Unleashing Nature's Allies: Comparing the Vertical Transmission Dynamics of Insect-Specific and Vertebrate-Infecting Flaviviruses in Mosquitoes.

Insect-specific viruses (ISVs) include viruses that are restricted to the infection of mosquitoes and are spread mostly through transovarial transmission. Despite using a distinct mode of transmission, ISVs are often phylogenetically related to arthropod-borne viruses (arboviruses) that are responsible for human diseases and able to infect both mosquitoes and vertebrates. ISVs can also induce a phenomenon called "superinfection exclusion", whereby a primary ISV infection in an insect inhibits subsequent viral infections of the insect. This has sparked interest in the use of ISVs for the control of pathogenic arboviruses transmitted by mosquitoes. In particular, insect-specific flaviviruses (ISFs) have been shown to inhibit infection of vertebrate-infecting flaviviruses (VIFs) both in vitro and in vivo. This has shown potential as a new and ecologically friendly biological approach to the control of arboviral disease. For this intervention to have lasting impacts for biological control, it is imperative that ISFs are maintained in mosquito populations with high rates of vertical transmission. Therefore, these strategies will need to optimise vertical transmission of ISFs in order to establish persistently infected mosquito lines for sustainable arbovirus control. This review compares recent observations of vertical transmission of arboviral and insect-specific flaviviruses and potential determinants of transovarial transmission rates to understand how the vertical transmission of ISFs may be optimised for effective arboviral control.

Vector competence of Culex quinquefasciatus for Tembusu virus and viral factors for virus transmission by mosquitoes.

The ongoing epidemic of flaviviruses worldwide has underscored the importance of studying flavivirus vector competence, considering their close association with mosquito vectors. Tembusu virus is an avian-related mosquito-borne flavivirus that has been an epidemic in China and Southeast Asia since 2010. However, the reason for the outbreak of Tembusu virus in 2010 remains unclear, and it is unknown whether changes in vector transmission played an essential role in this process. To address these questions, we conducted a study using Culex quinquefasciatus as a model for Tembusu virus infection, employing both oral infection and microinjection methods. Our findings confirmed that both vertical and venereal transmission collectively contribute to the cycle of Tembusu virus within the mosquito population, with persistent infections observed. Importantly, our data revealed that the prototypical Tembusu virus MM_1775 strain exhibited significantly greater infectivity and transmission rates in mosquitoes than did the duck Tembusu virus (CQW1 strain). Furthermore, we revealed that the viral E protein and 3' untranslated region are key elements responsible for these differences. In conclusion, our study sheds light on mosquito transmission of Tembusu virus and provides valuable insights into the factors influencing its infectivity and transmission rates. These findings contribute to a better understanding of Tembusu virus epidemiology and can potentially aid in the development of strategies to control its spread.

A conserved role for AKT in the replication of emerging flaviviruses in vertebrates and vectors.

One third of all emerging infectious diseases are vector-borne, with no licensed antiviral therapies available against any vector-borne viruses. Zika virus and Usutu virus are two emerging flaviviruses transmitted primarily by mosquitoes. These viruses modulate different host pathways, including the PI3K/AKT/mTOR pathway. Here, we report the effect on ZIKV and USUV replication of two AKT inhibitors, Miransertib (ARQ-092, allosteric inhibitor) and Capivasertib (AZD5363, competitive inhibitor) in different mammalian and mosquito cell lines. Miransertib showed a stronger inhibitory effect against ZIKV and USUV than Capivasertib in mammalian cells, while Capivasertib showed a stronger effect in mosquito cells. These findings indicate that AKT plays a conserved role in flavivirus infection, in both the vertebrate host and invertebrate vector. Nevertheless, the specific function of AKT may vary depending on the host species. These findings indicate that AKT may be playing a conserved role in flavivirus infection in both, the vertebrate host and the invertebrate vector. However, the specific function of AKT may vary depending on the host species. A better understanding of virus-host interactions is therefore required to develop new treatments to prevent human disease and new approaches to control transmission by insect vectors.

First detection of Bagaza virus in Common magpies (Pica pica), Portugal 2023.

