Identification and complete genome sequencing of paramyxoviruses in mallard ducks (Anas platyrhynchos) using random access amplification and next generation sequencing technologies

<p>Abstract</p> <p>Background</p> <p>During a wildlife screening program for avian influenza A viruses (AIV) and avian paramyxoviruses (APMV) in Belgium, we isolated two hemagglutinating agents from pools of cloacal swabs of wild mallards (<it>Anas platyrhynchos</it>) caught in a single sampling site at two different times. AIV and APMV1 were excluded using hemagglutination inhibition (HI) testing and specific real-time RT-PCR tests.</p> <p>Methods</p> <p>To refine the virological identification of APMV2-10 realized by HI subtyping tests and in lack of validated molecular tests for APMV2-10, random access amplification was used in combination with next generation sequencing for the sequence independent identification of the viruses and the determination of their genomes.</p> <p>Results</p> <p>Three different APMVs were identified. From one pooled sample, the complete genome sequence (15054 nucleotides) of an APMV4 was assembled from the random sequences. From the second pooled sample, the nearly complete genome sequence of an APMV6 (genome size of 16236 nucleotides) was determined, as well as a partial sequence for an APMV4. This APMV4 was closely related but not identical to the APMV4 isolated from the first sample. Although a cross-reactivity with other APMV subtypes did not allow formal identification, the HI subtyping revealed APMV4 and APMV6 in the respective pooled samples but failed to identify the co-infecting APMV4 in the APMV6 infected pool.</p> <p>Conclusions</p> <p>These data further contribute to the knowledge about the genetic diversity within the serotypes APMV4 and 6, and confirm the limited sensitivity of the HI subtyping test. Moreover, this study demonstrates the value of a random access nucleic acid amplification method in combination with massive parallel sequencing. Using only a moderate and economical sequencing effort, the characterization and full genome sequencing of APMVs can be obtained, including the identification of viruses in mixed infections.</p

The origin of biased sequence depth in sequence-independent nucleic acid amplification and optimization for efficient massive parallel sequencing

Sequence Independent Single Primer Amplification is one of the most widely used random amplification approaches in virology for sequencing template preparation. This technique relies on oligonucleotides consisting of a 39 random part used to prime complementary DNA synthesis and a 59 defined tag sequence for subsequent amplification. Recently, this amplification method was combined with next generation sequencing to obtain viral sequences. However, these studies showed a biased distribution of the resulting sequence reads over the analyzed genomes. The aim of this study was to elucidate the mechanisms that lead to biased sequence depth when using random amplification. Avian paramyxovirus type 8 was used as a model RNA virus to investigate these mechanisms. We showed, based on in silico analysis of the sequence depth in relation to GC-content, predicted RNA secondary structure and sequence complementarity to the 39 part of the tag sequence, that the tag sequence has the main contribution to the observed bias in sequence depth. We confirmed this finding experimentally using both fragmented and non-fragmented viral RNAs as well as primers differing in random oligomer length (6 or 12 nucleotides) and in the sequence of the amplification tag. The observed oligonucleotide annealing bias can be reduced by extending the random oligomer sequence and by in silico combining sequence data from SISPA experiments using different 59 defined tag sequences. These findings contribute to the optimization of random nucleic acid amplification protocols that are currently required for downstream applications such as viral metagenomics and microarray analysis

The Public Health Dimension of Disasters—Health Outcome Assessment of Disasters

A broad range of health problems are related to disasters. Insight into these health problems is needed for targeted disaster management. Disaster health outcome assessment can provide insight into the health effects of disasters. During the 15th World Congress on Disaster and Emergency Medicine in Amsterdam (2007), experts in the field of disaster epidemiology discussed important aspects of disaster health outcome assessment, such as: (1) what is meant by disaster health outcome assessment?; (2) why should one conduct a disaster health outcome assessment, and what are the objectives?, and (3) who benefits from the information obtained by a disaster health outcome assessment? A disaster health outcome assessment can be defined as a systematic assessment of the current and potential health problems in a population affected by a disaster. Different methods can be used to examine these health problems such as: (1) rapid assessment of health needs; (2) (longitudinal) epidemiological studies using questionnaires; (3) continuous surveillance of health problems using existing registration systems; (4) assessment of the use and distribution of health services; and (5) research into the etiology of the health effects of disasters. The public health impact of a disaster may not be immediately evident. Disaster health outcome assessment provides insight into the health related consequences of disasters. The information that is obtained by performing a disaster health outcome assessment can be used to initiate and adapt the provision of health care. Besides information for policy-makers, disaster health outcome assessments can contribute to the knowledge and evidence base of disaster health outcomes (scientific objective). Finally, disaster health outcome assessment might serve as a signal of recognition of the problems of the survivors. Several stakeholders may benefit from the information obtained from a disaster health outcome assessment. Disaster decision-makers and the public health community benefit from performing a disaster health outcome assessment, since it provides information that is useful for the different aspects of disaster management. Also, by providing information about the nature, prevalence, and course of health problems, (mental) health care workers can anticipate the health needs and requirements in the affected population. It is important to realize that the disaster is not over when the acute care has been provided. Instead, disasters will cause many other health problems and concerns such as infectious diseases and mental health problems. Disaster health outcome assessments provide insight into the public health impact of disaster

