COVID-19 Sniffer Dog experimental training: which protocol and which implications for reliable identification?

The introduction of trained sniffer dogs for COVID-19 disease detection could be an opportunity, as previously described for other diseases. Dogs could be trained to detect volatile organic compounds (VOCs), the whiff of COVDI-19 disease. Dogs involved in the study were three one male and two females from different breeds, Black German Shepherd, German Shepherd and Dutch Shepherd. The training was performed using sweat samples from COVID-19 positive apteints and from covid-19 free patients admitted at the University Hospital Campus Bio-medico of Rome. Gauze with sweat were collected in glass jar with metal top and put in metal boxes used for dog training. The dog training protocol was performed in two phase: the olfactory conditioning and the olfactory discrimintaion research. The training palnning was focused on the switch moment for the sniffer dog, the moment when the dog was able to identify VOCs specific for COVID-19 disease. At this time the dog was able to identify VOCs specific for COVID-19 disease with significant reliability, in terms of number of correct versus uncorrect (p<0.0001) reporting. In conclusion, this protocol could provide a useful tool for sniffer dogs training and their introduction in mass screening context, cheaper and faster than a conventional testing method.

SARS-CoV-2 B.1.617 Indian variants: are electrostatic potential changes responsible for a higher transmission rate?

Lineage B.1.617+, also known as G/452R.V3, is a recently described SARS-CoV-2 variant under investigation (VUI) firstly identified in October 2020 in India. As of May 2021, three sublineages labelled as B.1.617.1, B.1.617.2 and B.1.617.3 have been already identified, and their potential impact on the current pandemic is being studied. This variant has 13 amino acid changes, three in its spike protein, which are currently of particular concern: E484Q, L452R and P681R. Here we report a major effect of the mutations characterizing this lineage, represented by a marked alteration of the surface electrostatic potential (EP) of the Receptor Binding Domain (RBD) of the spike protein. Enhanced RBD-EP is particularly noticeable in the B.1.617.2 sublineage, which shows multiple replacements of neutral or negatively-charged amino acids with positively-charged amino acids. We here hypothesize that this EP change can favor the interaction between the B.1.617+RBD and the negatively-charged ACE2 thus conferring a potential increase in the virus transmission.

SARS-CoV-2 shifting transmission dynamics and hidden reservoirs limited the efficacy of public health interventions in Italy

We investigated SARS-CoV-2 transmission dynamics in Italy, one of the countries hit hardest by the pandemic, using phylodynamic analysis of viral genetic and epidemiological data. We observed the co-circulation of at least 13 different SARS-CoV-2 lineages over time, which were linked to multiple importations and characterized by large transmission clusters concomitant with a high number of infections. Subsequent implementation of a three-phase nationwide lockdown strategy greatly reduced infection numbers and hospitalizations. Yet we present evidence of sustained viral spread among sporadic clusters acting as "hidden reservoirs" during summer 2020. Mathematical modelling shows that increased mobility among residents eventually catalyzed the coalescence of such clusters, thus driving up the number of infections and initiating a new epidemic wave. Our results suggest that the efficacy of public health interventions is, ultimately, limited by the size and structure of epidemic reservoirs, which may warrant prioritization during vaccine deployment.

The 2019-new Coronavirus epidemic: evidence for virus evolution

There is concern about a new coronavirus, the 2019-nCoV, as a global public health threat. In this article, we provide a preliminary evolutionary and molecular epidemiological analysis of this new virus. A phylogenetic tree has been built using the 15 available whole genome sequence of 2019-nCoV and 12 whole genome sequences highly similar sequences available in gene bank (5 from SARS, 2 from MERS and 5 from Bat SARS-like Coronavirus). FUBAR analysis shows that the Nucleocapsid and the Spike Glycoprotein has some sites under positive pressure while homology modelling helped to explain some molecular and structural differences between the viruses. The phylogenetic tree showed that 2019.nCoV significantly clustered with Bat SARS-like Coronavirus sequence isolated in 2015, whereas structural analysis revealed mutation in S and nucleocapsid proteins. From these results, 2019nCoV could be considered a coronavirus distinct from SARS virus, probably transmitted from bats or another host where mutations conferred upon it the ability to infect humans.

Baseline haematological and biochemical reference values for healthy male adults from Mali.

