymorphic microsatellite loci to investigate population structure among 135 samples from across the geographic distribution of TcI. This DTU is the major cause of resurgent human disease in northern South America but also occurs in silvatic triatomine vectors and mammalian reservoir hosts throughout the continent. Based on a total dataset of 12,329 alleles, we demonstrate that silvatic TcI populations are extraordinarily genetically diverse, show spatial structuring on a continental scale, and have undergone recent biogeographic expansion into the southern United States of America. Conversely, the majority of human strains sampled are restricted to two distinct groups characterised by a considerable reduction in genetic diversity with respect to isolates from silvatic sources. In Venezuela, most human isolates showed little identity with known local silvatic strains, despite frequent invasion of the domestic setting by infected adult vectors. Multilocus linkage indices indicate predominantly clonal parasite propagation among all populations. However, excess homozygosity among silvatic strains and raised heterozygosity among domestic populations suggest that some level of genetic recombination cannot be ruled out. The epidemiological significance of these findings is discussed.

Trypanosoma cruzi IIc: phylogenetic and phylogeographic insights from sequence and microsatellite analysis and potential Impact on emergent chagas disease

Trypanosoma cruzi, the etiological agent of Chagas disease, is highly genetically diverse. Numerous lines of evidence point to the existence of six stable genetic lineages or DTUs: TcI, TcIIa, TcIIb, TcIIc, TcIId, and TcIIe. Molecular dating suggests that T. cruzi is likely to have been an endemic infection of neotropical mammalian fauna for many millions of years. Here we have applied a panel of 49 polymorphic microsatellite markers developed from the online T. cruzi genome to document genetic diversity among 53 isolates belonging to TcIIc, a lineage so far recorded almost exclusively in silvatic transmission cycles but increasingly a potential source of human infection. These data are complemented by parallel analysis of sequence variation in a fragment of the glucose-6-phosphate isomerase gene. New isolates confirm that TcIIc is associated with terrestrial transmission cycles and armadillo reservoir hosts, and demonstrate that TcIIc is far more widespread than previously thought, with a distribution at least from Western Venezuela to the Argentine Chaco. We show that TcIIc is truly a discrete T. cruzi lineage, that it could have an ancient origin and that diversity occurs within the terrestrial niche independently of the host species. We also show that spatial structure among TcIIc isolates from its principal host, the armadillo Dasypus novemcinctus, is greater than that among TcI from Didelphis spp. opossums and link this observation to differences in ecology of their respective niches. Homozygosity in TcIIc populations and some linkage indices indicate the possibility of recombination but cannot yet be effectively discriminated from a high genome-wide frequency of gene conversion. Finally, we suggest that the derived TcIIc population genetic data have a vital role in determining the origin of the epidemiologically important hybrid lineages TcIId and TcIIe.

Genetic typing and phylogeny of the Leishmania donovani complex by restriction analysis of PCR amplified gp63 intergenic regions

Protozoan parasites of the Leishmania donovani complex (L. donovani, L. infantum/L. chagasi) are causative agents of visceral leishmaniasis. To understand phylogeny and taxonomy within this group better we have developed 2 new polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) analyses of the major surface protease (msp or gp63) intergenic (ITG) regions. We have named this approach msp intergenic region RFLP typing (MIRT). One intergenic region lies between the constitutive msp (mspC) and stationary phase msp (mspS4) genes (ITG/CS) and the other between multicopy logarithmic phase msp (mspL) genes (ITG/L). The markers generated robust and congruent phylogenies, identifying 5 genetic clusters within L. donovani. One cluster was synonymous with L. infantum (L. chagasi); clusters strongly correlated with isoenzyme typing and some with geographical origin. These genetic groups may be important for epidemiological and clinical studies. The congruence of the groups identified indicates suitability of these genomic targets for taxonomic studies. Furthermore, subgroups of L. donovani were of equivalent phylogenetic status to L. infantum. No evidence was found to support the existence of L. archibaldi. It is likely to be necessary in future to re-evaluate the taxonomic status of L. donovani or of L. infantum, as discrete species.

Application of Comparative Phylogenomics To Study the Evolution of Yersinia enterocolitica and To Identify Genetic Differences Relating to Pathogenicity

Yersinia enterocolitica, an important cause of human gastroenteritis generally caused by the consumption of livestock, has traditionally been categorized into three groups with respect to pathogenicity, i.e., nonpathogenic (biotype 1A), low pathogenicity (biotypes 2 to 5), and highly pathogenic (biotype 1B). However, genetic differences that explain variation in pathogenesis and whether different biotypes are associated with specific nonhuman hosts are largely unknown. In this study, we applied comparative phylogenomics (whole-genome comparisons of microbes with DNA microarrays combined with Bayesian phylogenies) to investigate a diverse collection of 94 strains of Y. enterocolitica consisting of 35 human, 35 pig, 15 sheep, and 9 cattle isolates from nonpathogenic, low-pathogenicity, and highly pathogenic biotypes. Analysis confirmed three distinct statistically supported clusters composed of a nonpathogenic clade, a low-pathogenicity clade, and a highly pathogenic clade. Genetic differences revealed 125 predicted coding sequences (CDSs) present in all highly pathogenic strains but absent from the other clades. These included several previously uncharacterized CDSs that may encode novel virulence determinants including a hemolysin, a metalloprotease, and a type III secretion effector protein. Additionally, 27 CDSs were identified which were present in all 47 low-pathogenicity strains and Y. enterocolitica 8081 but absent from all nonpathogenic 1A isolates. Analysis of the core gene set for Y. enterocolitica revealed that 20.8% of the genes were shared by all of the strains, confirming this species as highly heterogeneous, adding to the case for the existence of three subspecies of Y. enterocolitica. Further analysis revealed that Y. enterocolitica does not cluster according to source (host).

