The alphabaculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) is the world... more The alphabaculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) is the world's most successful viral bioinsecticide. Through the 1980's and 1990's, this virus was extensively used for biological control of populations of Anticarsia gemmatalis (Velvetbean caterpillar) in soybean crops. During this period, genetic studies identified several variable loci in the AgMNPV, however, most of them were not characterized at the sequence level. In this study we report a full genome comparison among 17 wild-type isolates of AgMNPV. We found the pangenome of this virus to contain at least 167 hypothetical genes, 151 of which are shared by all genomes. The gene bro-a that might be involved in host specificity and carrying transporter, is absent in some genomes, and new hypothetical genes were observed. Among these genes there is a unique rnf12-like gene, probably implicated in ubiquitination. Events of gene fission and fusion are common, since four genes have been ob...
Tick-borne encephalitis virus (TBEV) is the most important arboviral agent causing disease of the... more Tick-borne encephalitis virus (TBEV) is the most important arboviral agent causing disease of the central nervous system in central Europe. In this study, 61 TBEV E gene sequences derived from 48 isolates from the Czech Republic, and four isolates and nine TBEV strains detected in ticks from Germany, covering more than half a century from 1954 to 2009, were sequenced and subjected to phylogenetic and Bayesian phylodynamic analysis to determine the phylogeography of TBEV in central Europe. The general Eurasian continental east-to-west pattern of the spread of TBEV was confirmed at the regional level but is interlaced with spreading that arises because of local geography and anthropogenic influence. This spread is reflected by the disease pattern in the Czech Republic that has been observed since 1991. The overall evolutionary rate was estimated to be approximately 8×10−4substitutions per nucleotide per year. The analysis of the TBEV E genes of 11 strains isolated at one natural focus...
The genome of the most virulent among 22 Brazilian geographical isolates of Spodoptera frugiperda... more The genome of the most virulent among 22 Brazilian geographical isolates of Spodoptera frugiperda nucleopolyhedrovirus, isolate 19 (SfMNPV-19), was completely sequenced and shown to comprise 132 565 bp and 141 open reading frames (ORFs). A total of 11 ORFs with no homology to genes in the GenBank database were found. Of those, four had typical baculovirus promoter motifs and polyadenylation sites. Computer-simulated restriction enzyme cleavage patterns of SfMNPV-19 were compared with published physical maps of other SfMNPV isolates. Differences were observed in terms of the restriction profiles and genome size. Comparison of SfMNPV-19 with the sequence of the SfMNPV isolate 3AP2 indicated that they differed due to a 1427 bp deletion, as well as by a series of smaller deletions and point mutations. The majority of genes of SfMNPV-19 were conserved in the closely related Spodoptera exigua NPV (SeMNPV) and Agrotis segetum NPV (AgseMNPV-A), but a few regions experienced major changes an...
Rift Valley fever virus (RVFV), a phlebovirus of the Bunyaviridae family, is an arthropod-borne v... more Rift Valley fever virus (RVFV), a phlebovirus of the Bunyaviridae family, is an arthropod-borne virus which emerges periodically throughout Africa, emphasizing that it poses a major threat for animal and human populations. To assess the genetic variability of RVFV, several isolates from diverse localities of Africa were investigated by means of reverse transcription-PCR followed by direct sequencing of a region of the small (S), medium (M), and large (L) genomic segments. Phylogenetic analysis showed the existence of three major lineages corresponding to geographic variants from West Africa, Egypt, and Central-East Africa. However, incongruences detected between the L, M, and S phylogenies suggested that genetic exchange via reassortment occurred between strains from different lineages. This hypothesis, depicted by parallel phylogenies, was further confirmed by statistical tests. Our findings, which strongly suggest exchanges between strains from areas of endemicity in West and East...
