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1.
J Gen Virol ; 105(5)2024 May.
Article in English | MEDLINE | ID: mdl-38809251

ABSTRACT

Tick-borne orthoflaviviruses (TBFs) are classified into three conventional groups based on genetics and ecology: mammalian, seabird and probable-TBF group. Recently, a fourth basal group has been identified in Rhipicephalus ticks from Africa: Mpulungu flavivirus (MPFV) in Zambia and Ngoye virus (NGOV) in Senegal. Despite attempts, isolating these viruses in vertebrate and invertebrate cell lines or intracerebral injection of newborn mice with virus-containing homogenates has remained unsuccessful. In this study, we report the discovery of Xinyang flavivirus (XiFV) in Haemaphysalis flava ticks from Xìnyáng, Henan Province, China. Phylogenetic analysis shows that XiFV was most closely related to MPFV and NGOV, marking the first identification of this tick orthoflavivirus group in Asia. We developed a reverse transcriptase quantitative PCR assay to screen wild-collected ticks and egg clutches, with absolute infection rates of 20.75 % in adult females and 15.19 % in egg clutches, suggesting that XiFV could be potentially spread through transovarial transmission. To examine potential host range, dinucleotide composition analyses revealed that XiFV, MPFV and NGOV share a closer composition to classical insect-specific orthoflaviviruses than to vertebrate-infecting TBFs, suggesting that XiFV could be a tick-only orthoflavivirus. Additionally, both XiFV and MPFV lack a furin cleavage site in the prM protein, unlike other TBFs, suggesting these viruses might exist towards a biased immature particle state. To examine this, chimeric Binjari virus with XIFV-prME (bXiFV) was generated, purified and analysed by SDS-PAGE and negative-stain transmission electron microscopy, suggesting prototypical orthoflavivirus size (~50 nm) and bias towards uncleaved prM. In silico structural analyses of the 3'-untranslated regions show that XiFV forms up to five pseudo-knot-containing stem-loops and a prototypical orthoflavivirus dumbbell element, suggesting the potential for multiple exoribonuclease-resistant RNA structures.


Subject(s)
Flavivirus , Ixodidae , Phylogeny , Animals , Flavivirus/genetics , Flavivirus/classification , Flavivirus/isolation & purification , China , Ixodidae/virology , Female
2.
Euro Surveill ; 29(20)2024 05.
Article in English | MEDLINE | ID: mdl-38757289

ABSTRACT

Aedes albopictus collected in 2023 in the greater Paris area (Île-de-France) were experimentally able to transmit five arboviruses: West Nile virus from 3 days post-infection (dpi), chikungunya virus and Usutu virus from 7 dpi, dengue virus and Zika virus from 21 dpi. Given the growing number of imported dengue cases reported in early 2024 in France, surveillance of Ae. albopictus should be reinforced during the Paris Olympic Games in July, when many international visitors including from endemic countries are expected.


Subject(s)
Aedes , Chikungunya virus , Dengue Virus , Zika Virus , Animals , Aedes/virology , Humans , Zika Virus/isolation & purification , Dengue Virus/isolation & purification , Chikungunya virus/isolation & purification , Paris , Mosquito Vectors/virology , West Nile virus/isolation & purification , Arboviruses/isolation & purification , Arbovirus Infections/transmission , Flavivirus/isolation & purification , France , Dengue/transmission , Dengue/epidemiology , Zika Virus Infection/transmission
3.
Proc Natl Acad Sci U S A ; 121(19): e2319400121, 2024 May 07.
Article in English | MEDLINE | ID: mdl-38687787

ABSTRACT

During their blood-feeding process, ticks are known to transmit various viruses to vertebrates, including humans. Recent viral metagenomic analyses using next-generation sequencing (NGS) have revealed that blood-feeding arthropods like ticks harbor a large diversity of viruses. However, many of these viruses have not been isolated or cultured, and their basic characteristics remain unknown. This study aimed to present the identification of a difficult-to-culture virus in ticks using NGS and to understand its epidemic dynamics using molecular biology techniques. During routine tick-borne virus surveillance in Japan, an unknown flaviviral sequence was detected via virome analysis of host-questing ticks. Similar viral sequences have been detected in the sera of sika deer and wild boars in Japan, and this virus was tentatively named the Saruyama virus (SAYAV). Because SAYAV did not propagate in any cultured cells tested, single-round infectious virus particles (SRIP) were generated based on its structural protein gene sequence utilizing a yellow fever virus-based replicon system to understand its nationwide endemic status. Seroepidemiological studies using SRIP as antigens have demonstrated the presence of neutralizing antibodies against SAYAV in sika deer and wild boar captured at several locations in Japan, suggesting that SAYAV is endemic throughout Japan. Phylogenetic analyses have revealed that SAYAV forms a sister clade with the Orthoflavivirus genus, which includes important mosquito- and tick-borne pathogenic viruses. This shows that SAYAV evolved into a lineage independent of the known orthoflaviviruses. This study demonstrates a unique approach for understanding the epidemiology of uncultured viruses by combining viral metagenomics and pseudoinfectious viral particles.


