Neanderthal behaviour, diet, and disease inferred from ancient DNA in dental calculus.

Recent genomic data have revealed multiple interactions between Neanderthals and modern humans, but there is currently little genetic evidence regarding Neanderthal behaviour, diet, or disease. Here we describe the shotgun-sequencing of ancient DNA from five specimens of Neanderthal calcified dental plaque (calculus) and the characterization of regional differences in Neanderthal ecology. At Spy cave, Belgium, Neanderthal diet was heavily meat based and included woolly rhinoceros and wild sheep (mouflon), characteristic of a steppe environment. In contrast, no meat was detected in the diet of Neanderthals from El Sidrón cave, Spain, and dietary components of mushrooms, pine nuts, and moss reflected forest gathering. Differences in diet were also linked to an overall shift in the oral bacterial community (microbiota) and suggested that meat consumption contributed to substantial variation within Neanderthal microbiota. Evidence for self-medication was detected in an El Sidrón Neanderthal with a dental abscess and a chronic gastrointestinal pathogen (Enterocytozoon bieneusi). Metagenomic data from this individual also contained a nearly complete genome of the archaeal commensal Methanobrevibacter oralis (10.2× depth of coverage)-the oldest draft microbial genome generated to date, at around 48,000 years old. DNA preserved within dental calculus represents a notable source of information about the behaviour and health of ancient hominin specimens, as well as a unique system that is useful for the study of long-term microbial evolution.

Phylodynamics of Enterovirus A71-Associated Hand, Foot, and Mouth Disease in Viet Nam.

UNLABELLED: Enterovirus A71 (EV-A71) is a major cause of hand, foot, and mouth disease (HFMD) and is particularly prevalent in parts of Southeast Asia, affecting thousands of children and infants each year. Revealing the evolutionary and epidemiological dynamics of EV-A71 through time and space is central to understanding its outbreak potential. We generated the full genome sequences of 200 EV-A71 strains sampled from various locations in Viet Nam between 2011 and 2013 and used these sequence data to determine the evolutionary history and phylodynamics of EV-A71 in Viet Nam, providing estimates of the effective reproduction number (Re) of the infection through time. In addition, we described the phylogeography of EV-A71 throughout Southeast Asia, documenting patterns of viral gene flow. Accordingly, our analysis reveals that a rapid genogroup switch from C4 to B5 likely took place during 2012 in Viet Nam. We show that the Re of subgenogroup C4 decreased during the time frame of sampling, whereas that of B5 increased and remained >1 at the end of 2013, corresponding to a rise in B5 prevalence. Our study reveals that the subgenogroup B5 virus that emerged into Viet Nam is closely related to variants that were responsible for large epidemics in Malaysia and Taiwan and therefore extends our knowledge regarding its associated area of endemicity. Subgenogroup B5 evidently has the potential to cause more widespread outbreaks across Southeast Asia. IMPORTANCE: EV-A71 is one of many viruses that cause HFMD, a common syndrome that largely affects infants and children. HFMD usually causes only mild illness with no long-term consequences. Occasionally, however, severe infection may arise, especially in very young children, causing neurological complications and even death. EV-A71 is highly contagious and is associated with the most severe HFMD cases, with large and frequent epidemics of the virus recorded worldwide. Although major advances have been made in the development of a potential EV-A71 vaccine, there is no current prevention and little is known about the patterns and dynamics of EV-A71 spread. In this study, we utilize full-length genome sequence data obtained from HFMD patients in Viet Nam, a geographical region where the disease has been endemic since 2003, to characterize the phylodynamics of this important emerging virus.

A complex mosaic of enteroviruses shapes community-acquired hand, foot and mouth disease transmission and evolution within a single hospital.