Usutu virus (USUV) is a Culex-associated mosquito-borne flavivirus of the Flaviviridae family. Si... more Usutu virus (USUV) is a Culex-associated mosquito-borne flavivirus of the Flaviviridae family. Since its discovery in 1959, the virus has been isolated from birds, arthropods and humans in Europe and Africa. An increasing number of Usutu virus infections in humans with neurological presentations have been reported. Recently, the virus has been detected in bats and horses, which deviates from the currently proposed enzootic cycle of USUV involving several different avian and mosquito species. Despite this increasing number of viral detections in different mammalian hosts, the existence of a non-avian reservoir remains unresolved. In Kedougou, a tropical region in the southeast corner of Senegal, Usutu virus was detected, isolated and sequenced from five asymptomatic small mammals: Two different rodent species and a single species of shrew. Additional molecular characterization and in vivo growth dynamics showed that these rodents/shrew-derived viruses are closely related to the refer...
We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for... more We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, saliva, urine and semen), development of immune responses (including antibodies, cytokines and chemokines), and virus genetic variation within samples collected from semen and urine during the infection course. The analysis was focused primarily on the two male patients who shed the virus for up to 158 days after the initial symptoms. ZIKV particles were detected in the spermatozoa cytoplasm and flagella, in immature sperm cells and could also be isolated from semen in cell culture, confirming that the virus is able to preserve integrity and infectivity during replication in the male reproductive system (MRS). Despite the damage caused by ZIKV infection within the MRS, our data showed that ZIKV infection did not result in infertility at...
Codon adaptation index (CAI) is a measure of synonymous codon usage biases given a usage referenc... more Codon adaptation index (CAI) is a measure of synonymous codon usage biases given a usage reference. Through mutation, selection, and drift, viruses can optimize their replication efficiency and produce more offspring, which could increase the chance of secondary transmission. To evaluate how higher CAI towards the host has been associated with higher viral titers, we explored temporal trends of several historic and extensively sequenced zoonotic flaviviruses and relationships within the genus itself. To showcase evolutionary and epidemiological relationships associated with silent, adaptive synonymous changes of viruses, we used codon usage tables from human housekeeping and antiviral immune genes, as well as tables from arthropod vectors and vertebrate species involved in the flavivirus maintenance cycle. We argue that temporal trends of CAI changes could lead to a better understanding of zoonotic emergences, evolutionary dynamics, and host adaptation. CAI appears to help illustrat...
Zika virus (ZIKV) was recognised as a zoonotic pathogen in Africa and southeastern Asia. Human in... more Zika virus (ZIKV) was recognised as a zoonotic pathogen in Africa and southeastern Asia. Human infections were infrequently reported until 2007, when the first known epidemic occurred in Micronesia. After 2013, the Asian lineage of ZIKV spread along the Pacific Islands and Americas, causing severe outbreaks with millions of human infections. The recent human infections of ZIKV were also associated with severe complications, such as an increase in cases of Guillain-Barre syndrome and the emergence of congenital Zika syndrome. To better understand the recent and rapid expansion of ZIKV, as well as the presentation of novel complications, we compared the genetic differences between the African sylvatic lineage and the Asian epidemic lineage that caused the recent massive outbreaks. The epidemic lineages have significant codon adaptation in NS1 gene to translate these proteins in human and Aedes aegypti mosquito cells compared to the African zoonotic lineage. Accordingly, a Brazilian ep...
The worldwide expansion of new emergent arboviruses such as Chikungunya and Zika reinforces the i... more The worldwide expansion of new emergent arboviruses such as Chikungunya and Zika reinforces the importance in understanding the role of mosquito species in spreading these pathogens in affected regions. This knowledge is essential for developing effective programs based on species specificity to avoid the establishment of endemic transmission cycles sustained by the identified local vectors. Although the first autochthonous transmission of Chikungunya virus was described in 2014 in the north of Brazil, the main outbreaks were reported in 2015 and 2016 in the northeast of Brazil. During 5 days of February 2016, we collected mosquitoes in homes of 6 neighborhoods of Aracaju city, the capital of Sergipe state. Four mosquito species were identified but Culex quinquefasciatus and Aedes aegypti were the most abundant. Field-caught mosquitoes were tested for Chikungunya (CHIKV), Zika (ZIKV) and Dengue viruses (DENV) by qRT-PCR and one CHIKV-infected Ae. aegypti female was detected. The com...