Subject(s)
Deer , Flavivirus , Metagenomics , Ticks , Animals , Metagenomics/methods , Japan/epidemiology , Deer/virology , Flavivirus/genetics , Flavivirus/isolation & purification , Flavivirus/classification , Ticks/virology , Phylogeny , Virome/genetics , Virion/genetics , Sus scrofa/virology , High-Throughput Nucleotide Sequencing , Humans , Seroepidemiologic Studies , Genome, Viral
4.
Viruses ; 16(4)2024 04 12.
Article in English | MEDLINE | ID: mdl-38675940

ABSTRACT

West Nile Virus (WNV) and Usutu Virus (USUV) are both neurotropic mosquito-borne viruses belonging to the Flaviviridae family. These closely related viruses mainly follow an enzootic cycle involving mosquitoes as vectors and birds as amplifying hosts, but humans and other mammals can also be infected through mosquito bites. WNV was first identified in Uganda in 1937 and has since spread globally, notably in Europe, causing periodic outbreaks associated with severe cases of neuroinvasive diseases such as meningitis and encephalitis. USUV was initially isolated in 1959 in Swaziland and has also spread to Europe, primarily affecting birds and having a limited impact on human health. There has been a recent expansion of these viruses' geographic range in Europe, facilitated by factors such as climate change, leading to increased human exposure. While sharing similar biological traits, ecology, and epidemiology, there are significant distinctions in their pathogenicity and their impact on both human and animal health. While WNV has been more extensively studied and is a significant public health concern in many regions, USUV has recently been gaining attention due to its emergence in Europe and the diversity of its circulating lineages. Understanding the pathophysiology, ecology, and transmission dynamics of these viruses is important to the implementation of effective surveillance and control measures. This perspective provides a brief overview of the current situation of these two viruses in Europe and outlines the significant challenges that need to be addressed in the coming years.


Subject(s)
Birds , Flavivirus Infections , Flavivirus , West Nile Fever , West Nile virus , Europe/epidemiology , West Nile virus/genetics , West Nile virus/physiology , West Nile virus/isolation & purification , Animals , Humans , Flavivirus/classification , Flavivirus/genetics , Flavivirus/pathogenicity , Flavivirus/isolation & purification , Flavivirus/physiology , Flavivirus Infections/epidemiology , Flavivirus Infections/virology , Flavivirus Infections/transmission , Flavivirus Infections/veterinary , West Nile Fever/epidemiology , West Nile Fever/virology , West Nile Fever/transmission , Birds/virology , Culicidae/virology , Mosquito Vectors/virology , Disease Outbreaks
5.
Virol Sin ; 39(2): 228-234, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38461965

ABSTRACT

Guaico Culex virus (GCXV) is a newly identified segmented Jingmenvirus from Culex spp. mosquitoes in Central and South America. The genome of GCXV is composed of four or five single-stranded positive RNA segments. However, the infection kinetics and transmission capability of GCXV in mosquitoes remain unknown. In this study, we used reverse genetics to rescue two GCXVs (4S and 5S) that contained four and five RNA segments, respectively, in C6/36 â€‹cells. Further in vitro characterization revealed that the two GCXVs exhibited comparable replication kinetics, protein expression and viral titers. Importantly, GCXV RNAs were detected in the bodies, salivary glands, midguts and ovaries of Culex quinquefasciatus at 4-10 days after oral infection. In addition, two GCXVs can colonize Cx. quinquefasciatus eggs, resulting in positive rates of 15%-35% for the second gonotrophic cycle. In conclusion, our results demonstrated that GCXVs with four or five RNA segments can be detected in Cx. quinquefasciatus eggs during the first and second gonotrophic cycles after oral infection.