Human enteroviruses (EV) pose a major risk to public health. This is especially so in the Asia-Pacific region where increasing numbers of hand, foot and mouth disease (HFMD) cases and large outbreaks of severe neurological disease associated with EV-A71 have occurred. Despite their importance, key aspects of the emergence, epidemiology and evolution of EVs remain unclear, and most studies of EV evolution have focused on a limited number of genes. Here, we describe the genomic-scale evolution of EV-A viruses sampled from pediatric patients with mild disease attending a single hospital in western Sydney, Australia, over an 18-month period. This analysis revealed the presence of eight viral serotypes-Coxsackievirus (CV) A2, A4, A5, A6, A8, A10, A16 and EV-A71-with up to four different serotypes circulating in any 1 month. Despite an absence of large-scale outbreaks, high levels of geographical and temporal mixing of serotypes were identified. Phylogenetic analysis revealed that multiple strains of the same serotype were present in the community, and that this diversity was shaped by multiple introductions into the Sydney population, with only a single lineage of CV-A6 exhibiting in situ transmission over the entire study period. Genomic-scale analyses also revealed the presence of novel and historical EV recombinants. Notably, our analysis revealed no association between viral phylogeny, including serotype, and patient age, sex, nor disease severity (for uncomplicated disease). This study emphasizes the contribution of EV-A viruses other than EV-A71 to mild EV disease including HFMD in Australia and highlights the need for greater surveillance of these viruses to improve strategies for outbreak preparedness and vaccine design.

Plasmodium falciparum malaria in 1st-2nd century CE southern Italy.

The historical record attests to the devastation malaria exacted on ancient civilizations, particularly the Roman Empire [1]. However, evidence for the presence of malaria during the Imperial period in Italy (1st-5th century CE) is based on indirect sources, such as historical, epigraphic, or skeletal evidence. Although these sources are crucial for revealing the context of this disease, they cannot establish the causative species of Plasmodium. Importantly, definitive evidence for the presence of malaria is now possible through the implementation of ancient DNA technology. As malaria is presumed to have been at its zenith during the Imperial period [1], we selected first or second molars from 58 adults from three cemeteries from this time: Isola Sacra (associated with Portus Romae, 1st-3rd century CE), Velia (1st-2nd century CE), and Vagnari (1st-4th century CE). We performed hybridization capture using baits designed from the mitochondrial (mtDNA) genomes of Plasmodium spp. on a prioritized subset of 11 adults (informed by metagenomic sequencing). The mtDNA sequences generated provided compelling phylogenetic evidence for the presence of P. falciparum in two individuals. This is the first genomic data directly implicating P. falciparum in Imperial period southern Italy in adults.

Fourteen types of co-circulating recombinant enterovirus were associated with hand, foot, and mouth disease in children from Wenzhou, China.

BACKGROUND: Although hand, foot, and mouth disease (HFMD) is a major public concern in China, the prevalence and clinical symptoms associated with the different agents of HFMD in this country remain poorly understood. OBJECTIVES: We investigated the clinical and molecular characteristics of enteroviruses in patients with HFMD from Wenzhou, China. STUDY DESIGN: Patients with laboratory-confirmed HFMD admitted to the Yuying Children's Hospital in Wenzhou, China during 2013 were included in this study. Viral RNA sequences were amplified using RT-PCR, determined by sequencing, and compared by phylogenetic analysis. RESULTS: A total of 955 clinically diagnosed HFMD cases were determined using PCR, with whole viral genomes obtained for each enterovirus type. 14 types of enterovirus belonging to two viral species were identified. Notably, Coxsackievirus A6 (CV-A6) was the most common species detected (77.8%), followed by EV-A71 (8.2%) and CV-A10 (8.1%). Phylogenetic analysis revealed multiple independent introductions of these viruses into Wenzhou. In addition, the enterovirus observed in Wenzhou had a recombinant history, with two or three recombination breakpoints. Although the illness associated with CV-A6 was milder than that of EV-A71, CV-A6 infection caused more widespread rash, larger blisters, and subsequent skin peeling and/or nail shedding. CONCLUSION: Our study revealed the co-circulation of 14 types of enteroviruses in a single location - Wenzhou, China - with CV-A6 virus the predominant agent of HFMD. This work highlights the need to perform larger-scale surveillance to fully understand the epidemiology of enteroviruses in China and the wider Asia-Pacific region.

A generic assay for whole-genome amplification and deep sequencing of enterovirus A71.

