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Viruses in the Flavivirus genus (e.g., Zika, yellow fever, dengue, West Nile, and Japanese encephalitis viruses) are arthropod-borne, globally distributed, and can cause a range of neurological or hemorrhagic diseases. The ongoing epidemics of flaviviral disease consistently demonstrate the need for new vaccines capable of outbreak control. However, safe, effective, and easy-to-produce vaccines remain relatively elusive due to limitations of conventional vaccine development that make it difficult to balance safety and efficacy. Insect-specific flaviviruses (ISFVs) are emerging as a novel method to overcome this challenge. Herein, we develop a new flavivirus vaccine platform based on the novel insect-specific flavivirus called Aripo virus, which we used to create a preclinical Zika virus (ZIKV) vaccine named Aripo/Zika virus (ARPV/ZIKV). ARPV/ZIKV is a live recombinant virus consisting of the ZIKV pre-membrane and envelope protein genes expressed on an Aripo virus backbone. In this work, we quantify the safety and efficacy of ARPV/ZIKV in multiple murine models, and begin to elucidate the mechanisms of humoral and cell-mediated immune induction for this new platform. Overall, the vaccine showed no evidence of pathogenicity in immunocompromised or suckling mice, and demonstrated a complete inability to replicate in various vertebrate cell lines. Despite this lack of replication, a single dose of live, unadjuvanted ARPV/ZIKV completely prevented ZIKV disease in mice and prevented in utero ZIKV transmission in gravid mice. Direct protection post-ZIKV challenge appears to be primarily mediated by neutralizing antibodies based on passive transfer, adoptive transfer, and T-cell depletion studies. However, vaccination studies in Rag1 KO, Tcra KO, and muMt- mice demonstrate the critical role of T-cell responses in developing immunity post-vaccination. In summary, ARPV/ZIKV is a promising vaccine candidate that induces robust adaptive immune responses, and this success is a p

Flaviviruses are a genus of vector-transmitted viruses that are nearly globally distributed, and flavivirus infections can result in life threatening diseases. Many flaviviruses such as Dengue, West Nile, yellow fever and Zika viruses are globally distributed. Zika virus (ZIKV) is a single strand positive-sense RNA virus, and its disease has been linked to Guillain Barré Syndrome (i.e., a debilitating autoimmune disorder that affects the nerves) in adults and congenital birth defects including microcephaly (i.e., a neurodevelopmental disorder due to impaired neural cell proliferation) in newborns. Insect-specific flaviviruses (ISFVs) are understudied given their apathogenic characteristics to humans and animals. However, given their close genetic relationship to vertebrate infectious flaviviruses, ISFVs can serve as a delivery system (i.e., vector) for flavivirus antigenic proteins. Aripo virus (ARPV) is a recently discovered ISFV isolated in Trinidad. We developed a chimeric Zika vaccine, Aripo-Zika, by substituting the pre-membrane and envelope genes of ZIKV into the ARPV genome. Here, we explored (i) the efficacy of Aripo-Zika (AZ) vaccination by evaluating passive transfer of maternal antibodies, (ii) the optimal dosage regimen, (iii) anti-vector immunity to the ARPV backbone, and (iv) the effects of boosters on vaccine efficacy. We also evaluated AZ safety via a co-infection study. Our results show a near linear relationship between increased dose and immunogenicity, with 1011 genome copies being the most effective minimum dose administered. Inclusion of boosters further increased the immunogenicity of AZ. Additionally, prior immunization with AZ showed minimal effects on subsequent immunization with an ARPV-West Nile virus (AWN) vaccine candidate, confirming the applicability of the ARPV backbone to multiple flavivirus vaccine candidates. In vitro co-infection of ZIKV with ARPV, and ZIKV with AZ in African green monkey kidney cells (i.e., Vero-76) indicated AR