Subject(s)
Culex , Mosquito Vectors , RNA, Viral , Virus Replication , Animals , Culex/virology , Mosquito Vectors/virology , RNA, Viral/genetics , Female , Cell Line , Flavivirus/genetics , Flavivirus/physiology , Flavivirus/isolation & purification , Kinetics , Viral Load , Genome, Viral , Salivary Glands/virology
6.
J Virol Methods ; 327: 114917, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38503367

ABSTRACT

Bagaza virus (BAGV) is a mosquito-borne orthoflavivirus known to occur in regions of southern Europe, Africa, India and the Middle East. The virus has been associated with neurological disease and fatalities in various wild bird species. Association with human disease is not confirmed although limited serological evidence has suggested human infection. Surveillance programs for screening mosquitoes for evidence of arbovirus infection play an important role in providing information regarding the circulation and spread of viruses in specific regions. BAGV was detected in a mosquito pool during surveillance of mosquitoes collected in central South Africa between November 2019 and March 2023. Homogenized mosquito pools were screened for flaviviral RNA using conventional RT-PCR and virus isolation was attempted on positive samples. BAGV was detected and subsequently isolated using cell culture. A multiplex tiling PCR method for targeted enrichment using a PCR based or amplicon sequencing approach of the complete genome of BAGV was developed and optimized. Primers were designed using alignment of complete genome sequence data retrieved from GenBank to identify suitable primer sites that would generate overlapping fragments spanning the complete genome. Six forward primers and eight reverse primers were identified that target the complete genome and amplified nine overlapping fragments, that ranged in length from 1954 to 2039 with an overlap ranging from 71 to 711 base pairs. The design strategy included multiple forward and reverse primer pairs for the 5' and 3' ends. Phylogenetic analysis with other isolates was performed and BAGV isolate VBD 74/23/3 was shown to share high similarity with previous BAGV isolates from all regions, with genetic distance ranging from 0.026 to 0.083. VBD 74/23/3 was most closely related to previous isolates from southern Africa, ZRU96/16/2 isolated from a post-mortem sample from a pheasant in 2016 and MP-314-NA-2018 isolated from mosquitoes in northwestern Namibia with genetic distance 0.0085 and 0.016 respectively. Currently there is limited complete genome sequence data available for many of the arboviruses circulating in Africa. The multiplex tiling method provided a simple and cost-effective method for obtaining complete genome sequence. This method can be readily applied to other viruses using sequence data from publicly available databases and would have important application facilitating genomic surveillance of arboviruses in low resource countries.


Subject(s)
Culicidae , Multiplex Polymerase Chain Reaction , Animals , South Africa , Culicidae/virology , Multiplex Polymerase Chain Reaction/methods , Flavivirus/genetics , Flavivirus/isolation & purification , Flavivirus/classification , RNA, Viral/genetics , Genome, Viral , Phylogeny , Mosquito Vectors/virology , Animals, Wild/virology
7.
J Virol ; 96(17): e0043922, 2022 09 14.
Article in English | MEDLINE | ID: mdl-35975997

ABSTRACT

Flaviviruses are positive-sense single-stranded RNA viruses, including some well-known human pathogens such as Zika, dengue, and yellow fever viruses, which are primarily associated with mosquito and tick vectors. The vast majority of flavivirus research has focused on terrestrial environments; however, recent findings indicate that a range of flaviviruses are also present in aquatic environments, both marine and freshwater. These flaviviruses are found in various hosts, including fish, crustaceans, molluscs, and echinoderms. Although the effects of aquatic flaviviruses on the hosts they infect are not all known, some have been detected in farmed species and may have detrimental effects on the aquaculture industry. Exploration of the evolutionary history through the discovery of the Wenzhou shark flavivirus in both a shark and crab host is of particular interest since the potential dual-host nature of this virus may indicate that the invertebrate-vertebrate relationship seen in other flaviviruses may have a more profound evolutionary root than previously expected. Potential endogenous viral elements and the range of novel aquatic flaviviruses discovered thus shed light on virus origins and evolutionary history and may indicate that, like terrestrial life, the origins of flaviviruses may lie in aquatic environments.


Subject(s)
Aquatic Organisms , Flavivirus Infections , Flavivirus , Animals , Aquaculture , Aquatic Organisms/isolation & purification , Aquatic Organisms/virology , Biological Evolution , Fishes/virology , Flavivirus/isolation & purification , Flavivirus Infections/virology , Humans
8.
Emerg Infect Dis ; 28(7): 1504-1506, 2022 07.
Article in English | MEDLINE | ID: mdl-35731200

ABSTRACT

Bagaza virus emerged in Spain in 2010 and was not reported in other countries in Europe until 2021, when the virus was detected by molecular methods in a corn bunting and several red-legged partridges in Portugal. Sequencing revealed high similarity between the 2021 strains from Portugal and the 2010 strains from Spain.


