Kyriakos Tsangaras

One Hundred Twenty Years of Koala Retrovirus Evolution Determined from Museum Skins

Although endogenous retroviruses are common across vertebrate genomes, the koala retrovirus (KoRV) is the only retrovirus known to be currently invading the germ line of its host. KoRV is believed to have first infected koalas in northern Australia less than two centuries ago. We examined KoRV in 28 koala museum skins collected in the late 19th and 20th centuries and deep sequenced the complete proviral envelope region from five northern Australian specimens. Strikingly, KoRV env sequences were conserved among koalas collected over the span of a century, and two functional motifs that affect viral infectivity were fixed across the museum koala specimens. We detected only 20 env polymorphisms among the koalas, likely representing derived mutations subject to purifying selection. Among northern Australian koalas, KoRV was already ubiquitous by the late 19th century, suggesting that KoRV evolved and spread among koala populations more slowly than previously believed. Given that museum and modern koalas share nearly identical KoRV sequences, it is likely that koala populations, for more than a century, have experienced increased susceptibility to diseases caused by viral pathogenesis.

A Potentially Fatal Mix of Herpes in Zoos

Pathogens often have a limited host range, but some can opportunistically jump to new species. Anthropogenic activities that mix reservoir species with novel, hence susceptible, species can provide opportunities for pathogens to spread beyond their normal host range. Furthermore, rapid evolution can produce new pathogens by mechanisms such as genetic recombination. Zoos unintentionally provide pathogens with a high diversity of species from different continents and habitats assembled within a confined space. Institutions alert to the problem of pathogen spread to unexpected hosts can monitor the emergence of pathogens and take preventative measures. However, asymptomatic infections can result in the causative pathogens remaining undetected in their reservoir host. Furthermore, pathogen spread to unexpected hosts may remain undiagnosed if the outcome of infection is limited, as in the case of compromised fertility, or if more severe outcomes are restricted to less charismatic species that prompt only limited investigation. We illustrate this problem here with a recombinant zebra herpesvirus infecting charismatic species including zoo polar bears over at least four years. The virus may cause fatal encephalitis and infects at least five mammalian orders, apparently without requiring direct contact with infected animals.

Hybridization Capture Reveals Evolution and Conservation across the Entire Koala Retrovirus Genome

The koala retrovirus (KoRV) is the only retrovirus known to be in the midst of invading the germ line of its host species. Hybridization capture and next generation sequencing were used on modern and museum DNA samples of koala (Phascolarctos cinereus) to examine ca. 130 years of evolution across the full KoRV genome. Overall, the entire proviral genome appeared to be conserved across time in sequence, protein structure and transcriptional binding sites. A total of 138 polymorphisms were detected, of which 72 were found in more than one individual. At every polymorphic site in the museum koalas, one of the character states matched that of modern KoRV. Among non-synonymous polymorphisms, radical substitutions involving large physiochemical differences between amino acids were elevated in env, potentially reflecting anti-viral immune pressure or avoidance of receptor interference. Polymorphisms were not detected within two functional regions believed to affect infectivity. Host sequences flanking proviral integration sites were also captured; with few proviral loci shared among koalas. Recently described variants of KoRV, designated KoRV-B and KoRV-J, were not detected in museum samples, suggesting that these variants may be of recent origin.

Museums and disease: using tissue archive and museum samples to study pathogens.

Molecular studies of archival and fossil samples have traditionally focused on the nucleic acids derived from the host species. However, there has recently been an increase in ancient DNA research on the identification and characterization of infectious agents within the hosts. The study of pathogens from the past provides great opportunities for discovering the causes of historical infection events, characterizing host-microorganism co-evolution and directly investigating the evolution of specific pathogens. Several research teams have been able to isolate and characterize a variety of different bacterial, parasite and viral microorganisms. However, this emerging field is not without obstacles. The diagenetic processes that make ancient DNA research generally difficult are also impediments to ancient pathogen research and perhaps more so given that their DNA may represent an even rarer proportion of the remaining nucleic acids in a fossil sample than host DNA. However, studies performed under controlled conditions and following stringent ancient DNA protocols can and have yielded reliable and often surprising results. This article reviews the advantages, problems, and failures of ancient microbiological research.