Flaviviruses are a genus of vector-transmitted viruses that are nearly globally distributed, and flavivirus infections can result in life threatening diseases. Many flaviviruses such as Dengue, West Nile, yellow fever and Zika viruses are globally distributed. Zika virus (ZIKV) is a single strand positive-sense RNA virus, and its disease has been linked to Guillain Barré Syndrome (i.e., a debilitating autoimmune disorder that affects the nerves) in adults and congenital birth defects including microcephaly (i.e., a neurodevelopmental disorder due to impaired neural cell proliferation) in newborns. Insect-specific flaviviruses (ISFVs) are understudied given their apathogenic characteristics to humans and animals. However, given their close genetic relationship to vertebrate infectious flaviviruses, ISFVs can serve as a delivery system (i.e., vector) for flavivirus antigenic proteins. Aripo virus (ARPV) is a recently discovered ISFV isolated in Trinidad. We developed a chimeric Zika vaccine, Aripo-Zika, by substituting the pre-membrane and envelope genes of ZIKV into the ARPV genome. Here, we explored (i) the efficacy of Aripo-Zika (AZ) vaccination by evaluating passive transfer of maternal antibodies, (ii) the optimal dosage regimen, (iii) anti-vector immunity to the ARPV backbone, and (iv) the effects of boosters on vaccine efficacy. We also evaluated AZ safety via a co-infection study. Our results show a near linear relationship between increased dose and immunogenicity, with 1011 genome copies being the most effective minimum dose administered. Inclusion of boosters further increased the immunogenicity of AZ. Additionally, prior immunization with AZ showed minimal effects on subsequent immunization with an ARPV-West Nile virus (AWN) vaccine candidate, confirming the applicability of the ARPV backbone to multiple flavivirus vaccine candidates. In vitro co-infection of ZIKV with ARPV, and ZIKV with AZ in African green monkey kidney cells (i.e., Vero-76) indicated AR

Vaccination remains critical for viral disease outbreak prevention and control, but conventional vaccine development typically involves trade-offs between safety and immunogenicity. We used a recently discovered insect-specific flavivirus as a vector in order to develop an exceptionally safe, flavivirus vaccine candidate with single-dose efficacy. To evaluate the safety and efficacy of this platform, we created a chimeric Zika virus (ZIKV) vaccine candidate, designated Aripo/Zika virus (ARPV/ZIKV). ZIKV has caused immense economic and public health impacts throughout the Americas and remains a significant public health threat. ARPV/ZIKV vaccination showed exceptional safety due to ARPV/ZIKV’s inherent vertebrate host-restriction. ARPV/ZIKV showed no evidence of replication or translation in vitro and showed no hematological, histological or pathogenic effects in vivo. A single-dose immunization with ARPV/ZIKV induced rapid and robust neutralizing antibody and cellular responses, which offered complete protection against ZIKV-induced morbidity, mortality and in utero transmission in immune-competent and -compromised murine models. Splenocytes derived from vaccinated mice demonstrated significant CD4+ and CD8+ responses and significant cytokine production post-antigen exposure. Altogether, our results further support that chimeric insect-specific flaviviruses are a promising strategy to restrict flavivirus emergence via vaccine development.

Vaccination remains critical for viral disease outbreak prevention and control, but conventional vaccine development typically involves trade-offs between safety and immunogenicity. We used a recently discovered insect-specific flavivirus as a vector in order to develop an exceptionally safe, flavivirus vaccine candidate with single-dose efficacy. To evaluate the safety and efficacy of this platform, we created a chimeric Zika virus (ZIKV) vaccine candidate, designated Aripo/Zika virus (ARPV/ZIKV). ZIKV has caused immense economic and public health impacts throughout the Americas and remains a significant public health threat. ARPV/ZIKV vaccination showed exceptional safety due to ARPV/ZIKV’s inherent vertebrate host-restriction. ARPV/ZIKV showed no evidence of replication or translation in vitro and showed no hematological, histological or pathogenic effects in vivo. A single-dose immunization with ARPV/ZIKV induced rapid and robust neutralizing antibody and cellular responses, which offered complete protection against ZIKV-induced morbidity, mortality and in utero transmission in immune-competent and -compromised murine models. Splenocytes derived from vaccinated mice demonstrated significant CD4+ and CD8+ responses and significant cytokine production post-antigen exposure. Altogether, our results further support that chimeric insect-specific flaviviruses are a promising strategy to restrict flavivirus emergence via vaccine development.

Vaccination remains critical for viral disease outbreak prevention and control, but conventional vaccine development typically involves trade-offs between safety and immunogenicity. We used a recently discovered insect-specific flavivirus as a vector in order to develop an exceptionally safe, flavivirus vaccine candidate with single-dose efficacy. To evaluate the safety and efficacy of this platform, we created a chimeric Zika virus (ZIKV) vaccine candidate, designated Aripo/Zika virus (ARPV/ZIKV). ZIKV has caused immense economic and public health impacts throughout the Americas and remains a significant public health threat. ARPV/ZIKV vaccination showed exceptional safety due to ARPV/ZIKV’s inherent vertebrate host-restriction. ARPV/ZIKV showed no evidence of replication or translation in vitro and showed no hematological, histological or pathogenic effects in vivo. A single-dose immunization with ARPV/ZIKV induced rapid and robust neutralizing antibody and cellular responses, which offered complete protection against ZIKV-induced morbidity, mortality and in utero transmission in immune-competent and -compromised murine models. Splenocytes derived from vaccinated mice demonstrated significant CD4+ and CD8+ responses and significant cytokine production post-antigen exposure. Altogether, our results further support that chimeric insect-specific flaviviruses are a promising strategy to restrict flavivirus emergence via vaccine development.