Subject(s)
Bird Diseases , Flavivirus Infections , Galliformes , Animals , Animals, Wild/virology , Bird Diseases/epidemiology , Bird Diseases/virology , Flavivirus/classification , Flavivirus/isolation & purification , Flavivirus Infections/epidemiology , Flavivirus Infections/veterinary , Galliformes/virology , Portugal/epidemiology , Spain
9.
PLoS Negl Trop Dis ; 16(4): e0010203, 2022 04.
Article in English | MEDLINE | ID: mdl-35427361

ABSTRACT

In Mauritania, several mosquito-borne viruses have been reported that can cause devastating diseases in animals and humans. However, monitoring data on their occurrence and local distribution are limited. Rift Valley fever virus (RVFV) is an arthropod-borne virus that causes major outbreaks throughout the African continent and the Arabian Peninsula. The first Rift Valley fever (RVF) epidemic in Mauritania occurred in 1987 and since then the country has been affected by recurrent outbreaks of the disease. To gain information on the occurrence of RVFV as well as other mosquito-borne viruses and their vectors in Mauritania, we collected and examined 4,950 mosquitoes, belonging to four genera and 14 species. The mosquitoes were captured during 2018 in the capital Nouakchott and in southern parts of Mauritania. Evidence of RVFV was found in a mosquito pool of female Anopheles pharoensis mosquitoes collected in December on a farm near the Senegal River. At that time, 37.5% of 16 tested Montbéliarde cattle on the farm showed RVFV-specific IgM antibodies. Additionally, we detected IgM antibodies in 10.7% of 28 indigenous cattle that had been sampled on the same farm one month earlier. To obtain information on potential RVFV reservoir hosts, blood meals of captured engorged mosquitoes were analyzed. The mosquitoes mainly fed on humans (urban areas) and cattle (rural areas), but also on small ruminants, donkeys, cats, dogs and straw-colored fruit bats. Results of this study demonstrate the circulation of RVFV in Mauritania and thus the need for further research to investigate the distribution of the virus and its vectors. Furthermore, factors that may contribute to its maintenance should be analyzed more closely. In addition, two mosquito pools containing Aedes aegypti and Culex quinquefasciatus mosquitoes showed evidence of dengue virus (DENV) 2 circulation in the city of Rosso. Further studies are therefore needed to also examine DENV circulation in Mauritania.


Subject(s)
Aedes , Dengue Virus , Feeding Behavior , Flavivirus , Rift Valley fever virus , Animals , Cattle , Female , Flavivirus/isolation & purification , Immunoglobulin M , Mauritania/epidemiology , Mosquito Vectors , Rift Valley fever virus/isolation & purification
10.
Cell Rep ; 37(11): 110091, 2021 12 14.
Article in English | MEDLINE | ID: mdl-34910910

ABSTRACT

Hematophagous arthropods, such as mosquitoes, naturally carry and transmit hundreds of arboviruses to humans. Blood meal is a predominant physical interface that shapes cross-species communications among humans, bloodsuckers, and arboviruses. Here, we identify a human-blood-derived microRNA, hsa-miR-150-5p, that interferes with a mosquito antiviral system to facilitate flavivirus infection and transmission. hsa-miR-150-5p is acquired with a blood meal into the mosquito hemocoel and persists for a prolonged time there. The agomir of hsa-miR-150-5p enhances, whereas the antagomir represses flaviviral infection in mosquitoes and transmission from mice to mosquitoes. Mechanistic studies indicate that hsa-miR-150-5p hijacks the mosquito Argonaute-1-mediated RNA interference system to suppress the expression of some chymotrypsins with potent virucidal activity. Mosquito chymotrypsins are essential for resisting systemic flavivirus infection in hemocoel tissues. Chymotrypsin homologs potentially targeted by miR-150-5p are also found in other hematophagous arthropods, demonstrating a conserved miR-150-5p-mediated cross-species RNAi mechanism that might determine flaviviral transmissibility in nature.