Currently the detection of Zika virus (ZIKV) in patient samples is done by real-time RT-PCR. Samp... more Currently the detection of Zika virus (ZIKV) in patient samples is done by real-time RT-PCR. Samples collected from rural area are sent to highly equipped laboratories for screening. A rapid point-of-care test is needed to detect the virus, especially at low resource settings. In this report, we describe the development of a reverse transcription isothermal recombinase polymerase amplification (RT-RPA) assay for the identification of ZIKV. RT-RPA assay was portable, sensitive (21 RNA molecules), and rapid (3-15 minutes). No cross-reactivity was detected to other flaviviruses, alphaviruses and arboviruses. Compared to real-time RT-PCR, the diagnostic sensitivity was 92%, while the specificity was 100%. The developed assay is a promising platform for rapid point of need detection of ZIKV in low resource settings and elsewhere (e.g. during mass gathering).
Zika virus (ZIKV) is responsible for a major ongoing epidemic in the Americas and has been causal... more Zika virus (ZIKV) is responsible for a major ongoing epidemic in the Americas and has been causally associated with fetal microcephaly. The development of a safe and effective ZIKV vaccine is therefore an urgent global health priority. Here we demonstrate that three different vaccine platforms protect against ZIKV challenge in rhesus monkeys. A purified inactivated virus vaccine induced ZIKV-specific neutralizing antibodies and completely protected monkeys against ZIKV strains from both Brazil and Puerto Rico. Purified immunoglobulin from vaccinated monkeys conferred passive protection in adoptive transfer studies. A plasmid DNA vaccine and a single-shot recombinant rhesus adenovirus serotype 52 vector expressing ZIKV prM-Env also elicited neutralizing antibodies and completely protected monkeys against ZIKV challenge. These data support the rapid clinical development of ZIKV vaccines for humans.
The alphabaculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) is the world... more The alphabaculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) is the world's most successful viral bioinsecticide. Through the 1980's and 1990's, this virus was extensively used for biological control of populations of Anticarsia gemmatalis (Velvetbean caterpillar) in soybean crops. During this period, genetic studies identified several variable loci in the AgMNPV, however, most of them were not characterized at the sequence level. In this study we report a full genome comparison among 17 wild-type isolates of AgMNPV. We found the pangenome of this virus to contain at least 167 hypothetical genes, 151 of which are shared by all genomes. The gene bro-a that might be involved in host specificity and carrying transporter, is absent in some genomes, and new hypothetical genes were observed. Among these genes there is a unique rnf12-like gene, probably implicated in ubiquitination. Events of gene fission and fusion are common, since four genes have been ob...
Tick-borne encephalitis virus (TBEV) is the most important arboviral agent causing disease of the... more Tick-borne encephalitis virus (TBEV) is the most important arboviral agent causing disease of the central nervous system in central Europe. In this study, 61 TBEV E gene sequences derived from 48 isolates from the Czech Republic, and four isolates and nine TBEV strains detected in ticks from Germany, covering more than half a century from 1954 to 2009, were sequenced and subjected to phylogenetic and Bayesian phylodynamic analysis to determine the phylogeography of TBEV in central Europe. The general Eurasian continental east-to-west pattern of the spread of TBEV was confirmed at the regional level but is interlaced with spreading that arises because of local geography and anthropogenic influence. This spread is reflected by the disease pattern in the Czech Republic that has been observed since 1991. The overall evolutionary rate was estimated to be approximately 8×10−4substitutions per nucleotide per year. The analysis of the TBEV E genes of 11 strains isolated at one natural focus...
