PhyloJS: Bridging phylogenetics and web development with a JavaScript utility library.

There is an increasing number of libraries devoted to parsing, manipulating and visualising phylogenetic trees in JavaScript. Many of these libraries bundle tree manipulation with visualisation, but have limited ability to manipulate trees and lack detailed documentation. As the number of web-based phylogenetic tools and the size of phylogenetics datasets increases, there is a need for a library that parses, writes and manipulates phylogenetic trees that is interoperable with other phylogenetic and data visualisation libraries. Here we introduce PhyloJS, a light zero-dependency TypeScript and JavaScript library for reading, writing and manipulating phylogenetic trees. PhyloJS allows for modification of and data-extraction from trees to integrate with other phylogenetics and data visualisation libraries. It can swiftly handle large trees, up to at least 10 6 tips in size, making it ideal for developing the next generation of more complex web-based phylogenetics applications handling ever larger datasets. The PhyloJS source code is available on GitHub (https://github.com/clockor2/phylojs) and can be installed via npm with the command npm install phylojs. Extensive documentation is available at https://clockor2.github.io/phylojs/.

No Ranaviral DNA Found in Australian Freshwater Turtles, 2014-19, Despite Previous Serologic Evidence.

Ranaviruses are pathogens of ectothermic vertebrates (fish, amphibians, and reptiles). Turtles are the most common group of reptiles reported with ranaviral infections. However, there have been no surveys for wild ranaviral infection in any turtles from the suborder Pleurodira, despite ranaviral distributions and experimentally susceptible pleurodiran turtle populations overlapping in several areas, including Australia. We assayed 397 pooled blood samples from six Australian freshwater turtle species collected from five different sites in northern Australia between 2014 and 2019. Historical serologic surveys in the area had found antiranaviral antibodies; however, we did not detect any ranaviral DNA in our samples. Discrepancies between historical serologic and our molecular results may be explained by low viral prevalence during the years that these samples were collected, survivorship bias, or possibly an age class bias in sampling.

Clockor2: Inferring global and local strict molecular clocks using root-to-tip regression.

Molecular sequence data from rapidly evolving organisms are often sampled at different points in time. Sampling times can then be used for molecular clock calibration. The root-to-tip (RTT) regression is an essential tool to assess the degree to which the data behave in a clock-like fashion. Here, we introduce Clockor2, a client-side web application for conducting RTT regression. Clockor2 allows users to quickly fit local and global molecular clocks, thus handling the increasing complexity of genomic datasets that sample beyond the assumption of homogeneous host populations. Clockor2 is efficient, handling trees of up to the order of 104 tips, with significant speed increases compared to other RTT regression applications. Although clockor2 is written as a web application, all data processing happens on the client-side, meaning that data never leaves the user's computer. Clockor2 is freely available at https : //clockor2.github.io/.

Pathogenesis of Bohle iridovirus infection in Krefft's freshwater turtle hatchlings (Emydura macquarii krefftii).

Ranaviruses have been detected in over 12 families of reptiles including many genera of turtles, tortoises, and terrapins, but the pathogenesis of these infections is still poorly understood. Krefft's river turtle hatchlings (N = 36; Emydura macquarii krefftii) were inoculated intramuscularly with Bohle iridovirus (BIV, Ranavirus, isolate) or saline, and euthanized at 9 timepoints (3 infected and 1 control per timepoint) over a 24-day period. Samples of lung, liver, kidney, and spleen were collected for quantitative polymerase chain reaction (PCR); internal organs, skin, and oral cavity samples were fixed for histopathological examination. The earliest lesions, at 8 days postinoculation (dpi), were lymphocytic inflammation of the skin and fibrinoid necrosis of regional vessels at the site of inoculation, and mild ulcerative necrosis with lymphocytic and heterophilic inflammation in the oral, nasal, and tongue mucosae. Fibrinonecrotic foci with heterophilic inflammation were detected in spleen and gonads at 16 dpi. Multifocal hepatic necrosis, heterophilic inflammation, and occasional basophilic intracytoplasmic inclusion bodies were observed at 20 dpi, along with ulcerative lymphocytic and heterophilic tracheitis and bronchitis. Tracheitis, bronchitis, and rare bone marrow necrosis were present at 24 dpi. Of the viscera tested for ranaviral DNA by PCR, the liver and spleen had the highest viral loads throughout infection, and thus appeared to be major targets of viral replication. Testing of whole blood by qPCR was the most-effective ante-mortem method for detecting ranaviral infection compared with oral swabs. This study represents the first time-dependent pathogenesis study of a ranaviral infection in turtles.