Subject(s)
Aedes/virology , Chymotrypsin/antagonists & inhibitors , Flavivirus Infections/virology , Flavivirus/isolation & purification , MicroRNAs/genetics , Mosquito Vectors/virology , Virus Replication , Animals , Female , Flavivirus/genetics , Flavivirus Infections/genetics , Flavivirus Infections/pathology , Genome, Viral , Humans , Male , Mice , Mice, Inbred C57BL , MicroRNAs/blood
11.
Viruses ; 13(12)2021 11 30.
Article in English | MEDLINE | ID: mdl-34960673

ABSTRACT

The surveillance for West Nile virus (WNV) in Catalonia (northeastern Spain) has consistently detected flaviviruses not identified as WNV. With the aim of characterizing the flaviviruses circulating in Catalonia, serum samples from birds and horses collected between 2010 and 2019 and positive by panflavivirus competition ELISA (cELISA) were analyzed by microneutralization test (MNT) against different flaviviruses. A third of the samples tested were inconclusive by MNT, highlighting the limitations of current diagnostic techniques. Our results evidenced the widespread circulation of flaviviruses, in particular WNV, but also Usutu virus (USUV), and suggest that chicken and horses could serve as sentinels for both viruses. In several regions, WNV and USUV overlapped, but no significant geographical aggregation was observed. Bagaza virus (BAGV) was not detected in birds, while positivity to tick-borne encephalitis virus (TBEV) was sporadically detected in horses although no endemic foci were observed. So far, no human infections by WNV, USUV, or TBEV have been reported in Catalonia. However, these zoonotic flaviviruses need to be kept under surveillance, ideally within a One Health framework.


Subject(s)
Bird Diseases/epidemiology , Flavivirus Infections/veterinary , Flavivirus/physiology , Horse Diseases/epidemiology , Animals , Antibodies, Viral/blood , Bird Diseases/blood , Bird Diseases/virology , Birds , Enzyme-Linked Immunosorbent Assay/veterinary , Flavivirus/genetics , Flavivirus/immunology , Flavivirus/isolation & purification , Flavivirus Infections/blood , Flavivirus Infections/epidemiology , Flavivirus Infections/virology , Horse Diseases/blood , Horse Diseases/virology , Horses , Seroepidemiologic Studies , Spain/epidemiology
12.
Viruses ; 13(11)2021 10 25.
Article in English | MEDLINE | ID: mdl-34834955

ABSTRACT

Mosquitoes in the Aedes and Culex genera are considered the main vectors of pathogenic flaviviruses worldwide. Entomological surveillance using universal flavivirus sets of primers in mosquitoes can detect not only pathogenic viruses but also insect-specific ones. It is hypothesized that insect-specific flaviviruses, which naturally infect these mosquitoes, may influence their vector competence for zoonotic arboviruses. Here, entomological surveillance was performed between January 2014 and May 2018 in five different provinces in the northeastern parts of South Africa, with the aim of identifying circulating flaviviruses. Mosquitoes were sampled using different carbon dioxide trap types. Overall, 64,603 adult mosquitoes were collected, which were screened by RT-PCR and sequencing. In total, 17 pools were found positive for insect-specific Flaviviruses in the mosquito genera Aedes (12/17, 70.59%) and Anopheles (5/17, 29.41%). No insect-specific viruses were detected in Culex species. Cell-fusing agent viruses were detected in Aedes aegypti and Aedes caballus. A range of anopheline mosquitoes, including Anopheles coustani, An. squamosus and An. maculipalpis, were positive for Culex flavivirus-like and Anopheles flaviviruses. These results confirm the presence of insect-specific flaviviruses in mosquito populations in South Africa, expands their geographical range and indicates potential mosquito species as vector species.


Subject(s)
Culicidae/virology , Flavivirus/classification , Flavivirus/isolation & purification , Mosquito Vectors/virology , Aedes/virology , Animals , Anopheles/virology , Arboviruses/classification , Arboviruses/genetics , Arboviruses/isolation & purification , Culex/virology , Flavivirus/genetics , Insect Viruses/isolation & purification , Phylogeny , South Africa
13.
Viruses ; 13(11)2021 11 16.
Article in English | MEDLINE | ID: mdl-34835097

ABSTRACT

BACKGROUND: Dengue virus and Japanese encephalitis virus are two common flaviviruses that are spread widely by Aedes and Culex mosquitoes. Livestock keeping is vital for cities; however, it can pose the risk of increasing the mosquito population. Our study explored how livestock keeping in and around a large city is associated with the presence of mosquitoes and the risk of them spreading flaviviruses. METHODS: An entomological study was conducted in 6 districts with 233 households with livestock, and 280 households without livestock, in Hanoi city. BG-Sentinel traps and CDC light traps were used to collect mosquitoes close to animal farms and human habitats. Adult mosquitoes were counted, identified to species level, and grouped into 385 pools, which were screened for flaviviruses using a pan-flavivirus qPCR protocol and sequencing. RESULTS: A total of 12,861 adult mosquitoes were collected at the 513 households, with 5 different genera collected, of which the Culex genus was the most abundant. Our study found that there was a positive association between livestock keeping and the size of the mosquito population-most predominantly between pig rearing and Culex species (p < 0.001). One pool of Cx. tritaeniorhynchus, collected in a peri-urban district, was found to be positive for Japanese encephalitis virus. CONCLUSIONS: The risk of flavivirus transmission in urban areas of Hanoi city due to the spread of Culex and Aedes mosquitoes could be facilitated by livestock keeping.