Vaccination remains critical for viral disease outbreak prevention and control, but conventional vaccine development typically involves trade-offs between safety and immunogenicity. We used a recently discovered insect-specific flavivirus as a vector in order to develop an exceptionally safe, flavivirus vaccine candidate with single-dose efficacy. To evaluate the safety and efficacy of this platform, we created a chimeric Zika virus (ZIKV) vaccine candidate, designated Aripo/Zika virus (ARPV/ZIKV). ZIKV has caused immense economic and public health impacts throughout the Americas and remains a significant public health threat. ARPV/ZIKV vaccination showed exceptional safety due to ARPV/ZIKV’s inherent vertebrate host-restriction. ARPV/ZIKV showed no evidence of replication or translation in vitro and showed no hematological, histological or pathogenic effects in vivo. A single-dose immunization with ARPV/ZIKV induced rapid and robust neutralizing antibody and cellular responses, which offered complete protection against ZIKV-induced morbidity, mortality and in utero transmission in immune-competent and -compromised murine models. Splenocytes derived from vaccinated mice demonstrated significant CD4+ and CD8+ responses and significant cytokine production post-antigen exposure. Altogether, our results further support that chimeric insect-specific flaviviruses are a promising strategy to restrict flavivirus emergence via vaccine development.

Metagenomic studies of mosquitoes have revealed that their virome is far more diverse and includes many more viruses than just the pathogenic arboviruses vectored by mosquitoes. In this study, the virome of 953 female mosquitoes collected in the summer of 2017, representing six mosquito species from two geographic locations in Mid-Eastern Sweden, were characterized. In addition, the near-complete genome of nine RNA viruses were characterized and phylogenetically analysed. These viruses showed association to the viral orders Bunyavirales, Picornavirales, Articulavirales, and Tymovirales, and to the realm Ribovira. Hence, through this study, we expand the knowledge of the virome composition of different mosquito species in Sweden. In addition, by providing viral reference genomes from wider geographic regions and different mosquito species, future in silico recognition and assembly of viral genomes in metagenomic datasets will be facilitated.

Metagenomic studies of mosquitoes have revealed that their virome is far more diverse and includes many more viruses than just the pathogenic arboviruses vectored by mosquitoes. In this study, the virome of 953 female mosquitoes collected in the summer of 2017, representing six mosquito species from two geographic locations in Mid-Eastern Sweden, were characterized. In addition, the near-complete genome of nine RNA viruses were characterized and phylogenetically analysed. These viruses showed association to the viral orders Bunyavirales, Picornavirales, Articulavirales, and Tymovirales, and to the realm Ribovira. Hence, through this study, we expand the knowledge of the virome composition of different mosquito species in Sweden. In addition, by providing viral reference genomes from wider geographic regions and different mosquito species, future in silico recognition and assembly of viral genomes in metagenomic datasets will be facilitated.

Metagenomic studies of mosquitoes have revealed that their virome is far more diverse and includes many more viruses than just the pathogenic arboviruses vectored by mosquitoes. In this study, the virome of 953 female mosquitoes collected in the summer of 2017, representing six mosquito species from two geographic locations in Mid-Eastern Sweden, were characterized. In addition, the near-complete genome of nine RNA viruses were characterized and phylogenetically analysed. These viruses showed association to the viral orders Bunyavirales, Picornavirales, Articulavirales, and Tymovirales, and to the realm Ribovira. Hence, through this study, we expand the knowledge of the virome composition of different mosquito species in Sweden. In addition, by providing viral reference genomes from wider geographic regions and different mosquito species, future in silico recognition and assembly of viral genomes in metagenomic datasets will be facilitated.