Phytest: quality control for phylogenetic analyses.

MOTIVATION: The ability to automatically conduct quality control checks on phylogenetic analyses is becoming more important with the increase in genetic sequencing and the use of real-time pipelines e.g. in the SARS-CoV-2 era. Implementations of real-time phylogenetic analyses require automated testing to make sure that problems in the data are caught automatically within analysis pipelines and in a timely manner. Here, we present Phytest (version 1.1) a tool for automating quality control checks on sequences, trees and metadata during phylogenetic analyses. RESULTS: Phytest is a phylogenetic analysis testing program that easily integrates into existing phylogenetic pipelines. We demonstrate the utility of Phytest with real-world examples. AVAILABILITY AND IMPLEMENTATION: Phytest source code is available on GitHub (https://github.com/phytest-devs/phytest) and can be installed via PyPI with the command 'pip install phytest'. Extensive documentation can be found at https://phytest-devs.github.io/phytest/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Real-Time and Remote MCMC Trace Inspection with Beastiary.

Bayesian phylogenetics has gained substantial popularity in the last decade, with most implementations relying on Markov chain Monte Carlo (MCMC). The computational demands of MCMC mean that remote servers are increasingly used. We present Beastiary, a package for real-time and remote inspection of log files generated by MCMC analyses. Beastiary is an easily deployed web-app that can be used to summarize and visualize the output of many popular software packages including BEAST, BEAST2, RevBayes, and MrBayes via a web browser. We describe the design and implementation of Beastiary and some typical use-cases, with a focus on real-time remote monitoring.

The Emergence of SARS-CoV-2 Variants of Concern Is Driven by Acceleration of the Substitution Rate.

The ongoing SARS-CoV-2 pandemic has seen an unprecedented amount of rapidly generated genome data. These data have revealed the emergence of lineages with mutations associated to transmissibility and antigenicity, known as variants of concern (VOCs). A striking aspect of VOCs is that many of them involve an unusually large number of defining mutations. Current phylogenetic estimates of the substitution rate of SARS-CoV-2 suggest that its genome accrues around two mutations per month. However, VOCs can have 15 or more defining mutations and it is hypothesized that they emerged over the course of a few months, implying that they must have evolved faster for a period of time. We analyzed genome sequence data from the GISAID database to assess whether the emergence of VOCs can be attributed to changes in the substitution rate of the virus and whether this pattern can be detected at a phylogenetic level using genome data. We fit a range of molecular clock models and assessed their statistical performance. Our analyses indicate that the emergence of VOCs is driven by an episodic increase in the substitution rate of around 4-fold the background phylogenetic rate estimate that may have lasted several weeks or months. These results underscore the importance of monitoring the molecular evolution of the virus as a means of understanding the circumstances under which VOCs may emerge.

Leech removal is not the primary driver of basking behavior in a freshwater turtle.

Leaving the water to bask (usually in the sun) is a common behavior for many freshwater turtles, with some species also engaging in "nocturnal basking." Ectoparasite removal is an obvious hypothesis to explain nocturnal basking and has also been proposed as a key driver of diurnal basking. However, the efficacy of basking, day or night, to remove leeches has not been experimentally tested. Therefore, we examined the number of leeches that were removed from Krefft's river turtles (Emydura macquarii krefftii) after experimentally making turtles bask at a range of times of day, durations, and temperatures. Turtles had high initial leech loads, with a mean of 32.1 leeches per turtle. Diurnal basking under a heat lamp for 3 hr at ~28°C significantly reduced numbers of leeches relative to controls. In diurnal trials, 90.9% of turtles lost leeches (mean loss of 7.1 leeches per turtle), whereas basking for 30 min under the same conditions was not effective (no turtles lost leeches, and all turtles were still visibly wet). Similarly, "nocturnal basking" at ~23°C for 3 hr was not effective at removing leeches. Only 18% of turtles lost leeches (one turtle lost one leech and another lost four leeches). Diurnal basking outdoors under direct sunlight for 20 min (mean temp = 34.5°C) resulted in a small reduction in leeches, with 50% of turtles losing leeches and an average loss of 0.7 leeches per turtle. These results indicate basking can remove leeches if temperatures are high or basking durations are long. However, it was only effective at unusually long basking durations in this system. Our data showed even the 20-min period was longer than 70.1% of natural diurnal basking events, many of which took place at cooler temperatures. Therefore, leech removal does not appear to be the purpose of the majority of basking events.

