Phylogenetic and phylodynamic approaches to understanding and combating the early SARS-CoV-2 pandemic.

Determining the transmissibility, prevalence and patterns of movement of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is central to our understanding of the impact of the pandemic and to the design of effective control strategies. Phylogenies (evolutionary trees) have provided key insights into the international spread of SARS-CoV-2 and enabled investigation of individual outbreaks and transmission chains in specific settings. Phylodynamic approaches combine evolutionary, demographic and epidemiological concepts and have helped track virus genetic changes, identify emerging variants and inform public health strategy. Here, we review and synthesize studies that illustrate how phylogenetic and phylodynamic techniques were applied during the first year of the pandemic, and summarize their contributions to our understanding of SARS-CoV-2 transmission and control.

Assignment of epidemiological lineages in an emerging pandemic using the pangolin tool.

The response of the global virus genomics community to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been unprecedented, with significant advances made towards the 'real-time' generation and sharing of SARS-CoV-2 genomic data. The rapid growth in virus genome data production has necessitated the development of new analytical methods that can deal with orders of magnitude of more genomes than previously available. Here, we present and describe Phylogenetic Assignment of Named Global Outbreak Lineages (pangolin), a computational tool that has been developed to assign the most likely lineage to a given SARS-CoV-2 genome sequence according to the Pango dynamic lineage nomenclature scheme. To date, nearly two million virus genomes have been submitted to the web-application implementation of pangolin, which has facilitated the SARS-CoV-2 genomic epidemiology and provided researchers with access to actionable information about the pandemic's transmission lineages.

Divergence across mitochondrial genomes of sympatric members of the Schistosoma indicum group and clues into the evolution of Schistosoma spindale.

Schistosoma spindale and Schistosoma indicum are ruminant-infecting trematodes of the Schistosoma indicum group that are widespread across Southeast Asia. Though neglected, these parasites can cause major pathology and mortality to livestock leading to significant welfare and socio-economic issues, predominantly amongst poor subsistence farmers and their families. Here we used mitogenomic analysis to determine the relationships between these two sympatric species of schistosome and to characterise S. spindale diversity in order to identify possible cryptic speciation. The mitochondrial genomes of S. spindale and S. indicum were assembled and genetic analyses revealed high levels of diversity within the S. indicum group. Evidence of functional changes in mitochondrial genes indicated adaptation to environmental change associated with speciation events in S. spindale around 2.5 million years ago. We discuss our results in terms of their theoretical and applied implications.

iPS-Cell Technology and the Problem of Genetic Instability-Can It Ever Be Safe for Clinical Use?

The use of induced Pluripotent Stem Cells (iPSC) as a source of autologous tissues shows great promise in regenerative medicine. Nevertheless, several major challenges remain to be addressed before iPSC-derived cells can be used in therapy, and experience of their clinical use is extremely limited. In this review, the factors affecting the safe translation of iPSC to the clinic are considered, together with an account of efforts being made to overcome these issues. The review draws upon experiences with pluripotent stem-cell therapeutics, including clinical trials involving human embryonic stem cells and the widely transplanted mesenchymal stem cells. The discussion covers concerns relating to: (i) the reprogramming process; (ii) the detection and removal of incompletely differentiated and pluripotent cells from the resulting medicinal products; and (iii) genomic and epigenetic changes, and the evolutionary and selective processes occurring during culture expansion, associated with production of iPSC-therapeutics. In addition, (iv) methods for the practical culture-at-scale and standardization required for routine clinical use are considered. Finally, (v) the potential of iPSC in the treatment of human disease is evaluated in the light of what is known about the reprogramming process, the behavior of cells in culture, and the performance of iPSC in pre-clinical studies.

Population genetic structure and geographical variation in Neotricula aperta (Gastropoda: Pomatiopsidae), the snail intermediate host of Schistosoma mekongi (Digenea: Schistosomatidae).