KAI407, a potent non-8-aminoquinoline compound that kills Plasmodium cynomolgi early dormant liver stage parasites in vitro.

Preventing relapses of Plasmodium vivax malaria through a radical cure depends on use of the 8-aminoquinoline primaquine, which is associated with safety and compliance issues. For future malaria eradication strategies, new, safer radical curative compounds that efficiently kill dormant liver stages (hypnozoites) will be essential. A new compound with potential radical cure activity was identified using a low-throughput assay of in vitro-cultured hypnozoite forms of Plasmodium cynomolgi (an excellent and accessible model for Plasmodium vivax). In this assay, primary rhesus hepatocytes are infected with P. cynomolgi sporozoites, and exoerythrocytic development is monitored in the presence of compounds. Liver stage cultures are fixed after 6 days and stained with anti-Hsp70 antibodies, and the relative proportions of small (hypnozoite) and large (schizont) forms relative to the untreated controls are determined. This assay was used to screen a series of 18 known antimalarials and 14 new non-8-aminoquinolines (preselected for blood and/or liver stage activity) in three-point 10-fold dilutions (0.1, 1, and 10 μM final concentrations). A novel compound, designated KAI407 showed an activity profile similar to that of primaquine (PQ), efficiently killing the earliest stages of the parasites that become either primary hepatic schizonts or hypnozoites (50% inhibitory concentration [IC50] for hypnozoites, KAI407, 0.69 μM, and PQ, 0.84 μM; for developing liver stages, KAI407, 0.64 μM, and PQ, 0.37 μM). When given as causal prophylaxis, a single oral dose of 100 mg/kg of body weight prevented blood stage parasitemia in mice. From these results, we conclude that KAI407 may represent a new compound class for P. vivax malaria prophylaxis and potentially a radical cure

Risk assessment of SARS-CoV-2 infection in free-ranging wild animals in Belgium.

The aim of this review paper is to evaluate the putative susceptibilities of different free-ranging wild animal species in Belgium to SARS-CoV-2 and provide a risk assessment of SARS-CoV-2 infection in those animals. Since the onset of the Covid-19 pandemic, natural SARS-CoV-2 infections have mainly been confirmed in domestic and production animals, and in wild animals kept in captivity, although the numbers remain limited when compared to human cases. Recently, the first SARS-CoV-2 infections in presumably escaped minks found in the wild have been detected, further addressing the much-feared scenario of transmission of the virus to animals living in the wild and its consequences. Considering the most likely origin of the virus being a wild animal and the putative susceptibilities of free-ranging wild animal species to SARS-CoV-2, the risk of infection with possible establishment of the virus in these populations has to be investigated closely. The authors conclude that most attention should be given to surveillance and awareness raising activities for SARS-CoV-2 infection in wild mustelids, bats, wild canids and felids, particularly these collected in wildlife rescue centres. People involved in frequent and close contact with wild animals should take all necessary precautionary measures to protect wild animals against exposure to the virus. One year after the first detection of SARS-CoV-2 in humans, the time has come to increase investments in research and surveillance activities in animals, including in free-ranging wild animals, as part of a One Health control of this pandemic. This study focusing on Belgium could be helpful for other countries with similar animal densities and ecosystems

Highly Pathogenic H5N1 Influenza Virus in Smuggled Thai Eagles, Belgium

We report the isolation and characterization of a highly pathogenic avian influenza A/H5N1 virus from Crested Hawk-Eagles smuggled into Europe by air travel. A screening performed in human and avian contacts indicated no dissemination occurred. Illegal movements of birds are a major threat for the introduction of highly pathogenic avian influenza

Quantification of Calcyclin and Heat Shock Protein 90 in Sera from Women with and without Preeclampsia by Mass Spectrometry