Bagaza virus (BAGV) is a mosquito-borne flavivirus of the family Flaviviridae, genus Orthoflavivirus, Ntaya serocomplex. Like other viruses of the Ntaya and Japanese encephalitis serocomplexes, it is maintained in nature in transmission cycles involving viremic wild bird reservoirs and Culex spp. mosquitoes. The susceptibility of red-legged partridge, ring-necked pheasant, Himalayan monal and common wood pigeon is well known. Determining whether other species are susceptible to BAGV infection is fundamental to understanding the dynamics of disease transmission and maintenance. In September 2023, seven Eurasian magpies were found dead in a rural area in the Mértola district (southern Portugal) where a BAGV-positive cachectic red-legged partridge had been found two weeks earlier. BAGV had also been detected in several red-legged partridges in the same area in September 2021. Three of the magpies were tested for Bagaza virus, Usutu virus, West Nile virus, Avian influenza virus and Avian paramyxovirus serotype 1, and were positive for BAGV only. Sequencing data confirmed the specificity of the molecular detection. Our results indicate that BAGV is circulating in southern Portugal and confirm that Eurasian magpie is potential susceptible to BAGV infection. The inclusion of the abundant Eurasian magpie in the list of BAGV hosts raises awareness of the potential role of this species as as an amplifying host.

Molecular detection of Usutu virus in pools of Culex pipiens mosquitoes in Greece.

Usutu virus (USUV) is a mosquito-borne flavivirus originating from Africa, that belongs to the Japanese encephalitis virus (JEV) complex. In nature, USUV involves Culex spp. mosquitoes acting as vectors and birds as amplifying hosts. The virus has recently spread in Europe and is considered an emerging human pathogen. This is the first research study performed in Greece revealing the presence and circulation of USUV in Culex spp. mosquito populations. Out of the 1,500 mosquito pools tested with real-time RT-PCR, four (Roesch et al., 2019) were positive for USUV. All four pools were collected from the region of Central Macedonia, Northern Greece.

Ixodes ricinus as potential vector for Usutu virus.

Usutu virus (USUV) is an emerging flavivirus that is maintained in an enzootic cycle with mosquitoes as vectors and birds as amplifying hosts. In Europe, the virus has caused mass mortality of wild birds, mainly among Common Blackbird (Turdus merula) populations. While mosquitoes are the primary vectors for USUV, Common Blackbirds and other avian species are exposed to other arthropod ectoparasites, such as ticks. It is unknown, however, if ticks can maintain and transmit USUV. We addressed this question using in vitro and in vivo experiments and field collected data. USUV replicated in IRE/CTVM19 Ixodes ricinus tick cells and in injected ticks. Moreover, I. ricinus nymphs acquired the virus via artificial membrane blood-feeding and maintained the virus for at least 70 days. Transstadial transmission of USUV from nymphs to adults was confirmed in 4.9% of the ticks. USUV disseminated from the midgut to the haemocoel, and was transmitted via the saliva of the tick during artificial membrane blood-feeding. We further explored the role of ticks by monitoring USUV in questing ticks and in ticks feeding on wild birds in the Netherlands between 2016 and 2019. In total, 622 wild birds and the Ixodes ticks they carried were tested for USUV RNA. Of these birds, 48 (7.7%) carried USUV-positive ticks. The presence of negative-sense USUV RNA in ticks, as confirmed via small RNA-sequencing, showed active virus replication. In contrast, we did not detect USUV in 15,381 questing ticks collected in 2017 and 2019. We conclude that I. ricinus can be infected with USUV and can transstadially and horizontally transmit USUV. However, in comparison to mosquito-borne transmission, the role of I. ricinus ticks in the epidemiology of USUV is expected to be minor.

Is Culex modestus a New Usutu virus vector?