The Concurrent Detection of Chelonid Alphaherpesvirus 5 and Chelonia mydas Papillomavirus 1 in Tumoured and Non-Tumoured Green Turtles.

Characterised by benign tumours, fibropapillomatosis (FP) is a debilitating disease that predominantly afflicts the endangered green turtle (Chelonia mydas). A growing body of histological and molecular evidence has associated FP tumours with Chelonid alphaherpesvirus 5 (ChHV5). However, a recent study which detected both ChHV5 and Chelonia mydas papillomavirus 1 (CmPV1) DNA in FP tumour tissues has challenged this hypothesis. The present study aimed to establish a probe-based qPCR to assess the wider prevalence of CmPV1 and co-occurrence with ChHV5 in 275 marine turtles foraging in waters adjacent to the east coast of Queensland, Australia: three categories: Group A (FP tumours), Group B (non-tumoured skin from FP turtles) and Group C (non-tumoured skin from turtles without FP). Concurrent detection of ChHV5 and CmPV1 DNA is reported for all three categories, where Group A had the highest rate (43.5%). ChHV5 viral loads in Group A were significantly higher than loads seen in Group B and C. This was not the case for CmPV1 where the loads in Group B were highest, followed by Group A. However, the mean CmPV1 load for Group A samples was not significantly different to the mean load reported from Group B or C samples. Collectively, these results pivot the way we think about FP; as an infectious disease where two separate viruses may be at play.

Cutaneous Lesions in Freshwater Turtles (Emydura macquarii krefftii and Myuchelys latisternum) in a Rainforest Creek in North Queensland, Australia.

Freshwater turtles inhabit most rivers and creeks on the east coast of Australia, but some species are only found in specific catchments, which makes them vulnerable to extinction. During annual fieldtrips to Alligator Creek, North Queensland, the resident population of Myuchelys latisternum and Emydura macquarii krefftii in a natural pond, just outside Bowling Green National Park, have been surveyed for a number of years and demographic data recorded against tagged turtles. Rounded, cutaneous lesions on individual animals were first noted in August 2016, three years after the first survey of the population. Turtles living in the upstream sections of the creek were not affected. An initial investigation into the cause of the lesions ruled out pollutants and although the bacterial communities appeared to be different on turtles with lesions, a causative agent was not identified. Attempts to isolate virus in culture was not successful and specific PCRs for ranavirus, papillomavirus, adenovirus and herpesvirus did not identify their presence. Blood biochemical parameters, body condition and activity levels were not significantly different between affected turtles and those without lesions. The turtles in this pond were monitored regularly over the following three years with 249 M. latisternum and 192 E. m. krefftii captured, tagged and released. The prevalence of the lesions fluctuated with season from 0 to 77 and 68% respectively, but did not vary significantly between species or sex in adults. There was a tendency for larger animals to be more likely to have lesions. The position of the lesions on the turtles was mostly on dorsal surfaces, distally on the legs and proximal on the tales of males, indicating that the initial lesion may have been associated with a behaviourally induced trauma. Recaptured animals (n = 43) during this period, provided records of lesion progression over time and while some healed up between capture events, others persisted for up to 24 months. Some turtles were repeatedly captured without lesions. Intra-species aggression associated with seasonal behaviours could potentially be the primary cause of skin trauma, followed by a secondary invasion of an unusual pathogen present in the environment.