The genome of the most virulent among 22 Brazilian geographical isolates of Spodoptera frugiperda... more The genome of the most virulent among 22 Brazilian geographical isolates of Spodoptera frugiperda nucleopolyhedrovirus, isolate 19 (SfMNPV-19), was completely sequenced and shown to comprise 132 565 bp and 141 open reading frames (ORFs). A total of 11 ORFs with no homology to genes in the GenBank database were found. Of those, four had typical baculovirus promoter motifs and polyadenylation sites. Computer-simulated restriction enzyme cleavage patterns of SfMNPV-19 were compared with published physical maps of other SfMNPV isolates. Differences were observed in terms of the restriction profiles and genome size. Comparison of SfMNPV-19 with the sequence of the SfMNPV isolate 3AP2 indicated that they differed due to a 1427 bp deletion, as well as by a series of smaller deletions and point mutations. The majority of genes of SfMNPV-19 were conserved in the closely related Spodoptera exigua NPV (SeMNPV) and Agrotis segetum NPV (AgseMNPV-A), but a few regions experienced major changes an...
Rift Valley fever virus (RVFV), a phlebovirus of the Bunyaviridae family, is an arthropod-borne v... more Rift Valley fever virus (RVFV), a phlebovirus of the Bunyaviridae family, is an arthropod-borne virus which emerges periodically throughout Africa, emphasizing that it poses a major threat for animal and human populations. To assess the genetic variability of RVFV, several isolates from diverse localities of Africa were investigated by means of reverse transcription-PCR followed by direct sequencing of a region of the small (S), medium (M), and large (L) genomic segments. Phylogenetic analysis showed the existence of three major lineages corresponding to geographic variants from West Africa, Egypt, and Central-East Africa. However, incongruences detected between the L, M, and S phylogenies suggested that genetic exchange via reassortment occurred between strains from different lineages. This hypothesis, depicted by parallel phylogenies, was further confirmed by statistical tests. Our findings, which strongly suggest exchanges between strains from areas of endemicity in West and East...
Usutu virus (USUV) is a Culex-associated mosquito-borne flavivirus of the Flaviviridae family. Si... more Usutu virus (USUV) is a Culex-associated mosquito-borne flavivirus of the Flaviviridae family. Since its discovery in 1959, the virus has been isolated from birds, arthropods and humans in Europe and Africa. An increasing number of Usutu virus infections in humans with neurological presentations have been reported. Recently, the virus has been detected in bats and horses, which deviates from the currently proposed enzootic cycle of USUV involving several different avian and mosquito species. Despite this increasing number of viral detections in different mammalian hosts, the existence of a non-avian reservoir remains unresolved. In Kedougou, a tropical region in the southeast corner of Senegal, Usutu virus was detected, isolated and sequenced from five asymptomatic small mammals: Two different rodent species and a single species of shrew. Additional molecular characterization and in vivo growth dynamics showed that these rodents/shrew-derived viruses are closely related to the refer...
We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for... more We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, saliva, urine and semen), development of immune responses (including antibodies, cytokines and chemokines), and virus genetic variation within samples collected from semen and urine during the infection course. The analysis was focused primarily on the two male patients who shed the virus for up to 158 days after the initial symptoms. ZIKV particles were detected in the spermatozoa cytoplasm and flagella, in immature sperm cells and could also be isolated from semen in cell culture, confirming that the virus is able to preserve integrity and infectivity during replication in the male reproductive system (MRS). Despite the damage caused by ZIKV infection within the MRS, our data showed that ZIKV infection did not result in infertility at...
Codon adaptation index (CAI) is a measure of synonymous codon usage biases given a usage referenc... more Codon adaptation index (CAI) is a measure of synonymous codon usage biases given a usage reference. Through mutation, selection, and drift, viruses can optimize their replication efficiency and produce more offspring, which could increase the chance of secondary transmission. To evaluate how higher CAI towards the host has been associated with higher viral titers, we explored temporal trends of several historic and extensively sequenced zoonotic flaviviruses and relationships within the genus itself. To showcase evolutionary and epidemiological relationships associated with silent, adaptive synonymous changes of viruses, we used codon usage tables from human housekeeping and antiviral immune genes, as well as tables from arthropod vectors and vertebrate species involved in the flavivirus maintenance cycle. We argue that temporal trends of CAI changes could lead to a better understanding of zoonotic emergences, evolutionary dynamics, and host adaptation. CAI appears to help illustrat...