Several mosquito-borne diseases affecting humans are emerging or reemerging in the United States. The early detection of pathogens in mosquito populations is essential to prevent and control the spread of these diseases. In this study, we tested the potential applicability of the Lawrence Livermore Microbial Detection Array (LLMDA) to enhance biosurveillance by detecting microbes present in Aedes aegypti, Aedes albopictus, and Culex mosquitoes, which are major vector species globally, including in Texas. The sensitivity and reproducibility of the LLMDA were tested in mosquito samples spiked with different concentrations of dengue virus (DENV), revealing a detection limit of >100 but <1,000 PFU/ml. Additionally, field-collected mosquitoes from Chicago, IL, and College Station, TX, of known infection status (West Nile virus [WNV] and Culex flavivirus [CxFLAV] positive) were tested on the LLMDA to confirm its efficiency. Mosquito field samples of unknown infection status, collected in San Antonio, TX, and the Lower Rio Grande Valley (LRGV), TX, were run on the LLMDA and further confirmed by PCR or quantitative PCR (qPCR). The analysis of the field samples with the LLMDA revealed the presence of cell-fusing agent virus (CFAV) in A. aegypti populations. Wolbachia was also detected in several of the field samples (A. albopictus and Culex spp.) by the LLMDA. Our findings demonstrated that the LLMDA can be used to detect multiple arboviruses of public health importance, including viruses that belong to the Flavivirus, Alphavirus, and Orthobunyavirus genera. Additionally, insect-specific viruses and bacteria were also detected in field-collected mosquitoes. Another strength of this array is its ability to detect multiple viruses in the same mosquito pool, allowing for the detection of cocirculating pathogens in an area and the identification of potential ecological associations between different viruses. This array can aid in the biosurveillance of mosquito-borne virus

Metagenomic studies of mosquitoes have revealed that their virome is far more diverse and includes many more viruses than just the pathogenic arboviruses vectored by mosquitoes. In this study, the virome of 953 female mosquitoes collected in the summer of 2017, representing six mosquito species from two geographic locations in Mid-Eastern Sweden, were characterized. In addition, the near-complete genome of nine RNA viruses were characterized and phylogenetically analysed. These viruses showed association to the viral orders Bunyavirales, Picornavirales, Articulavirales, and Tymovirales, and to the realm Ribovira. Hence, through this study, we expand the knowledge of the virome composition of different mosquito species in Sweden. In addition, by providing viral reference genomes from wider geographic regions and different mosquito species, future in silico recognition and assembly of viral genomes in metagenomic datasets will be facilitated.

Metagenomic studies of mosquitoes have revealed that their virome is far more diverse and includes many more viruses than just the pathogenic arboviruses vectored by mosquitoes. In this study, the virome of 953 female mosquitoes collected in the summer of 2017, representing six mosquito species from two geographic locations in Mid-Eastern Sweden, were characterized. In addition, the near-complete genome of nine RNA viruses were characterized and phylogenetically analysed. These viruses showed association to the viral orders Bunyavirales, Picornavirales, Articulavirales, and Tymovirales, and to the realm Ribovira. Hence, through this study, we expand the knowledge of the virome composition of different mosquito species in Sweden. In addition, by providing viral reference genomes from wider geographic regions and different mosquito species, future in silico recognition and assembly of viral genomes in metagenomic datasets will be facilitated.

Background: Many arboviral outbreaks have occurred in various locations in Kenya. Entomological surveys are suitable methods for revealing information about circulating arboviruses before human outbreaks are recognized. Therefore, mosquitoes were collected in Kenya to determine the distribution of arboviruses. Methods: Various species of mosquitoes were sampled from January to July 2012 using several collection methods. Mosquito homogenates were directly tested by reverse transcription-polymerase chain reaction (RT-PCR) using various arbovirus-targeted primer pairs. Results: We collected 12,569 mosquitoes. Although no human-related arboviruses were detected, Culex flavivirus (CxFV), an insect-specific arbovirus, was detected in 54 pools of 324 Culex quinquefasciatus individuals collected during the rainy season. Of these 54 positive pools, 96.3% (52/54) of the mosquitoes were collected in Busia, on the border of western Kenya and Uganda. The remaining two CxFV-positive pools were collected in Mombasa and Kakamega, far from Busia. Phylogenetic analysis revealed minimal genetic diversity among the CxFVs collected in Mombasa, Kakamega, and Busia, even though these cities are in geographically different regions. Additionally, CxFV was detected in one mosquito pool collected in Mombasa during the dry season. In addition to Culex mosquitoes, Aedes (Stegomyia) and Anopheles mosquitoes were also positive for the Flavivirus genus. Cell fusing agent virus was detected in one pool of Aedes aegypti. Mosquito flavivirus was detected in three pools of Anopheles gambiae s.l. collected in the dry and rainy seasons. Conclusions: Although no mosquitoes were positive for human-related arbovirus, insect-specific viruses were detected in various species of mosquitoes. The heterogeneity observed in the number of CxFVs in Culex mosquitoes in different locations in Kenya suggests that the abundance of human-related viruses might differ depending on the abundance of insect-specific viruses., Tropical Medicine and Health, 46(1), 19; 2018