Recent, independent and anthropogenic origins of Trypanosoma cruzi hybrids.

The single celled eukaryote Trypanosoma cruzi, a parasite transmitted by numerous species of triatomine bug in the Americas, causes Chagas disease in humans. T. cruzi generally reproduces asexually and appears to have a clonal population structure. However, two of the six major circulating genetic lineages, TcV and TcVI, are TcII-TcIII inter-lineage hybrids that are frequently isolated from humans in regions where chronic Chagas disease is particularly severe. Nevertheless, a prevalent view is that hybridisation events in T. cruzi were evolutionarily ancient and that active recombination is of little epidemiological importance. We analysed genotypes of hybrid and non-hybrid T. cruzi strains for markers representing three distinct evolutionary rates: nuclear GPI sequences (n = 88), mitochondrial COII-ND1 sequences (n = 107) and 28 polymorphic microsatellite loci (n = 35). Using Maximum Likelihood and Bayesian phylogenetic approaches we dated key evolutionary events in the T. cruzi clade including the emergence of hybrid lineages TcV and TcVI, which we estimated to have occurred within the last 60,000 years. We also found evidence for recent genetic exchange between TcIII and TcIV and between TcI and TcIV. These findings show that evolution of novel recombinants remains a potential epidemiological risk. The clearly distinguishable microsatellite genotypes of TcV and TcVI were highly heterozygous and displayed minimal intra-lineage diversity indicative of even earlier origins than sequence-based estimates. Natural hybrid genotypes resembled typical meiotic F1 progeny, however, evidence for mitochondrial introgression, absence of haploid forms and previous experimental crosses indicate that sexual reproduction in T. cruzi may involve alternatives to canonical meiosis. Overall, the data support two independent hybridisation events between TcII and TcIII and a recent, rapid spread of the hybrid progeny in domestic transmission cycles concomitant with, or as a result of, disruption of natural transmission cycles by human activities.

Transmission of louping ill virus between infected and uninfected ticks co‐feeding on mountain hares

Abstract. Most of the data on oral infection of ticks by louping ill virus have been obtained from experiments in which animals were infected by syringe inoculation with infectious material. Using infected ticks to mimic the natural situation, we have demonstrated that louping ill (LI) virus transmission can occur from infected to uninfected Ixodes acinus feeding in close proximity on mountain hares (Lepus timidus). Under these conditions the hares developed either low or undetectable viraemias. Highest prevalence of LI virus infection was observed in recipient nymphs which had fed to repletion between days 3 and 7 post‐attachment of virus‐infected adults; following engorgement, 56% of nymphs acquired virus. These results demonstrate the efficient transmission of LI virus between co‐feeding ticks on naive mountain hares. However, when ticks were allowed to co‐feed on virus‐immune hares a significant reduction in the frequency of infection was observed. Neither red deer (Cervus elaphus) nor New Zealand White rabbits supported transmission of LI virus. The significance of virus transmission between cofeeding ticks on LI virus epidemiology is discussed.

Flow cytometric analysis and microsatellite genotyping reveal extensive DNA content variation in Trypanosoma cruzi populations and expose contrasts between natural and experimental hybrids

Trypanosoma cruzi exhibits remarkable genetic heterogeneity. This is evident at the nucleotide level but also structurally, in the form of karyotypic variation and DNA content differences between strains. Although natural populations of T. cruzi are predominantly clonal, hybrid lineages (TcIId and TcIIe) have been identified and hybridisation has been demonstrated in vitro, raising the possibility that genetic exchange may continue to shape the evolution of this pathogen. The mechanism of genetic exchange identified in the laboratory is unusual, apparently involving fusion of diploid parents followed by genome erosion. We investigated DNA content diversity in natural populations of T. cruzi in the context of its genetic subdivisions by using flow cytometric analysis and multilocus microsatellite genotyping to determine the relative DNA content and estimate the ploidy of 54 cloned isolates. The maximum difference observed was 47.5% between strain Tu18 cl2 (TcIIb) and strain C8 cl1 (TcI), which we estimated to be equivalent to ∼73 Mb of DNA. Large DNA content differences were identified within and between discrete typing units (DTUs). In particular, the mean DNA content of TcI strains was significantly less than that for TcII strains (P < 0.001). Comparisons of hybrid DTUs TcIId/IIe with corresponding parental DTUs TcIIb/IIc indicated that natural hybrids are predominantly diploid. We also measured the relative DNA content of six in vitro-generated TcI hybrid clones and their parents. In contrast to TcIId/IIe hybrid strains these experimental hybrids comprised populations of sub-tetraploid organisms with mean DNA contents 1.65–1.72 times higher than the parental organisms. The DNA contents of both parents and hybrids were shown to be relatively stable after passage through a mammalian host, heat shock or nutritional stress. The results are discussed in the context of hybridisation mechanisms in both natural and in vitro settings.