Purpose: The objective of present study is to determine serum levels and placental distribution of two interacting proteins calcyclin and heat shock protein 90 in preeclampsia. Experimental design: Maternal serum levels of calcyclin and heat shock protein 90 are compared throughout pregnancy from the first trimester till term among women with preeclampsia (n = 43) and age-matched normotensive pregnant controls (n = 46). A serum-based 2D LC-MS assay using Parallel Reaction Monitoring is applied to quantify both calcyclin and heat shock protein 90. Results: Serum levels of calcyclin are significantly lower in patients with preeclampsia in the second trimester of pregnancy as compared to controls (p < 0.05). Serum levels of heat shock protein 90 are significantly higher in patients with preeclampsia in the third trimester as compared to controls (p < 0.001). Conclusion and clinical relevance: Both interacting proteins calcyclin and heat shock protein 90 are notably changed in preeclamptic patients compared to controls. Calcyclin is already decreased before the onset of preeclampsia in the second trimester and HSP90 is strongly increased in the third trimester. This suggests that these proteins may play a role in the pathogenesis of preeclampsia and ought to be investigated in large cohort studies as molecular biomarkers

The coevolution of juvenile play-fighting and adult competition

This is the author accepted manuscript. The final version is available from Wiley via the DOI in this recordAlthough play-fighting is widespread among juvenile mammals, its adaptive significance remains unclear. It has been proposed that play is beneficial for developing skills to improve success in adult contests (motor training hypothesis), but the links between juvenile play-fighting and adult aggression are complex and not well understood. In this theoretical study, we investigate the coevolution between juvenile play-fighting and adult aggression using evolutionary computer simulations. We consider a simple life history with two sequential stages: a juvenile phase in which individuals play-fight with other juveniles to develop their fighting skills; and an adult phase in which individuals engage in potentially aggressive contests over access to resources and ultimately mating opportunities, leading to reproductive success. The simulations track genetic evolution in key traits affecting adult contests, such as the level of aggression, as well as juvenile investment in play-fighting, capturing the coevolutionary feedbacks between juvenile and adult decisions. We find that coevolution leads to one of two outcomes: a high-play, high-aggression situation with highly aggressive adult contests preceded by a prolonged period of juvenile play-fighting to improve fighting ability, or a low-play, low-aggression situation in which adult contests are resolved without fighting and there is minimal investment in play-fighting before individuals mature. Which of these outcomes is favoured depends on the mortality costs and on the type of 27 societal structure: societies with strong reproductive skew, favouring monopolisation of resources, show high levels of adult aggression and high investment in juvenile play-fighting, whereas societies with low reproductive skew have both low adult aggression and low levels of play-fighting. A review of empirical evidence, particularly in the primate genus Macaca, highlights some limitations of our model and suggests that other, complementary functional explanations are needed to account for the full range of competitive and cooperative forms of play fighting. Our study illustrates the power of evolutionary simulations to shed light on the long-standing puzzle of animal play

Preclinical feasibility of robot-assisted sentinel lymph node biopsy using multi-modality magnetic and fluorescence guidance in the head and neck

First published: 08 September 2022Background: Sentinel lymph node biopsy (SLNB) is a staging procedure dependent on accurate mapping of draining lymphatics via tracers. Robotassisted SLNB enables access to multiple neck levels with a single incision and intraoperative fluorescence guidance to the SLN. Methods: Lymphatic mapping in swine was done using a magnetic tracer and fluorescent dye, injected into the tongue. MRI preoperatively mapped lymphatic spread of the magnetic tracer. Dissection was performed using a da Vinci Xi robot guided by fluorescence-imaging of the dye. Results: Robot-assisted SLNB was successfully performed in all animals (n = 5). A novel MRI protocol differentiated SLNs (n = 6) from lower echelon nodes (n = 11) based on flow progression. Fluorescence imaging provided valuable intraoperative guidance and correlated with magnetic-positive nodes. Conclusions: This study demonstrates preclinical feasibility of a robot-assisted approach to SLNB using magnetic and fluorescent tracers in the head and neck, enabling both preoperative mapping and intraoperative guidance.Giri Krishnan, Aidan Cousins, Nguyen Pham, Valentina Milanova, Melanie Nelson, Shridhar Krishnan, Nynke S. van den Berg, Anil Shetty, Eben L. Rosenthal, Peter-John Wormald, Benjamin Thierry, Andrew Foreman, Suren Krishna