Biogeography and taxonomy of extinct and endangered monk seals illuminated by ancient DNA and skull morphology

Abstract Extinctions and declines of large marine vertebrates have major ecological impacts and are of critical concern in marine environments. The Caribbean monk seal, Monachus tropicalis, last definitively reported in 1952, was one of the few marine mammal species to become extinct in historical times. Despite its importance for understanding the evolutionary biogeography of southern phocids, the relationships of M. tropicalis to the two living species of critically endangered monk seals have not been resolved. In this study we present the first molecular data for M. tropicalis, derived from museum skins. Phylogenetic analysis of cytochrome b sequences indicates that M. tropicalis was more closely related to the Hawaiian rather than the Mediterranean monk seal. Divergence time estimation implicates the formation of the Panamanian Isthmus in the speciation of Caribbean and Hawaiian monk seals. Molecular, morphological and temporal divergence between the Mediterranean and “New World monk seals” (Hawaiian and Caribbean) is profound, equivalent to or greater than between sister genera of phocids. As a result, we classify the Caribbean and Hawaiian monk seals together in a newly erected genus, Neomonachus. The two genera of extant monk seals (Monachus and Neomonachus) represent old evolutionary lineages each represented by a single critically endangered species, both warranting continuing and concerted conservation attention and investment if they are to avoid the fate of their Caribbean relative.

Historically low mitochondrial DNA diversity in koalas (Phascolarctos cinereus)

BackgroundThe koala (Phascolarctos cinereus) is an arboreal marsupial that was historically widespread across eastern Australia until the end of the 19th century when it suffered a steep population decline. Hunting for the fur trade, habitat conversion, and disease contributed to a precipitous reduction in koala population size during the late 1800s and early 1900s. To examine the effects of these reductions in population size on koala genetic diversity, we sequenced part of the hypervariable region of mitochondrial DNA (mtDNA) in koala museum specimens collected in the 19th and 20th centuries, hypothesizing that the historical samples would exhibit greater genetic diversity.ResultsThe mtDNA haplotypes present in historical museum samples were identical to haplotypes found in modern koala populations, and no novel haplotypes were detected. Rarefaction analyses suggested that the mtDNA genetic diversity present in the museum samples was similar to that of modern koalas.ConclusionsLow mtDNA diversity may have been present in koala populations prior to recent population declines. When considering management strategies, low genetic diversity of the mtDNA hypervariable region may not indicate recent inbreeding or founder events but may reflect an older historical pattern for koalas.

Evolutionary Relationships among Extinct and Extant Sloths: The Evidence of Mitogenomes and Retroviruses

Macroevolutionary trends exhibited by retroviruses are complex and not entirely understood. The sloth endogenized foamy-like retrovirus (SloEFV), which demonstrates incongruence in virus–host evolution among extant sloths (Order Folivora), has not been investigated heretofore in any extinct sloth lineages and its premodern history within folivorans is therefore unknown. Determining retroviral coevolutionary trends requires a robust phylogeny of the viral host, but the highly reduced modern sloth fauna (6 species in 2 genera) does not adequately represent what was once a highly diversified clade (∼100 genera) of placental mammals. At present, the amount of molecular data available for extinct sloth taxa is limited, and analytical results based on these data tend to conflict with phylogenetic inferences made on the basis of morphological studies. To augment the molecular data set, we applied hybridization capture and next-generation Illumina sequencing to two extinct and three extant sloth species to retrieve full mitochondrial genomes (mitogenomes) from the hosts and the polymerase gene of SloEFV. The results produced a fully resolved and well-supported phylogeny that supports dividing crown families into two major clades: 1) The three-toed sloth, Bradypus, and Nothrotheriidae and 2) Megalonychidae, including the two-toed sloth, Choloepus, and Mylodontidae. Our calibrated time tree indicates that the Miocene epoch (23.5 Ma), particularly its earlier part, was an important interval for folivoran diversification. Both extant and extinct sloths demonstrate multiple complex invasions of SloEFV into the ancestral sloth germline followed by subsequent introgressions across different sloth lineages. Thus, sloth mitogenome and SloEFV evolution occurred separately and in parallel among sloths.