INTRODUCTION: Haematological reference values are very important for diagnostic orientation and treatment decision. The aim of this study was to establish haematological reference values for Malian healthy adults. METHODS: A cross-sectional study including 161 male Malians aged between 19 and 54 years old was performed. Median and reference ranges were calculated for haematological and biochemical parameters. Parametric student's t-test was used to determine any statistically significant differences by age, smoker status, body mass index (BMI) and occupation. Ranges were further compared with those reported for other African, Afro-American and Caucasian populations. RESULTS: Increased levels of MCV, MCH, PLT and EOS were found in younger Malians who had abnormal BMI and altered platelets parameters. Notably, significantly lower eosinophil and monocyte counts were observed in Malians compared to Europeans The smoking status did not seem to directly affect RIs. CONCLUSION: This is the first study to determine normal laboratory parameters in Malian adult males. Our results underscore the necessity of establishing region-specific clinical reference ranges that would allow clinicians and practitioners to manage laboratory tests, diagnosis and therapies. These data are useful not only for the management of patients in Mali, but also to support European and American clinicians in the health management of asylum seekers and migrants from Mali.

Phylogeny of Culex theileri virus flavivirus in Spain, Myanmar, Portugal and Turkey

Objective: To study the genetic diversity of Culex theileri flavivirus and the spread of this virus among Spain, Portugal and Turkey. Methods: A database consisting of 55 sequences of the NS5/3'UTR region of Culex theileri flavivirus group downloaded from GenBank were aligned and manual edited with Bioedit. ModelTest v. 3.7 was used to select the simplest evolutionary model that adequately fitted the sequence data. Maximum likelihood analysis was performed using MEGA7. The phylogenetic signal of the dataset was investigated by the likelihood mapping analysis. Results: The phylogenetic tree showed three clusters. Myanmar sequences clusterd together with Turkish sequences, Spain and Portugal strains grouped together and two Turkish sequences grouped separately. Selective pressure analysis showed a moderate percentage of sites (22.5%) under pervasive negative selection and only 1% under pervasive positive selection. The sites subject to selective pressure in CTFV RdRp NS5 fragments have been located onto the predicted three-dimensional structure. Conclusions: Phylogenetic and evolutionary analysis can be an important tool for understanding the evolutionary impact of the probable contemporary existence between non-pathogenic and pathogenic flaviviruses among these vectors.

Mayaro virus infection, the next epidemic wave after Zika? Evolutionary and structural analysis

Objective:To evaluate the evolution of the pathogen Mayaro virus, causing Mayaro fever (a mosquito-borne disease) and to perform selective pressure analysis and homology modelling.Methods:Nine different datasets were built, one for each protein (from protein C to non-structural protein 4) and the last one for the complete genome. Selective pressure and homology modelling analyses were applied.Results:Two main clades (A and B) were pointed in the maximum likelihood tree. The clade A included five Brazilian sequences sampled from 1955 to 2015. The Brazilian sequence sampled in 2014 significantly clustered with the Haitian sequence sampled in 2015. The clade B included the remaining 27 sequences sampled in the Central and Southern America from 1957 to 2013. Selective pressure analysis revealed several sites under episodic diversifying selection in envelope surface glycoprotein E1, non-structural protein 1 and non- structural protein 3 with a posterior probability P≤0.01. Homology modelling showed different sites modified by selective pressure and some protein-protein interaction sites at high interaction propensity.Conclusion:Maximum likelihood analysis confirmed the Mayaro virus previous circulation in Haiti and the successful spread to the Caribbean and USA. Selective pressure analysis revealed a strong presence of negatively selected sites, suggesting a probable purging of deleterious polymorphisms in functional genes. Homology model showed the position 31, under selective pressure, located in the edge of the ADP-ribose binding site predicting to possess a high potential of protein-protein interaction and suggesting the possible chance for a protective vaccine, thus preventing Mayaro virus urbanization as with Chikungunya virus.