Usutu virus is an emerging pathogen transmitted by mosquitoes. Culex modestus mosquitoes are widespread in Europe, but their role in disease transmission is poorly understood. Recent data from a single infectious mosquito suggested that Culex modestus could be an unrecognized vector for Usutu virus. In this study, our aim was to corroborate this finding using a larger sample size. We collected immature Culex modestus from a reedbed pond in Flemish Brabant, Belgium, and reared them in the laboratory until the third generation. Adult females were then experimentally infected with Usutu virus in a blood meal and incubated at 25 °C for 14 days. The presence of Usutu virus in the saliva, head and body of each female was determined by plaque assay and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). The transmission efficiency was 54% (n = 15/28), confirming that Belgian Culex modestus can experimentally transmit Usutu virus.

Virulence and transmission vary between Usutu virus lineages in Culex pipiens.

Usutu virus (USUV) is a zoonotic arbovirus infecting mainly wild birds. It is transmitted by ornithophilic mosquitoes, mainly of the genus Culex from birds to birds and to several vertebrate dead-end hosts. Several USUV lineages, differing in their virulence have emerged in the last decades and now co-circulate in Europe, impacting human populations. However, their relative transmission and effects on their mosquito vectors is still not known. We thus compared the vector competence and survival of Culex pipiens mosquitoes experimentally infected with two distinct USUV lineages, EU2 and EU3, that are known to differ in their virulence and replication in vertebrate hosts. Infection rate was variable among blood feeding assays but variations between EU2 and EU3 lineages were consistent suggesting that Culex pipiens was equally susceptible to infection by both lineages. However, EU3 viral load increased with viral titer in the blood meal while EU2 viral load was high at all titers which suggest a greater replication of EU2 than EU3 in mosquito. While their relative transmission efficiencies are similar, at least at low blood meal titer, positive correlation between transmission and blood meal titer was observed for EU3 only. Contrary to published results in vertebrates, EU3 induced a higher mortality to mosquitoes (i.e. virulence) than EU2 whatever the blood meal titer. Therefore, we found evidence of lineage-specific differences in vectorial capacity and virulence to both the vector and vertebrate host which lead to balanced propagation of both viral lineages. These results highlight the need to decipher the interactions between vectors, vertebrate hosts, and the diversity of arbovirus lineages to fully understand transmission dynamics.

The microbiota's role in flavivirus control: Rosenbergiella_YN46.

The transmission of flaviviruses, such as dengue virus (DENV) and Zika virus (ZIKV), poses a significant threat to global public health. Zhang et al. recently showed that Rosenbergiella sp. YN46 (Rosenbergiella_YN46), a bacterium from the mosquito gut, inhibits flavivirus transmission and thus offers a potential biocontrol strategy with broad public health implications.

Advancements in Research on Duck Tembusu Virus Infections.

Duck Tembusu Virus (DTMUV) is a pathogen of the Flaviviridae family that causes infections in poultry, leading to significant economic losses in the duck farming industry in recent years. Ducks infected with this virus exhibit clinical symptoms such as decreased egg production and neurological disorders, along with serious consequences such as ovarian hemorrhage, organ enlargement, and necrosis. Variations in morbidity and mortality rates exist across different age groups of ducks. It is worth noting that DTMUV is not limited to ducks alone; it can also spread to other poultry such as chickens and geese, and antibodies related to DTMUV have even been found in duck farm workers, suggesting a potential risk of zoonotic transmission. This article provides a detailed overview of DTMUV research, delving into its genomic characteristics, vaccines, and the interplay with host immune responses. These in-depth research findings contribute to a more comprehensive understanding of the virus's transmission mechanism and pathogenic process, offering crucial scientific support for epidemic prevention and control.

Circulation of West Nile Virus and Usutu Virus in Europe: Overview and Challenges.

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.

Duck Tembusu virus NS3 protein induces apoptosis by activating the PERK/PKR pathway and mitochondrial pathway.

IMPORTANCE: Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that replicates well in mosquito, bird, and mammalian cells. An in vivo study revealed that BALB/c mice and Kunming mice were susceptible to DTMUV after intracerebral inoculation. Moreover, there are no reports about DTMUV-related human disease, but antibodies against DTMUV and viral RNA were detected in the serum samples of duck industry workers. This information implies that DTMUV has expanded its host range and poses a threat to mammalian health. Thus, understanding the pathogenic mechanism of DTMUV is crucial for identifying potential antiviral targets. In this study, we discovered that NS3 can induce the mitochondria-mediated apoptotic pathway through the PERK/PKR pathway; it can also interact with voltage-dependent anion channel 2 to induce apoptosis. Our findings provide a theoretical basis for understanding the pathogenic mechanism of DTMUV infection and identifying potential antiviral targets and may also serve as a reference for exploring the pathogenesis of other flaviviruses.