BACKGROUND: Neotricula aperta is the snail-intermediate host of the parasitic blood-fluke Schistosoma mekongi which causes Mekong schistosomiasis in Cambodia and the Lao PDR. Despite numerous phylogenetic studies only one DNA-sequence based population-genetic study of N. aperta had been published, and the origin, structure and persistence of N. aperta were poorly understood. Consequently, a phylogenetic and population genetic study was performed, with addition of new data to pre-existing DNA-sequences for N. aperta from remote and inaccessible habitats, including one new taxon from Laos and 505 bp of additional DNA-sequence for all sampled taxa,. PRINCIPAL FINDINGS: Spatial Principal Component Analysis revealed the presence of significant spatial-genetic clustering. Genetic-distance-based clustering indicated four populations with near perfect match to a priori defined ecogeographical regions. Spring-dwelling taxa were found to form an ecological isolate relative to other N. aperta. The poor dispersal capabilities suggested by spatial-genetic analyses were confirmed by Bayesian inference of migration rates. Population divergence time estimation implied a mid-Miocene colonisation of the present range, with immediate and rapid radiation in each ecogeographical region. Estimated effective population sizes were large (120-310 thousand). CONCLUSIONS: The strong spatial-genetic structure confirmed the poor dispersal capabilities of N. aperta-suggesting human-mediated reintroduction of disease to controlled areas as the primary reason for control failure. The isolation of the spring-dwelling taxa and ecogeographical structure suggests adaptation of sub-populations to different habitats; the epidemiological significance of this needs investigation. The large effective population sizes indicate that the high population densities observed in surveyed habitats are also present in inaccessible areas; affording great potential for recrudescence driven by animal-reservoir transmission in remote streams. Mid-Miocene colonisation implies heterochronous evolution of these snails and associated schistosomes and suggests against coevolution of snail and parasite. Heterochronicity favours ecological factors as shapers of host-parasite specificity and greater potential for escape from schistosomiasis control through host-switching.

An investigation into the potential effects of infrapopulation structure and other sources of sampling error, on population genetic studies of the transmission of Schistosoma japonicum (Trematoda: Digenea).

BACKGROUND: Schistosoma japonicum remains a major challenge to human and animal health. Earlier microsatellite-based studies reported possible definitive-host-specific private alleles within S. japonicum, opening the possibility that different definitive hosts might harbour different parasite strains. Previous investigations have also detected near-identical multilocus genotypes in populations of adult worms - possibly the result of mutations occurring during the asexual (intramolluscan) phase of clonal expansion. Research has also revealed extensive deviations from Hardy-Weinberg Proportions (HWP) and conflicting results among studies. The present study was performed to examine some of the potential effects of infrapopulation structure on microsatellite-based studies of the transmission ecology of S. japonicum. Potential sources of bias considered included organotropic distribution of worms, non-random mating and corrections for clonal expansion. RESULTS: Stool samples from naturally infected hosts were used to infect snails in the laboratory and thereby expose mice. 274 individual worms were typed at seven microsatellite loci. Removal of individuals bearing duplicate MLGs (as a correction for presumed clonal expansion) had an impact on both HWP and organotropic genetic differentiation. The study found no evidence that heterozygote deficiencies were caused by a Wahlund effect. Female-male pairings appeared to be random and there was no evidence for mate choice by heterozygosity. There was some indication that excess heterozygosity, induced by clonal expansion, can offset heterozygote deficiencies caused by small population size or populations fragmented by parasite control efforts. CONCLUSIONS: The view is supported that miracidia are preferable to adult worms in investigations into host-specific parasite lineages. Where adults must be used, extreme care should be taken with regard to sampling if infrapopulations of small animals are compared with those of larger animals; this is because of organotropic patterns in genetic variation and the tendency to sample from different organs in differently sized hosts. As corrections for clones may accentuate signals of population subdivision, corrections should only be made if tests for clonal expansion prove positive. Finally, evidence for heterozygote deficiency caused by small sample size, calls for carefully designed random and comprehensive sampling strategies for S. japonicum in China, where control efforts have greatly fragmented parasite populations.

Comparative Phylogenetic Studies on Schistosoma japonicum and Its Snail Intermediate Host Oncomelania hupensis: Origins, Dispersal and Coevolution.