Mayaro virus infection, the next epidemic wave after Zika? Evolutionary and structural analysis

Objective: To evaluate the evolution of the pathogen Mayaro virus, causing Mayaro fever (a mosquito-borne disease) and to perform selective pressure analysis and homology modelling. Methods: Nine different datasets were built, one for each protein (from protein C to non-structural protein 4) and the last one for the complete genome. Selective pressure and homology modelling analyses were applied. Results: Two main clades (A and B) were pointed in the maximum likelihood tree. The clade A included five Brazilian sequences sampled from 1955 to 2015. The Brazilian sequence sampled in 2014 significantly clustered with the Haitian sequence sampled in 2015. The clade B included the remaining 27 sequences sampled in the Central and Southern America from 1957 to 2013. Selective pressure analysis revealed several sites under episodic diversifying selection in envelope surface glycoprotein E1, non-structural protein 1 and non- structural protein 3 with a posterior probability P≤0.01. Homology modelling showed different sites modified by selective pressure and some protein-protein interaction sites at high interaction propensity. Conclusion: Maximum likelihood analysis confirmed the Mayaro virus previous circulation in Haiti and the successful spread to the Caribbean and USA. Selective pressure analysis revealed a strong presence of negatively selected sites, suggesting a probable purging of deleterious polymorphisms in functional genes. Homology model showed the position 31, under selective pressure, located in the edge of the ADP-ribose binding site predicting to possess a high potential of protein-protein interaction and suggesting the possible chance for a protective vaccine, thus preventing Mayaro virus urbanization as with Chikungunya virus.

Genetic diversity in Ebola virus: Phylogenetic and in silico structural studies of Ebola viral proteins

OBJECTIVE@#To explore the genetic diversity and the modification of antibody response in the recent outbreak of Ebola Virus.@*METHODS@#Sequences retrieved from public databases, the selective pressure analysis and the homology modeling based on the all protein (nucleoprotein, VP35, VP40, soluble glycoprotein, small soluble glycoprotein, VP30, VP24 and polymerase) were used.@*RESULTS@#Structural proteins VP24, VP30, VP35 and VP40 showed relative conserved sequences making them suitable target candidates for antiviral treatment. On the contrary, nucleoprotein, polymerase and soluble glycoprotein have high mutation frequency.@*CONCLUSIONS@#Data from this study point out important aspects of Ebola virus sequence variability that for epitope and vaccine design should be considered for appropriate targeting of conserved protein regions.

Phylogeny of Murray Valley encephalitis virus in Australia and Papua New Guinea

OBJECTIVE@#To study the genetic diversity of Murray Valley encephalitis virus (MVEV) in Australia and Papua New Guinea.@*METHODS@#MVEV envelope gene sequences were aligned using Clustal X and manual editing was performed with Bioedit. ModelTest v. 3.7 was used to select the simplest evolutionary model that adequately fitted the sequence data. Maximum likelihood analysis was performed using PhyML. The phylogenetic signal of the dataset was investigated by the likelihood mapping analysis. The Bayesian phylogenetic tree was built using BEAST.@*RESULTS@#The phylogenetic trees showed two main clades. The clade Ⅰ including eight strains isolated from West Australia. The clade Ⅱ was characterized by at least four epidemic entries, three of which localized in Northern West Australia and one in Papua New Guinea. The estimated mean evolutionary rate value of the MVEV envelope gene was 0.407 × 10(-3) substitution/site/year (95% HPD: 0.623 × 10(-4)-0.780 × 10(-3)). Population dynamics defines a relative constant population until the year 2000, when a reduction occurred, probably due to a bottleneck.@*CONCLUSIONS@#This study has been useful in supporting the probable connection between climate changes and viral evolution also by the vector point of view; multidisciplinary monitoring studies are important to prevent new viral epidemics inside and outside new endemic areas.

Zika Virus spreading in South America: Evolutionary analysis of emerging neutralizing resistant Phe279Ser strains

OBJECTIVE@#To investigate the genetic diversity of Zika Virus (ZIKV) and the relationships existing among these circulating viruses worldwide. To evaluate the genetic polymorphisms harbored from ZIKV that can have an influence on the virus circulation.@*METHODS@#Three different ZIKV dataset were built. The first dataset included 63 E gene sequences, the second one 22 NS3 sequences and the third dataset was composed of 108 NS5 gene sequences. Phylogenetic and selective pressure analysis was performed. The edited nucleic acid alignment from the Envelope dataset was used to generate a conceptual translation to the corresponding peptide sequences through UGene software.@*RESULTS@#The phylogeographic reconstruction was able to discriminate unambiguously that the Brazilian strains are belonged to the Asian lineage. The structural analysis reveals instead the presence of the Ser residue in the Brazilian sequences (however already observed in other previously reported ZIKV infections) that could suggest the presence of a neutralization-resistant population of viruses.@*CONCLUSIONS@#Phylogenetic, evolutionary and selective pressure analysis contributed to improve the knowledge on the circulation of ZIKV.

