1 Role of Molecular Phylogenetics in Studying Disease Epidemiology and Evolution Jyotsana Dixit Praveen Karanth Lab

2 Molecular Phylogenetics and Infectious diseases  Evaluation of pathways of transmission among several hosts and polarity of transmission events.  Can address whether the genomic record has evidence to support a hypothesis for a particular transmission pathway.  Can reconstruct genomic changes at the level of each nucleotide and unravel parental and descendent strains. Influenza virus AIDS virus Malaria Parasite

3 Role of Molecular Phylogenetics in understanding Influenza Evolution  Influenza A virus is an enveloped, single stranded, negative-sense RNA virus of the family Orthomyxoviridae, with a genome consisting of eight gene segments.  It has been classified into at least 16 HA and 9 NA subtypes according to the distinct antigenic properties of the viral HA and NA surface proteins Influenza viruses are divided into three main types: influenza A, B, and C Webster et al., 1992; Fouchier et al., 2005

4 Influenza A virus becomes pandemic for Humans: Through Zoonotics

5 Influenza A escapes immune response in host population to cause Pandemic Virus infects the host cell Point mutations produces new antigenic variants Due to host immune system pressure new antigenic variants are selected Antigenic Drift Antigenic Shift

6 Highly pathogenic Avian influenza A Caused economic burden through excessive poultry deaths Human influenza pandemic involving avian influenza virus if this virus exchanges genetic material with co-circulating seasonal human influenza viruses Recent emergence of the novel influenza A(H1N1/2009) virus of swine origin Tsan-Yuk Lam et al., 2010

7 Genetic origins of Human pandemic Influenza Molecular phylogenetic analysis of influenza virus in 1980s led to the conclusions about possible origins of human pandemic influenza strains causing 1918, 1957 and 1968 pandemics. Reid and Toubenberger 2003; Fanning et al., 2002 Fig: Phylogenetic tree showing H1 sequences, including the 1918 pandemic strains and the 1917 Brant goose (arrows) (Fanning et al., 2002).

8 Origin of novel pandemic influenza A(H1N1/2009) GJD Smith et al. Nature 459, (2009)

9 Phylogenetic analysis a useful tool Elucidate genetic origin, selection pressures, evolutionary rates, reassortment histories, population dynamics, and migration patterns of influenza viruses in different host populations. Proved to be an essential tool in monitoring influenza virus infection in birds, pigs and humans. In future with the availability of sequence data from larger viral populations will reveal the intricacies of influenza virus evolution at even finer temporal and spatial resolutions.

10 Human Immunodeficiency virus  HIV is an enveloped, diploid, positive-sense, single stranded RNA retrovirus (family Retroviridae) with a 9.75 kb genome.  The virus has an extraordinarily high mutation rate (around 1 error per 3x105 bases replicated), which gives rise to a very diverse viral population, even within one host (often referred to as quasispecies).  This diversity and rapid mutation rate makes it attractive to analyze HIV sequences using phylogenetic techniques, and its sequence diversity has been the focus of various aspects of both clinical and research studies and applications

11 Evolution of HIV HIV belongs to lentivirus subgroup of retroviruses. Sharp and Hahn 2011

12 Origin of AIDS Virus Sharp and Hahn 2011

13  Wertherin and Worobey 2009, used the extent SIV sequences to derive the molecular clocks and estimate the timing of ancestral SIVs.  Found that it is only few 100 Years Ago.  However, recent study by Worobey et al., 2010, taking SIV samples from a wider regions, substantiated the earlier conclusion and found the infection to be present at least 30,000 years ago. Dating SIVs origin

14 Evolutionary history of primate lentivirus is still Incomplete  So far SIV infections only found in African monkeys and apes.  It seems likely that primate lentivirus emerged in Africa sometime after the split between lineages of African and Asian old world monkeys i.e. ~6-10 MYA.  Since neither Asian nor new world primates have been sampled exhaustively, the conclusion that SIVs are restricted to African primates remain tentative. © Zinner et al., 2011

15 Origin of HIV-1Origin of HIV-2 Sharp & Hahn 2011

16 Phylogenetics: an effective tool for tracking disease transmission Transmission of HIV: Identifying and tracking the transmission of HIV infection between individuals in a population is one of the most widely used applications of phylogenetics. One of the most famous, earlier examples of this type of investigation is the case of the HIV-infected Florida dentist who, somehow, infected five of his patients with HIV. Yih Ou et al., 1992