BACKGROUND: Schistosoma japonicum causes major public health problems in China and the Philippines; this parasite, which is transmitted by freshwater snails of the species Oncomelania hupensis, causes the disease intestinal schistosomiasis in humans and cattle. Researchers working on Schistosoma in Africa have described the relationship between the parasites and their snail intermediate hosts as coevolved or even as an evolutionary arms race. In the present study this hypothesis of coevolution is evaluated for S. japonicum and O. hupensis. The origins and radiation of the snails and the parasite across China, and the taxonomic validity of the sub-species of O. hupensis, are also assessed. METHODOLOGY/PRINCIPAL FINDINGS: The findings provide no evidence for coevolution between S. japonicum and O. hupensis, and the phylogeographical analysis suggests a heterochronous radiation of the parasites and snails in response to different palaeogeographical and climatic triggers. The results are consistent with a hypothesis of East to West colonisation of China by Oncomelania with a re-invasion of Japan by O. hupensis from China. The Taiwan population of S. japonicum appears to be recently established in comparison with mainland Chinese populations. CONCLUSIONS/SIGNIFICANCE: The snail and parasite populations of the western mountain region of China (Yunnan and Sichuan) appear to have been isolated from Southeast Asian populations since the Pleistocene; this has implications for road and rail links being constructed in the region, which will breach biogeographical barriers between China and Southeast Asia. The results also have implications for the spread of S. japonicum. In the absence of coevolution, the parasite may more readily colonise new snail populations to which it is not locally adapted, or even new intermediate host species; this can facilitate its dispersal into new areas. Additional work is required to assess further the risk of spread of S. japonicum.

Update on the distribution and phylogenetics of Biomphalaria (Gastropoda: Planorbidae) populations in Guangdong Province, China.

In 1973 planorbid snails then identified as Biomphalaria straminea were discovered in Hong Kong, China. It was assumed that these snails had been introduced to Hong Kong via the import of tropical fish by air from South America. In 2012 Biomphalaria were found for the first time in Guangdong Province, China. In view of the renewed interest in these invasive snails, a morphological and DNA-sequence based phylogenetic study was undertaken for seven populations of Biomphalaria snails collected in Guangdong. Morphologically and phylogenetically, five of the populations clustered more closely with Biomphalaria kuhniana than with B. straminea. Levels of genetic diversity among the populations were about half those of autochthonous populations in Brazil, the phylogenetic relationships did not correlate with a radiation from any one international port in China, and different lineages appeared associated with different ports. Consequently in explaining the current distribution of the snails, multiple colonization events, each establishing a new local snail population near to maritime international container ports, were considered more likely than the spread of snails from Hong Kong to China. The displacement of B. straminea by B. kuhniana in Guangdong is considered as an explanation for the habitat changes observed among the snails between Hong Kong in the 1980s and the present. The conclusions of the study are that any risk of Schistosoma mansoni transmission in China is more likely to come from parasite importation in the intramolluscan stage, than from transmission by migrant workers from South America or Africa. In addition, although likely to be rare, sporadic outbreaks of imported schistosomiasis (caused by invading infected snails) could be a threat to public health in the vicinity of International container ports (not only in Guangdong Province). Further work is called for to investigate further the presence of B. kuhniana and its potential interactions with B. straminea (the former is thought to be incompatible with S. mansoni), and the responses of Chinese Biomphalaria to potential competitors such as Thiaridae. The current expansion of container ports in Brazil and Venezuela, and the increase in trade with China, is likely to accentuate any current risk of imported schistosomiasis, and surveillance around ports in China, together with further research, are necessary.

A phylogeny for the pomatiopsidae (Gastropoda: Rissooidea): a resource for taxonomic, parasitological and biodiversity studies.