Molecular epidemiology and phylogeny of Nipah virus infection: A mini review

Nipah virus (NiV) is a member of the genus Henipavirus of the family Paramyxoviridae, characterized by high pathogenicity and endemic in South Asia. It is classified as a Biosafety Level-4 (BSL-4) agent. The case-fatality varies from 40% to 70% depending on the severity of the disease and on the availability of adequate healthcare facilities. At present no antiviral drugs are available for NiV disease and the treatment is just supportive. Phylogenetic and evolutionary analyses can be used to help in understanding the epidemiology and the temporal origin of this virus. This review provides an overview of evolutionary studies performed on Nipah viruses circulating in different countries. Thirty phylogenetic studies have been published from 2000 to 2015 years, searching on pub-med using the key words 'Nipah virus AND phylogeny' and twenty-eight molecular epidemiological studies from 2006 to 2015 have been performed, typing the key words 'Nipah virus AND molecular epidemiology'. Overall data from the published study demonstrated as phylogenetic and evolutionary analysis represent promising tools to evidence NiV epidemics, to study their origin and evolution and finally to act with effective preventive measure.

The phylogenetic and evolutionary history of Kokobera virus

OBJECTIVE@#To estimate the genetic diversity of Kokobera virus, the date of origin and the spread among different viruses in the endemic regions of Australia.@*METHODS@#Two datasets were built. The first consisting of 29 sequences of the NS5/3' UTR region of Kokobera group downloaded from GenBank, the second including only 24 sequences of Kokobera viruses, focus is on this group.@*RESULTS@#Bayesian time analysis revealed two different entries in Australia of Kokobera virus in the 50s years with the dated ancestor in 1861 year. Clades A and B showed a clear separation of the Kokobera sequences according to the geographic region.@*CONCLUSIONS@#Data from the study showed as Kokobera virus, despite of its ancient origin and its circulation before the European colonization, remained limited to the Australian country and nowadays limited mostly to the regions were Australian marsupials are mostly found.

Phylogeny of Murray Valley encephalitis virus in Australia and Papua New Guinea

Objective: To study the genetic diversity of Murray Valley encephalitis virus (MVEV) in Australia and Papua New Guinea. Methods: MVEV envelope gene sequences were aligned using Clustal X and manual editing was performed with Bioedit. ModelTest v. 3.7 was used to select the simplest evolutionary model that adequately fitted the sequence data. Maximum likelihood analysis was performed using PhyML. The phylogenetic signal of the dataset was investigated by the likelihood mapping analysis. The Bayesian phylogenetic tree was built using BEAST. Results: The phylogenetic trees showed two main clades. The clade I including eight strains isolated from West Australia. The clade II was characterized by at least four epidemic entries, three of which localized in Northern West Australia and one in Papua New Guinea. The estimated mean evolutionary rate value of the MVEV envelope gene was 0.407 × 10

Genetic diversity in Ebola virus: Phylogenetic and in silico structural studies of Ebola viral proteins

Objective: To explore the genetic diversity and the modification of antibody response in the recent outbreak of Ebola Virus. Methods: Sequences retrieved from public databases, the selective pressure analysis and the homology modeling based on the all protein (nucleoprotein, VP35, VP40, soluble glycoprotein, small soluble glycoprotein, VP30, VP24 and polymerase) were used. Results: Structural proteins VP24, VP30, VP35 and VP40 showed relative conserved sequences making them suitable target candidates for antiviral treatment. On the contrary, nucleoprotein, polymerase and soluble glycoprotein have high mutation frequency. Conclusions: Data from this study point out important aspects of Ebola virus sequence variability that for epitope and vaccine design should be considered for appropriate targeting of conserved protein regions.