Zika virus (ZIKV) was recognised as a zoonotic pathogen in Africa and southeastern Asia. Human in... more Zika virus (ZIKV) was recognised as a zoonotic pathogen in Africa and southeastern Asia. Human infections were infrequently reported until 2007, when the first known epidemic occurred in Micronesia. After 2013, the Asian lineage of ZIKV spread along the Pacific Islands and Americas, causing severe outbreaks with millions of human infections. The recent human infections of ZIKV were also associated with severe complications, such as an increase in cases of Guillain-Barre syndrome and the emergence of congenital Zika syndrome. To better understand the recent and rapid expansion of ZIKV, as well as the presentation of novel complications, we compared the genetic differences between the African sylvatic lineage and the Asian epidemic lineage that caused the recent massive outbreaks. The epidemic lineages have significant codon adaptation in NS1 gene to translate these proteins in human and Aedes aegypti mosquito cells compared to the African zoonotic lineage. Accordingly, a Brazilian ep...
The worldwide expansion of new emergent arboviruses such as Chikungunya and Zika reinforces the i... more The worldwide expansion of new emergent arboviruses such as Chikungunya and Zika reinforces the importance in understanding the role of mosquito species in spreading these pathogens in affected regions. This knowledge is essential for developing effective programs based on species specificity to avoid the establishment of endemic transmission cycles sustained by the identified local vectors. Although the first autochthonous transmission of Chikungunya virus was described in 2014 in the north of Brazil, the main outbreaks were reported in 2015 and 2016 in the northeast of Brazil. During 5 days of February 2016, we collected mosquitoes in homes of 6 neighborhoods of Aracaju city, the capital of Sergipe state. Four mosquito species were identified but Culex quinquefasciatus and Aedes aegypti were the most abundant. Field-caught mosquitoes were tested for Chikungunya (CHIKV), Zika (ZIKV) and Dengue viruses (DENV) by qRT-PCR and one CHIKV-infected Ae. aegypti female was detected. The com...
Currently the detection of Zika virus (ZIKV) in patient samples is done by real-time RT-PCR. Samp... more Currently the detection of Zika virus (ZIKV) in patient samples is done by real-time RT-PCR. Samples collected from rural area are sent to highly equipped laboratories for screening. A rapid point-of-care test is needed to detect the virus, especially at low resource settings. In this report, we describe the development of a reverse transcription isothermal recombinase polymerase amplification (RT-RPA) assay for the identification of ZIKV. RT-RPA assay was portable, sensitive (21 RNA molecules), and rapid (3-15 minutes). No cross-reactivity was detected to other flaviviruses, alphaviruses and arboviruses. Compared to real-time RT-PCR, the diagnostic sensitivity was 92%, while the specificity was 100%. The developed assay is a promising platform for rapid point of need detection of ZIKV in low resource settings and elsewhere (e.g. during mass gathering).
Zika virus (ZIKV) is responsible for a major ongoing epidemic in the Americas and has been causal... more Zika virus (ZIKV) is responsible for a major ongoing epidemic in the Americas and has been causally associated with fetal microcephaly. The development of a safe and effective ZIKV vaccine is therefore an urgent global health priority. Here we demonstrate that three different vaccine platforms protect against ZIKV challenge in rhesus monkeys. A purified inactivated virus vaccine induced ZIKV-specific neutralizing antibodies and completely protected monkeys against ZIKV strains from both Brazil and Puerto Rico. Purified immunoglobulin from vaccinated monkeys conferred passive protection in adoptive transfer studies. A plasmid DNA vaccine and a single-shot recombinant rhesus adenovirus serotype 52 vector expressing ZIKV prM-Env also elicited neutralizing antibodies and completely protected monkeys against ZIKV challenge. These data support the rapid clinical development of ZIKV vaccines for humans.
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