BACKGROUND: The Pomatiopsidae are reported from northern India into southern China and Southeast Asia, with two sub-families, the Pomatiopsinae (which include freshwater, amphibious, terrestrial and marine species) and the freshwater Triculinae. Both include species acting as intermediate host for species of the blood-fluke Schistosoma which cause a public health problem in East Asia. Also, with around 120 species, triculine biodiversity exceeds that of any other endemic freshwater molluscan fauna. Nevertheless, the origins of the Pomatiopsidae, the factors driving such a diverse radiation and aspects of their co-evolution with Schistosoma are not fully understood. Many taxonomic questions remain; there are problems identifying medically relevant species. The predicted range is mostly unsurveyed and the true biodiversity of the family is underestimated. Consequently, the aim of the study was to collect DNA-sequence data for as many pomatiopsid taxa as possible, as a first step in providing a resource for identification of epidemiologically significant species (by non-malacologists), for use in resolving taxonomic confusion and for testing phylogeographical hypotheses. RESULTS: The evolutionary radiation of the Triculinae was shown to have been rapid and mostly post late Miocene. Molecular dating indicated that the radiation of these snails was driven first by the uplift of the Himalaya and onset of a monsoon system, and then by late-Pliocene global warming. The status of Erhaia as Anmicolidae is supported. The genera Tricula and Neotricula are shown to be non-monophyletic and the tribe Jullieniini may be polyphyletic (based on convergent characters). Triculinae from northern Vietnam could be derived from Gammatricula of Fujian/Yunnan, China. CONCLUSIONS: The molecular dates and phylogenetic estimates in this study are consistent with an Australasian origin for the Pomatiopsidae and an East to West radiation via Oligocene Borneo-Philippines island hopping to Japan and then China (Triculinae arising mid-Miocene in Southeast China), and less so with a triculine origin in Tibet. The lack of monophyly in the medically important genera and indications of taxonomic inaccuracies, call for further work to identify epidemiologically significant taxa (e.g., Halewisia may be potential hosts for Schistosoma mekongi) and highlight the need for surveys to determine the true biodiversity of the Triculinae.

A population growth trend analysis for Neotricula aperta, the snail intermediate host of Schistosoma mekongi, after construction of the Pak-Mun dam.

BACKGROUND: The Pak-Mun dam is a controversial hydro-power project on the Mun River in Northeast Thailand. The dam is sited in a habitat of the freshwater snail Neotricula aperta, which is the intermediate host for the parasitic blood-fluke Schistosoma mekongi causing Mekong schistosomiasis in humans in Cambodia and Laos. Few data are available which can be used to assess the effects of water resource development on N. aperta. The aim of this study was to obtain data and to analyze the possible impact of the dam on N. aperta population growth. METHODOLOGY/PRINCIPAL FINDINGS: Estimated population densities were recorded for an N. aperta population in the Mun River 27 km upstream of Pak-Mun, from 1990 to 2011. The Pak-Mul dam began to operate in 1994. Population growth was modeled using a linear mixed model expression of a modified Gompertz stochastic state-space exponential growth model. The N. aperta population was found to be quite stable, with the estimated growth parameter not significantly different from zero. Nevertheless, some marked changes in snail population density were observed which were coincident with changes in dam operation policy. CONCLUSIONS/SIGNIFICANCE: The study found that there has been no marked increase in N. aperta population growth following operation of the Pak-Mun dam. The analysis did indicate a large and statistically significant increase in population density immediately after the dam came into operation; however, this increase was not persistent. The study has provided the first vital baseline data on N. aperta population behavior near to the Pak-Mun dam and suggests that the operation policy of the dam may have an impact on snail population density. Nevertheless, additional studies are required for other N. aperta populations in the Mun River and for an extended time series, to confirm or refine the findings of this work.

Observations on Neotricula aperta (Gastropoda: Pomatiopsidae) population densities in Thailand and central Laos: implications for the spread of Mekong schistosomiasis.