Zika Virus spreading in South America: Evolutionary analysis of emerging neutralizing resistant Phe279Ser strains

Objective To investigate the genetic diversity of Zika Virus (ZIKV) and the relationships existing among these circulating viruses worldwide. To evaluate the genetic polymorphisms harbored from ZIKV that can have an influence on the virus circulation. Methods Three different ZIKV dataset were built. The first dataset included 63 E gene sequences, the second one 22 NS3 sequences and the third dataset was composed of 108 NS5 gene sequences. Phylogenetic and selective pressure analysis was performed. The edited nucleic acid alignment from the Envelope dataset was used to generate a conceptual translation to the corresponding peptide sequences through UGene software. Results The phylogeographic reconstruction was able to discriminate unambiguously that the Brazilian strains are belonged to the Asian lineage. The structural analysis reveals instead the presence of the Ser residue in the Brazilian sequences (however already observed in other previously reported ZIKV infections) that could suggest the presence of a neutralization-resistant population of viruses. Conclusions Phylogenetic, evolutionary and selective pressure analysis contributed to improve the knowledge on the circulation of ZIKV.

Molecular epidemiology and phylogeny of Nipah virus infection: A mini review

Nipah virus (NiV) is a member of the genus Henipavirus of the family Paramyxoviridae, characterized by high pathogenicity and endemic in South Asia. It is classified as a Biosafety Level-4 (BSL-4) agent. The case-fatality varies from 40% to 70% depending on the severity of the disease and on the availability of adequate healthcare facilities. At present no antiviral drugs are available for NiV disease and the treatment is just supportive. Phylogenetic and evolutionary analyses can be used to help in understanding the epidemiology and the temporal origin of this virus. This review provides an overview of evolutionary studies performed on Nipah viruses circulating in different countries. Thirty phylogenetic studies have been published from 2000 to 2015 years, searching on pub-med using the key words ‘Nipah virus AND phylogeny’ and twenty-eight molecular epidemiological studies from 2006 to 2015 have been performed, typing the key words ‘Nipah virus AND molecular epidemiology’. Overall data from the published study demonstrated as phylogenetic and evolutionary analysis represent promising tools to evidence NiV epidemics, to study their origin and evolution and finally to act with effective preventive measure.

The phylogenetic and evolutionary history of Kokobera virus

Objective To estimate the genetic diversity of Kokobera virus, the date of origin and the spread among different viruses in the endemic regions of Australia. Methods Two datasets were built. The first consisting of 29 sequences of the NS5/3′ UTR region of Kokobera group downloaded from GenBank, the second including only 24 sequences of Kokobera viruses, focus is on this group. Results Bayesian time analysis revealed two different entries in Australia of Kokobera virus in the 50s years with the dated ancestor in 1861 year. Clades A and B showed a clear separation of the Kokobera sequences according to the geographic region. Conclusions Data from the study showed as Kokobera virus, despite of its ancient origin and its circulation before the European colonization, remained limited to the Australian country and nowadays limited mostly to the regions were Australian marsupials are mostly found.

Molecular epidemiology and phylogenetic analysis of Hepatitis B virus in a group of migrants in Italy.

BACKGROUND: Hepatitis B virus infection (HBV) is widespread and it is considered a major health problem worldwide. The global distribution of HBV varies significantly between countries and between regions of the world. Among the many factors contributing to the changing epidemiology of viral hepatitis, the movement of people within and between countries is a potentially important one. In Italy, the number of migrant individuals has been increasing during the past 25 years. HBV genotype D has been found throughout the world, although its highest prevalence is in the Mediterranean area, the Middle East and southern Asia. We describe the molecular epidemiology of HBV in a chronically infected population of migrants (living in Italy), by using the phylogenetic analysis. METHODS: HBV-DNA was amplified and sequenced from 43 HBV chronically infected patients. Phylogenetic and evolutionary analysis were performed using both maximum Likelihood and Bayesian methods. RESULTS AND CONCLUSION: Of the 43 HBV S gene isolates from migrants, 25 (58.1 %) were classified as D genotype. Maximum Likelihood analysis showed an intermixing between Moldavian and foreigners sequences mostly respect to Italian ones. Italian sequences clustered mostly together in a main clade separately from all others. The estimation of the time of the tree's root gave a mean value of 17 years ago, suggesting the origin of the tree back to 1992 year. The skyline plot showed that the number of infections softly increased until the early 2005s, after which reached a plateau. Comparing phylogenetic data to the migrants date of arrival in Italy, it should be possible that migrants arrived in Italy yet infected from their country of origin. In conclusion, this is the first paper where phylogenetic analysis and genetic evolution has been used to characterize HBV sub genotypes D1 circulation in a selected and homogenous group of migrants coming from a restricted area of Balkans and to approximately define the period of infection besides the migration date.