Cell-Free DNA Analysis of Targeted Genomic Regions in Maternal Plasma for Non-Invasive Prenatal Testing of Trisomy 21, Trisomy 18, Trisomy 13, and Fetal Sex

BACKGROUND There is great need for the development of highly accurate cost effective technologies that could facilitate the widespread adoption of noninvasive prenatal testing (NIPT). METHODS We developed an assay based on the targeted analysis of cell-free DNA for the detection of fetal aneuploidies of chromosomes 21, 18, and 13. This method enabled the capture and analysis of selected genomic regions of interest. An advanced fetal fraction estimation and aneuploidy determination algorithm was also developed. This assay allowed for accurate counting and assessment of chromosomal regions of interest. The analytical performance of the assay was evaluated in a blind study of 631 samples derived from pregnancies of at least 10 weeks of gestation that had also undergone invasive testing. RESULTS Our blind study exhibited 100% diagnostic sensitivity and specificity and correctly classified 52/52 (95% CI, 93.2%-100%) cases of trisomy 21, 16/16 (95% CI, 79.4%-100%) cases of trisomy 18, 5/5 (95% CI, 47.8%-100%) cases of trisomy 13, and 538/538 (95% CI, 99.3%-100%) normal cases. The test also correctly identified fetal sex in all cases (95% CI, 99.4%-100%). One sample failed prespecified assay quality control criteria, and 19 samples were nonreportable because of low fetal fraction. CONCLUSIONS The extent to which free fetal DNA testing can be applied as a universal screening tool for trisomy 21, 18, and 13 depends mainly on assay accuracy and cost. Cell-free DNA analysis of targeted genomic regions in maternal plasma enables accurate and cost-effective noninvasive fetal aneuploidy detection, which is critical for widespread adoption of NIPT.

Hybridization Capture Using Short PCR Products Enriches Small Genomes by Capturing Flanking Sequences (CapFlank)

Solution hybridization capture methods utilize biotinylated oligonucleotides as baits to enrich homologous sequences from next generation sequencing (NGS) libraries. Coupled with NGS, the method generates kilo to gigabases of high confidence consensus targeted sequence. However, in many experiments, a non-negligible fraction of the resulting sequence reads are not homologous to the bait. We demonstrate that during capture, the bait-hybridized library molecules add additional flanking library sequences iteratively, such that baits limited to targeting relatively short regions (e.g. few hundred nucleotides) can result in enrichment across entire mitochondrial and bacterial genomes. Our findings suggest that some of the off-target sequences derived in capture experiments are non-randomly enriched, and that CapFlank will facilitate targeted enrichment of large contiguous sequences with minimal prior target sequence information.

Sequence variation of koala retrovirus transmembrane protein p15E among koalas from different geographic regions

The koala retrovirus (KoRV), which is transitioning from an exogenous to an endogenous form, has been associated with high mortality in koalas. For other retroviruses, the envelope protein p15E has been considered a candidate for vaccine development. We therefore examined proviral sequence variation of KoRV p15E in a captive Queensland and three wild southern Australian koalas. We generated 163 sequences with intact open reading frames, which grouped into 39 distinct haplotypes. Sixteen distinct haplotypes comprising 139 of the sequences (85%) coded for the same polypeptide. Among the remaining 23 haplotypes, 22 were detected only once among the sequences, and each had 1 or 2 non-synonymous differences from the majority sequence. Several analyses suggested that p15E was under purifying selection. Important epitopes and domains were highly conserved across the p15E sequences and in previously reported exogenous KoRVs. Overall, these results support the potential use of p15E for KoRV vaccine development.