Enterovirus A71 (EV-A71) has emerged as the most important cause of large outbreaks of severe and sometimes fatal hand, foot and mouth disease (HFMD) across the Asia-Pacific region. EV-A71 outbreaks have been associated with (sub)genogroup switches, sometimes accompanied by recombination events. Understanding EV-A71 population dynamics is therefore essential for understanding this emerging infection, and may provide pivotal information for vaccine development. Despite the public health burden of EV-A71, relatively few EV-A71 complete-genome sequences are available for analysis and from limited geographical localities. The availability of an efficient procedure for whole-genome sequencing would stimulate effort to generate more viral sequence data. Herein, we report for the first time the development of a next-generation sequencing based protocol for whole-genome sequencing of EV-A71 directly from clinical specimens. We were able to sequence viruses of subgenogroup C4 and B5, while RNA from culture materials of diverse EV-A71 subgenogroups belonging to both genogroup B and C was successfully amplified. The nature of intra-host genetic diversity was explored in 22 clinical samples, revealing 107 positions carrying minor variants (ranging from 0 to 15 variants per sample). Our analysis of EV-A71 strains sampled in 2013 showed that they all belonged to subgenogroup B5, representing the first report of this subgenogroup in Vietnam. In conclusion, we have successfully developed a high-throughput next-generation sequencing-based assay for whole-genome sequencing of EV-A71 from clinical samples.

Biodiversity and evolution of Imjin virus and Thottapalayam virus in Crocidurinae shrews in Zhejiang Province, China.

The recent discovery of numerous hantaviruses in insectivores has provided a new view of hantavirus biodiversity and evolution. To determine the presence and genetic diversity of Imjin virus (MJNV) and Thottapalayam virus (TPMV) in insectivores in Zhejiang Province, China, we captured and performed virus screening of 32 Ussuri white-toothed shrews (Crocidura lasiura) and 105 Asian house shrews (Suncus murinus) in different coastal regions. Hantavirus genome (S, M, and L segments) sequences were successfully recovered from one Ussuri white-toothed shrew and seven Asian house shrews. Phylogenetic analysis revealed that the virus carried by the Ussuri white-toothed shrew was most closely related to MJNV, but with >15% nucleotide sequence difference, suggesting that it represents a new subtype. The hantaviruses carried by Asian house shrews were closely related to the TPMV variants found in the same geographic area, but more distantly related to those sampled in India and Nepal. Additionally, the TPMV sequences obtained in this study, as well as those found previously in this area, could be divided into three lineages reflecting their geographic origins, indicative of largely allopatric evolution. Overall, our data highlights the high genetic diversity of insectivore-borne hantaviruses in China, suggesting that more may be discovered in the future.

Yersinia pestis and the plague of Justinian 541-543 AD: a genomic analysis.

BACKGROUND: Yersinia pestis has caused at least three human plague pandemics. The second (Black Death, 14-17th centuries) and third (19-20th centuries) have been genetically characterised, but there is only a limited understanding of the first pandemic, the Plague of Justinian (6-8th centuries). To address this gap, we sequenced and analysed draft genomes of Y pestis obtained from two individuals who died in the first pandemic. METHODS: Teeth were removed from two individuals (known as A120 and A76) from the early medieval Aschheim-Bajuwarenring cemetery (Aschheim, Bavaria, Germany). We isolated DNA from the teeth using a modified phenol-chloroform method. We screened DNA extracts for the presence of the Y pestis-specific pla gene on the pPCP1 plasmid using primers and standards from an established assay, enriched the DNA, and then sequenced it. We reconstructed draft genomes of the infectious Y pestis strains, compared them with a database of genomes from 131 Y pestis strains from the second and third pandemics, and constructed a maximum likelihood phylogenetic tree. FINDINGS: Radiocarbon dating of both individuals (A120 to 533 AD [plus or minus 98 years]; A76 to 504 AD [plus or minus 61 years]) places them in the timeframe of the first pandemic. Our phylogeny contains a novel branch (100% bootstrap at all relevant nodes) leading to the two Justinian samples. This branch has no known contemporary representatives, and thus is either extinct or unsampled in wild rodent reservoirs. The Justinian branch is interleaved between two extant groups, 0.ANT1 and 0.ANT2, and is distant from strains associated with the second and third pandemics. INTERPRETATION: We conclude that the Y pestis lineages that caused the Plague of Justinian and the Black Death 800 years later were independent emergences from rodents into human beings. These results show that rodent species worldwide represent important reservoirs for the repeated emergence of diverse lineages of Y pestis into human populations. FUNDING: McMaster University, Northern Arizona University, Social Sciences and Humanities Research Council of Canada, Canada Research Chairs Program, US Department of Homeland Security, US National Institutes of Health, Australian National Health and Medical Research Council.