Subject(s)
Aedes/virology , Culex/virology , Flavivirus/isolation & purification , Livestock/virology , Mosquito Vectors/virology , Animals , Cities , Family Characteristics , Humans , Vietnam
14.
Sci Rep ; 11(1): 19031, 2021 09 24.
Article in English | MEDLINE | ID: mdl-34561471

ABSTRACT

Nucleic acid test (NAT), most typically quantitative PCR, is one of the standard methods for species specific flavivirus diagnosis. Semi-comprehensive NATs such as pan-flavivirus PCR which covers genus Flavivirus are also available; however, further specification by sequencing is required for species level differentiation. In this study, a semi-comprehensive detection system that allows species differentiation of flaviviruses was developed by integration of the pan-flavivirus PCR and Nanopore sequencing. In addition, a multiplexing method was established by adding index sequences through the PCR with a streamlined bioinformatics pipeline. This enables defining cut-off values for observed read counts. In the laboratory setting, this approach allowed the detection of up to nine different flaviviruses. Using clinical samples collected in Vietnam and Brazil, seven different flaviviruses were also detected. When compared to a commercial NAT, the sensitivity and specificity of our system were 66.7% and 95.4%, respectively. Conversely, when compared to our system, the sensitivity and specificity of the commercial NAT were 57.1% and 96.9%, respectively. In addition, Nanopore sequencing detected more positive samples (n = 8) compared to the commercial NAT (n = 6). Collectively, our study has established a semi-comprehensive sequencing-based diagnostic system for the detection of flaviviruses at extremely affordable costs, considerable sensitivity, and only requires simple experimental methods.


Subject(s)
Flavivirus Infections/diagnosis , Flavivirus Infections/virology , Flavivirus/isolation & purification , Nanopore Sequencing/methods , Brazil , Computational Biology/methods , Flavivirus/genetics , Humans , Polymerase Chain Reaction/methods , Sensitivity and Specificity , Vietnam
15.
Viruses ; 13(8)2021 07 28.
Article in English | MEDLINE | ID: mdl-34452347

ABSTRACT

The Usutu virus (USUV) is a mosquito-borne zoonotic flavivirus. Despite its continuous circulation in Europe, knowledge on the pathology, cellular and tissue tropism and pathogenetic potential of different circulating viral lineages is still fragmentary. Here, macroscopic and microscopic evaluations are performed in association with the study of cell and tissue tropism and comparison of lesion severity of two circulating virus lineages (Europe 3; Africa 3) in 160 Eurasian blackbirds (Turdus merula) in the Netherlands. Results confirm hepatosplenomegaly, coagulative necrosis and lymphoplasmacytic inflammation as major patterns of lesions and, for the first time, vasculitis as a novel virus-associated lesion. A USUV and Plasmodium spp. co-infection was commonly identified. The virus was associated with lesions by immunohistochemistry and was reported most commonly in endothelial cells and blood circulating and tissue mononucleated cells, suggesting them as a major route of entry and spread. A tropism for mononuclear phagocytes cells was further supported by viral labeling in multinucleated giant cells. The involvement of ganglionic neurons and epithelial cells of the gastrointestinal tract suggests a possible role of oral transmission, while the involvement of feather follicle shafts and bulbs suggests their use as a diagnostic sample for live bird testing. Finally, results suggest similar pathogenicity for the two circulating lineages.