17 Phylogenetics and study of Human malaria parasite evolution  Malaria caused due to infection by protozoan parasite of genus Plasmodium.  Five Human malaria causing species: Plasmodium falciparum Plasmodium vivax Plasmodium ovale Plasmodium malariae Plasmodium knowlesi  P. falciparum and P. vivax has widest distribution, P. falciparum is highest in causing mortality and morbidity.  P. Ovale has most limited distribution of all, endemic to New Guinea and Philippines. Fig: P. falciparum attacking blood cells

18 Molecular Phylogenetics: Study of Human Malaria Parasite Evolution  Human Plasmodium is a polyphylectic genus.  The human malaria parasite species are remotely related to each other.  Suggesting that adaptation to humans has occurred several times independently during the history of the genus.  It is still unclear, however, when these associations began and from where they came. Fig: Phylogenetic relationship among the 17 Plasmodium species inferred from the gene encoding cytochrome b. Escalante et al., 1998

19 Origin of P. falciparum: A highly debated topic Fig: phylogenetic tree of Plasmodium genus based on SSU rRNA gene sequence data.  P. falciparum clustered with two avian parasites rather than with those infecting mammals.  Thus suggesting that P. falciparum was the result of a transfer from birds to humans. Early molecular phylogenetic studies Plasmodium genus Waters et al., 1991 and 1993

Subsequent analyses demonstrated that the closest sister taxon of P. falciparum was Plasmodium reichenowi, a parasite isolated from a chimpanzee. P. falciparum did not directly originate from an avian malarial parasite escalante et al., 1998

21 The results of subsequent studies were contradictory Part of the confusion concerning the origin of P. falciparum arose because of: Biases in the representation of certain taxa, Small number of loci analysed and/or Improper rooting. Closer to rodent or primate Plasmodium Closer to Avian Plasmodium Perkins and Schall 2002; McCutchanet al., 1996

22 Liu et al., 2010 Study: Revealed true evolutionary history of P. falciparum  Utilized non invasive technique  A large collection of fecal samples from: Three subspecies of chimpanzees (P. t. troglodytes, P. troglodytes ellioti), bonobos, and Two subspecies of gorillas (subspecies G. gorilla gorilla and G. gorilla graueri).  Method of single template amplification to sequence mitochondrial, apicoplastic, and nuclear genes of Plasmodium isolates from mixed infections.

23 Phylogenetic evidences points to gorilla origin of Human P. falciparum Liu et al., 2010 Single gorilla to human cross-species transmission

Non-Laverania parasites form a separate clad Liu et al., 2010

25 Plasmodium vivax Origin Fig: The world malaria map showing the distributional pattern of P. vivax malaria across the continents (Guerra, et al., 2010). The black dots are indicating the areas of high populations at risk of P. vivax infections. Distribution correlated with the occurrence of Duffy negative phenotype Fig: Spatial distribution of Duffy negative phenotype

26 P.vivax Origin T-33C point mutation leads to loss of expression of the gene in Erythrocytes thus Duffy Negative phenotype. P. vivax infecti on No P. vivax infection The Duffy antigen receptor for chemokines (DARC) is used by P. vivax trophozoits to invade RBCs

27 The consensus view has thus been that P. vivax emerged in Southeast Asia following the cross-species transmission of a macaque parasite Escalante et al., 2005

Liu et al., 2014 : Large scale Molecular Phylogenetic study to reveal the true history of P. vivax Ape samples: highly endangered and live in remote forests regions thus utilized fecal samples for detection and amplification of malaria parasite DNA fecal samples from non-habituated Apes Wild living Chimpanzees (P. troglodytus) Western Gorillas (G. gorilla) Eastern Gorillas (G. beringei) Bonobos (P. paniscus) 998 Blood samples from Macaques Liu et al., 2014

29 Ape P. vivax Human P. vivax Human P. vivax arose from within a Plasmodium species that infects chimpanzees and gorilla and indicate an origin in Africa rather than Asia (as previously assumed). Liu et al., 2014

30 Few Limitations of Molecular Phylogenetics Available sequence data Length of sequences and the choice of genomic regions to be used in the analysis Continuous updation and reanalysis of existing Phylogenetic relationships Computing power required to handle and analyze longer and longer sequences involving more complex Phylogenetic techniques

31 Yet despite all these limitations Phylogenetics is currently a rapidly expanding specialty, contributing useful insights to the understanding of how diseases have evolved and how they continue to evolve…. (Yuk Lam et al., 2010)

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