BACKGROUND: The snail Neotricula aperta transmits Mekong schistosomiasis in southern Laos and Cambodia, with about 1.5 million people at risk of infection. Plans are under consideration for at least 12 hydroelectric power dams on the lower Mekong river and much controversy surrounds predictions of their environmental impacts. Unfortunately, there are almost no ecological data (such as long-term population trend studies) available for N. aperta which could be used in impact assessment. Predictions currently assume that the impacts will be the same as those observed in Africa (i.e., a worsening of the schistosomiasis problem); however, marked ecological differences between the snails involved suggest that region specific models are required. The present study was performed as an initial step in providing data, which could be useful in the planning of water resource development in the Mekong. Snail population density records were analyzed for populations close to, and far downstream of, the Nam Theun 2 (NT2) project in Laos in order to detect any changes that might be attributable to impoundment. RESULTS: The population immediately downstream of NT2 and that sampled 400 km downstream in Thailand both showed a long-term trend of slow growth from 1992 to 2005; however, both populations showed a marked decline in density between 2005 and 2011. The decline in Thailand was to a value significantly lower than that predicted by a linear mixed model for the data, whilst the population density close to NT2 fell to undetectable levels in 2011 from densities of over 5000 m(-2) in 2005. The NT2 dam began operation in 2010. CONCLUSIONS: The impact of the NT2 dam on N. aperta population density could be more complex than first thought and may reflect the strict ecological requirements of this snail. There was no indication that responses of N. aperta populations to dam construction are similar to those observed with Bulinus and Schistosoma haematobium in Africa, for example. In view of the present findings, more ecological data (in particular population density monitoring and surveillance for new habitats) are urgently required in order to understand properly the likely impacts of water resource development on Mekong schistosomiasis.

Studies on the parasitology, phylogeography and the evolution of host-parasite interactions for the snail intermediate hosts of medically important trematode genera in Southeast Asia.

In this chapter a review of research published since 2000 on the biology of the snail intermediate hosts of trematode parasites of medical importance in Southeast Asia, and related taxa is presented. Recent taxonomic revisions of the first intermediate hosts of Paragonimus in the region are considered and an account of changes in current perspectives regarding the evolution of intermediate-host:parasite associations for both Paragonimus and Schistosoma is given. The latest phylogeographical hypotheses for Schistosoma, Paragonimus, Fasciola and Fasciolopsis are also reviewed and compared. Work performed in the region on the snail intermediate hosts of other less studied parasites, such as Opisthorchis/Clonorchis and haplorchids, is also described.

The phylogeography of Indoplanorbis exustus (Gastropoda: Planorbidae) in Asia.

BACKGROUND: The freshwater snail Indoplanorbis exustus is found across India, Southeast Asia, central Asia (Afghanistan), Arabia and Africa. Indoplanorbis is of economic importance in that it is responsible for the transmission of several species of the genus Schistosoma which infect cattle and cause reduced livestock productivity. The snail is also of medical importance as a source of cercarial dermatitis among rural workers, particularly in India. In spite of its long history and wide geographical range, it is thought that Indoplanorbis includes only a single species. The aims of the present study were to date the radiation of Indoplanorbis across Asia so that the factors involved in its dispersal in the region could be tested, to reveal potential historical biogeographical events shaping the phylogeny of the snail, and to look for signs that I. exustus might be polyphyletic. RESULTS: The results indicated a radiation beginning in the late Miocene with a divergence of an ancestral bulinine lineage into Assam and peninsular India clades. A Southeast Asian clade diverged from the peninsular India clade late-Pliocene; this clade then radiated at a much more rapid pace to colonize all of the sampled range of Indoplanorbis in the mid-Pleistocene. CONCLUSIONS: The phylogenetic depth of divergences between the Indian clades and Southeast Asian clades, together with habitat and parasitological differences suggest that I. exustus may comprise more than one species. The timescale estimated for the radiation suggests that the dispersal to Arabia and to Southeast Asia was facilitated by palaeogeographical events and climate change, and did not require human involvement. Further samples from Afghanistan, Africa and western India are required to refine the phylogeographical hypothesis and to include the African Recent dispersal.

Molecular phylogenetics of Triculine snails (Gastropoda: Pomatiopsidae) from southern China.