Subject(s)
Bird Diseases/virology , Flavivirus Infections/veterinary , Flavivirus/physiology , Passeriformes/virology , Animals , Bird Diseases/pathology , Flavivirus/genetics , Flavivirus/isolation & purification , Flavivirus/pathogenicity , Flavivirus Infections/pathology , Flavivirus Infections/virology , Netherlands , Phagocytes/virology , Virulence
16.
Virology ; 562: 50-62, 2021 10.
Article in English | MEDLINE | ID: mdl-34256244

ABSTRACT

We describe the isolation and characterization of a novel insect-specific flavivirus (ISFV), tentatively named Aripo virus (ARPV), that was isolated from Psorophora albipes mosquitoes collected in Trinidad. The ARPV genome was determined and phylogenetic analyses showed that it is a dual host associated ISFV, and clusters with the main mosquito-borne flaviviruses. ARPV antigen was significantly cross-reactive with Japanese encephalitis virus serogroup antisera, with significant cross-reactivity to Ilheus and West Nile virus (WNV). Results suggest that ARPV replication is limited to mosquitoes, as it did not replicate in the sandfly, culicoides or vertebrate cell lines tested. We also demonstrated that ARPV is endocytosed into vertebrate cells and is highly immunomodulatory, producing a robust innate immune response despite its inability to replicate in vertebrate systems. We show that prior infection or coinfection with ARPV limits WNV-induced disease in mouse models, likely the result of a robust ARPV-induced type I interferon response.


Subject(s)
Flavivirus/immunology , Immunomodulation , Insect Viruses/immunology , Vertebrates/immunology , Animals , Antigens, Viral/immunology , Cross Reactions , Culicidae/virology , Disease Models, Animal , Flavivirus/genetics , Flavivirus/isolation & purification , Flavivirus/pathogenicity , Genome, Viral/genetics , Host Specificity , Immunity, Innate , Insect Viruses/genetics , Insect Viruses/isolation & purification , Insect Viruses/pathogenicity , Macrophages/immunology , Mice , Phylogeny , Vertebrates/virology , Viral Interference , Virus Replication , West Nile Fever/immunology , West Nile virus/immunology , West Nile virus/pathogenicity
17.
J Gen Virol ; 102(7)2021 07.
Article in English | MEDLINE | ID: mdl-34236957

ABSTRACT

Mosquito-borne flaviviruses are significant contributors to the arboviral disease burdens both in Australia and globally. While routine arbovirus surveillance remains a vital exercise to identify known flaviviruses in mosquito populations, novel or divergent and emerging species can be missed by these traditional methods. The MAVRIC (monoclonal antibodies to viral RNA intermediates in cells) system is an ELISA-based method for broad-spectrum isolation of positive-sense and double-stranded RNA (dsRNA) viruses based on detection of dsRNA in infected cells. While the MAVRIC ELISA has successfully been used to detect known and novel flaviviruses in Australian mosquitoes, we previously reported that dsRNA could not be detected in dengue virus-infected cells using this method. In this study we identified additional flaviviruses which evade detection of dsRNA by the MAVRIC ELISA. Utilising chimeric flaviviruses we demonstrated that this outcome may be dictated by the non-structural proteins and/or untranslated regions of the flaviviral genome. In addition, we report a modified fixation method that enables improved detection of flavivirus dsRNA and inactivation of non-enveloped viruses from mosquito populations using the MAVRIC system. This study demonstrates the utility of anti-dsRNA monoclonal antibodies for identifying viral replication in insect and vertebrate cell systems and highlights a unique characteristic of flavivirus replication.


Subject(s)
Culicidae/virology , Flavivirus/isolation & purification , Flavivirus/physiology , RNA, Double-Stranded/analysis , RNA, Viral/analysis , Aedes/virology , Animals , Antibodies, Monoclonal , Australia , Cell Line , Dengue Virus/genetics , Dengue Virus/isolation & purification , Dengue Virus/physiology , Enzyme-Linked Immunosorbent Assay , Flavivirus/genetics , RNA, Double-Stranded/immunology , RNA, Viral/immunology , Viral Envelope Proteins/analysis , Viral Envelope Proteins/metabolism , Viral Nonstructural Proteins/analysis , Viral Nonstructural Proteins/metabolism , Virus Replication
18.
Virol J ; 18(1): 150, 2021 07 19.
Article in English | MEDLINE | ID: mdl-34281569