Dose-dependent morbidity of freshwater turtle hatchlings, Emydura macquarii krefftii, inoculated with Ranavirus isolate (Bohle iridovirus, Iridoviridae).

Ranaviral infections cause mass die-offs in wild and captive turtle populations. Two experimental studies were performed to first determine the susceptibility of an Australian turtle species (Emydura macquarii krefftii) to different routes of infection and second examine the effect of viral titre on the morbidity in hatchlings. All inoculation routes (intracoelomic, intramuscular and oral) produced disease, but the clinical signs, histopathology and time to onset of disease varied with the route. The median infectious and lethal doses for intramuscularly inoculated hatchlings were 102.52 (1.98-2.93) and 104.43 (3.81-5.19) TCID50 ml-1, respectively. Clinical signs began 14 to 29 days post-inoculation and the median survival time was 22 days (16-25) across all dose groups. For every 10-fold increase in dose, the odds of developing any clinical signs or severe clinical signs increased by 3.39 [P<0.01, 95 % confidence interval (CI): 1.81-6.36] and 3.71 (P<0.01, 95 % CI: 1.76-7.80), respectively. Skin lesions, previously only reported in ranaviral infection in lizards, were observed in the majority of intramuscularly inoculated hatchlings that developed ranaviral disease. The histological changes were consistent with those in previous reports for reptiles and consisted of necrosis at or near the site of injection, in the spleen, liver and oral cavity. Systemic inflammation was also observed, predominantly affecting necrotic organs. The estimates reported here can be used to model ranaviral disease and quantify and manage at-risk populations.

Ranaviruses and reptiles.

Ranaviruses can infect many vertebrate classes including fish, amphibians and reptiles, but for the most part, research has been focused on non-reptilian hosts, amphibians in particular. More recently, reports of ranaviral infections of reptiles are increasing with over 12 families of reptiles currently susceptible to ranaviral infection. Reptiles are infected by ranaviruses that are genetically similar to, or the same as, the viruses that infect amphibians and fish; however, physiological and ecological differences result in differences in study designs. Although ranaviral disease in reptiles is often influenced by host species, viral strain and environmental differences, general trends in pathogenesis are emerging. More experimental studies using a variety of reptile species, life stages and routes of transmission are required to unravel the complexity of wild ranavirus transmission. Further, our understanding of the reptilian immune response to ranaviral infection is still lacking, although the considerable amount of work conducted in amphibians will serve as a useful guide for future studies in reptiles.

Clinical signs, pathology and dose-dependent survival of adult wood frogs, Rana sylvatica, inoculated orally with frog virus 3 Ranavirus sp., Iridoviridae.

Amphibian populations suffer massive mortalities from infection with frog virus 3 FV3, genus Ranavirus, family Iridoviridae, a pathogen also involved in mortalities of fish and reptiles. Experimental oral infection with FV3 in captive-raised adult wood frogs, Rana sylvatica Lithobates sylvaticus, was performed as the first step in establishing a native North American animal model of ranaviral disease to study pathogenesis and host response. Oral dosing was successful LD50 was 10(2.93 2.423.44) p.f.u. for frogs averaging 35mm in length. Onset of clinical signs occurred 614days post-infection p.i. median 11 days p.i. and time to death was 1014 days p.i. median 12 days p.i.. Each tenfold increase in virus dose increased the odds of dying by 23-fold and accelerated onset of clinical signs and death by approximately 15. Ranavirus DNA was demonstrated in skin and liver of all frogs that died or were euthanized because of severe clinical signs. Shedding of virus occurred in faeces 710 days p.i. 34.5days before death and skin sheds 10 days p.i. 01.5days before death of some frogs dead from infection. Most common lesions were dermal erosion and haemorrhages haematopoietic necrosis in bone marrow, kidney, spleen and liver and necrosis in renal glomeruli, tongue, gastrointestinal tract and urinary bladder mucosa. Presence of ranavirus in lesions was confirmed by immunohistochemistry. Intracytoplasmic inclusion bodies probably viral were present in the bone marrow and the epithelia of the oral cavity, gastrointestinal tract, renal tubules and urinary bladder. Our work describes a ranaviruswood frog model and provides estimates that can be incorporated into ranavirus disease ecology models.