Triculine (Gastropoda: Rissooidea: Pomatiopsidae) snails are involved in the transmission of schistosomiasis and paragonimiasis; their distributions are mainly across southeastern Asia and southern China. In the present investigation, partial sequences of COI, 16S, and 28S were examined to infer the phylogenetic relationships among the species rich and poorly understood gastropod. Samples were collected from 12 geographic locations in six provinces of southern China. Several methods such as maximum parsimony, maximum likelihood and distance analysis were used in phylogenetic analyses among these taxa. The resultant phylogenetic trees showed a similar topology irrespective of the phylogenetic methods used. The taxa fell into two clades, with those from Fujian, Guangxi, and Zhejiang Provinces in one clade and those from Hunan, Sichuan and Hubei in the other. Among the taxa in Hubei Province, five formed a monophyletic clade, but Tricula sp. H-SHY fell into a sister clade of Tricula hortensis of Sichuan, whilst Tricula hongshanensis formed a single clade. Sister taxa Tricula pingi and Tricula hsiangi formed well-supported clade within almost all the trees. These results, while preliminary, represent the first attempt to reconstruct a phylogeny for Triculinae across China.

The distribution of Mekong schistosomiasis, past and future: preliminary indications from an analysis of genetic variation in the intermediate host.

Neotricula aperta is the only known intermediate host of Schistosoma mekongi which infects humans in Cambodia and the southern tip of Lao PDR. DNA-sequence data (partial rrnL, i.e., mitochondrial 16S large ribosomal-RNA gene) were obtained for 359 N. aperta snails sampled at 31 localities in Cambodia, Lao PDR and Thailand. A nested clade analysis was performed to detect and evaluate any geographical patterns in the observed variation and to identify genetic subpopulations or clades. Coalescent simulations were used to compare different historical biogeographical hypotheses for N. aperta and S. mekongi. A coalescent based method was also used to provide maximum likelihood estimates (MLEs) for effective populations sizes and historical growth and migration rates. Dates were also estimated for phylogenetic events on the gene tree reconstructed for the sampled haplotypes (e.g. the time to most recent common ancestor). N. aperta was found to be divided into two monophyletic clades, a spring-dwelling form of northern Lao PDR and a more widespread larger-river dwelling form of southern Lao PDR and Cambodia; this divergence was dated at 9.3 Ma. The populations with the largest estimated population sizes were found in the Mekong River of Lao PDR and Cambodia; these, together with those of the rivers of eastern Cambodia, appeared to have been the fastest growing populations. Dominant levels of gene-flow (migration) were apparent in a South to North direction, particularly out of seeder populations in the Cambodian Mekong River. The radiation of N. aperta into sub-clades across Cambodia and Lao PDR is dated at around 5 Ma. The findings suggest that historical events, rather than ecology, might best explain the absence of S. mekongi from most of Lao PDR. The public health implications of these findings are discussed, as are pointers for future studies and surveillance.

DNA-sequence variation among Schistosoma mekongi populations and related taxa; phylogeography and the current distribution of Asian schistosomiasis.

BACKGROUND: Schistosomiasis in humans along the lower Mekong River has proven a persistent public health problem in the region. The causative agent is the parasite Schistosoma mekongi (Trematoda: Digenea). A new transmission focus is reported, as well as the first study of genetic variation among S. mekongi populations. The aim is to confirm the identity of the species involved at each known focus of Mekong schistosomiasis transmission, to examine historical relationships among the populations and related taxa, and to provide data for use (a priori) in further studies of the origins, radiation, and future dispersal capabilities of S. mekongi. METHODOLOGY/PRINCIPAL FINDINGS: DNA sequence data are presented for four populations of S. mekongi from Cambodia and southern Laos, three of which were distinguishable at the COI (cox1) and 12S (rrnS) mitochondrial loci sampled. A phylogeny was estimated for these populations and the other members of the Schistosoma sinensium group. The study provides new DNA sequence data for three new populations and one new locus/population combination. A Bayesian approach is used to estimate divergence dates for events within the S. sinensium group and among the S. mekongi populations. CONCLUSIONS/SIGNIFICANCE: The date estimates are consistent with phylogeographical hypotheses describing a Pliocene radiation of the S. sinensium group and a mid-Pleistocene invasion of Southeast Asia by S. mekongi. The date estimates also provide Bayesian priors for future work on the evolution of S. mekongi. The public health implications of S. mekongi transmission outside the lower Mekong River are also discussed.