ABSTRACT

BACKGROUND: Mosquito-borne flaviviruses are prime pathogens and have been a major hazard to humans and animals. They comprise several arthropod-borne viruses, including dengue virus, yellow fever virus, Japanese encephalitis virus, and West Nile virus. Culex flavivirus (CxFV) is a member of the insect-specific flavivirus (ISF) group belonging to the genus Flavivirus, which is widely distributed in a variety of mosquito populations. METHODS: Viral nucleic acid was extracted from adult mosquito pools and subjected to reverse transcriptase nested polymerase chain reaction (PCR) using target-specific primers for detecting CxFV nonstructural protein 5 (NS5). The PCR-positive samples were then sequenced, and a phylogenetic tree was constructed, including reference sequences obtained from GenBank. RESULTS: 21 pools, belonging to Culex pipiens pallens (Cx. p. pallens) were found to be positive for the CxFV RNA sequence, with a minimum infection rate of 14.5/1000 mosquitoes. The phylogenetic analysis of the NS5 protein sequences indicated that the detected sequences were closely related to strains identified in China, with 95-98% sequence similarities. CONCLUSION: Our findings highlight the presence of CxFV in Cx. p. pallens mosquito species in Jeju province, Republic of Korea. This is the first study reporting the prevalence of CxFV in Culex Pipiens (Cx. pipiens) host in the Jeju province, which can create possible interaction with other flaviviruses causing human and animal diseases. Although, mosquito-borne disease causing viruses were not identified properly, more detailed surveillance and investigation of both the host and viruses are essential to understand the prevalence, evolutionary relationship and genetic characteristic with other species.


Subject(s)
Culex , Flavivirus , Animals , Culex/virology , Flavivirus/genetics , Flavivirus/isolation & purification , Phylogeny , Republic of Korea
19.
J Med Entomol ; 58(6): 2406-2411, 2021 11 09.
Article in English | MEDLINE | ID: mdl-33939805

ABSTRACT

Diseases caused by flaviviruses are a major public health burden across the world. In the past decades, South America has suffered dengue epidemics, the re-emergence of yellow fever and St. Louis encephalitis viruses, and the introduction of West Nile and Zika viruses. Many insect-specific flaviviruses (ISFs) that cannot replicate in vertebrate cells have recently been described. In this study, we analyzed field-collected mosquito samples from six different ecoregions of Argentina to detect flaviviruses. We did not find any RNA belonging to pathogenic flaviviruses or ISFs in adults or immature stages. However, flaviviral-like DNA similar to flavivirus NS5 region was detected in 83-100% of Aedes aegypti (L.). Despite being previously described as an ancient element in the Ae. aegypti genome, the flaviviral-like DNA sequence was not detected in all Ae. aegypti samples and sequences obtained did not form a monophyletic group, possibly reflecting the genetic diversity of mosquito populations in Argentina.


Subject(s)
Aedes/virology , DNA, Viral/analysis , Flavivirus/isolation & purification , Animals , Argentina , Flavivirus/genetics
20.
Parasit Vectors ; 14(1): 243, 2021 May 07.
Article in English | MEDLINE | ID: mdl-33962673

ABSTRACT

BACKGROUND: West Nile (WNV) and Usutu (USUV) are emerging vector-borne zoonotic flaviviruses. They are antigenically very similar, sharing the same life cycle with birds as amplification host, Culicidae as vector, and man/horse as dead-end host. They can co-circulate in an overlapping geographic range. In Europe, surveillance plans annually detect several outbreaks. METHODS: In Italy, a WNV/USUV surveillance plan is in place through passive and active surveillance. After a 2018 WNV outbreak, a reinforced integrated risk-based surveillance was performed in four municipalities through clinical and serological surveillance in horses, Culicidae catches, and testing on human blood-based products for transfusion. RESULTS: Eight WNV cases in eight equine holdings were detected. Twenty-three mosquitoe catches were performed and 2367 specimens of Culex pipiens caught; 17 pools were USUV positive. A total of 8889 human blood donations were tested, and two asymptomatic donors were USUV positive. CONCLUSIONS: Different surveillance components simultaneously detected WNV only in horses and USUV only in humans and mosquitoes. While in endemic areas (i.e. northern Italy) entomological surveillance is successfully used as an early detection warning, this method in central Italy seems ineffective. To achieve a high level of sensitivity, the entomological trapping effort should probably exceed a reasonable balance between cost and performance. Besides, WNV/USUV early detection can be addressed by horses and birds. Further research is needed to adapt the surveillance components in different epidemiological contexts.


Subject(s)
Culex/virology , Flavivirus Infections/veterinary , Flavivirus Infections/virology , Flavivirus/isolation & purification , Mosquito Vectors/virology , West Nile Fever/veterinary , West Nile Fever/virology , West Nile virus/isolation & purification , Animals , Culex/physiology , Epidemiological Monitoring , Flavivirus/classification , Flavivirus/genetics , Flavivirus Infections/epidemiology , Flavivirus Infections/transmission , Horse Diseases/epidemiology , Horse Diseases/transmission , Horse Diseases/virology , Horses , Humans , Italy/epidemiology , Mosquito Vectors/physiology , West Nile Fever/epidemiology , West Nile Fever/transmission , West Nile virus/classification , West Nile virus/genetics
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