Toll-like receptor 4 and lipopolysaccharide from commensal microbes regulate Tembusu virus infection.

Unlike most flaviviruses transmitted by arthropods, Tembusu virus (TMUV) is still active during winter and causes outbreaks in some areas, indicating vector-independent spread of the virus. Gastrointestinal transmission might be one of the possible routes of vector-free transmission, which also means that the virus has to interact with more intestinal bacteria. Here, we found evidence that TMUV indeed can transmit through the digestive tract. Interestingly, using an established TMUV disease model by oral gavage combined with an antibiotic treatment, we revealed that a decrease in intestinal bacteria significantly reduced local TMUV proliferation in the intestine, revealing that the bacterial microbiome is important in TMUV infection. We found that lipopolysaccharide (LPS) present in the outer membrane of Gram-negative bacteria enhanced TMUV proliferation by promoting its attachment. Toll-like receptor 4 (TLR4), a cell surface receptor, can transmit signal from LPS. We confirmed colocalization of TLR4 with TMUV envelope (E) protein as well as their interaction in infected cells. Coherently, TMUV infection of susceptible cells was inhibited by an anti-TLR4 antibody, purified soluble TLR4 protein, and knockdown of TLR4 expression. LPS-enhanced TMUV proliferation could also be blocked by a TLR4 inhibitor. Meanwhile, pretreatment of duck primary cells with TMUV significantly impaired LPS-induced interleukin 6 production. Collectively, our study provides first insights into vector-free transmission mechanisms of flaviviruses.

Flaviviruses hijack the host microbiota to facilitate their transmission.

Flaviviruses, such as Dengue and Zika viruses, infect millions of people worldwide using mosquitos as vectors. In this issue of Cell, Zhang et al. reveal how these viruses manipulate the skin microbiome of infected hosts in a way that increases vector recruitment and viral spread. They propose vitamin A as a way to counteract the virus and decrease transmission.

Roles of ESCRT Proteins ALIX and CHMP4A and Their Interplay with Interferon-Stimulated Gene 15 during Tick-Borne Flavivirus Infection.

Flaviviruses are usually transmitted to humans via mosquito or tick bites. During infection, virus replication and assembly, whose cellular sites are relatively close, are controlled by virus proteins and a diverse range of host proteins. By siRNA-mediated gene silencing, we showed that ALIX and CHMP4A, two members of the host endosomal sorting complex required for transport (ESCRT) protein machinery, are required during flavivirus infection. Using cell lines expressing subgenomic replicons and replicon virus-like particles, we demonstrated specific roles for ALIX and CHMP4A in viral replication and assembly, respectively. Employing biochemical and imaging methodology, we showed that the ESCRT proteins are recruited by a putative specific late (L) domain motif LYXLA within the NS3 protein of tick-borne flaviviruses. Furthermore, to counteract the recruitment of ESCRT proteins, the host cells may elicit defense mechanisms. We found that ectopic expression of the interferon-stimulated gene 15 (ISG15) or the E3 ISG15-protein ligase (HERC5) reduced virus replication by suppressing the positive effects of ALIX and CHMP4A. Collectively, these results have provided new insights into flavivirus-host cell interactions that function as checkpoints, including the NS3 and the ESCRT proteins, the ISG15 and the ESCRT proteins, at essential stages of the virus life cycle. IMPORTANCE Flaviviruses are important zoonotic viruses with high fatality rates worldwide. Here, we report that during infection, the virus employs members of ESCRT proteins for virus replication and assembly. Among the ESCRT proteins, ALIX acts during virus replication, while CHMP4A is required during virus assembly. Another important ESCRT protein, TSG101, is not required for virus production. The ESCRT, complex, ALIX-CHMP4A, is recruited to NS3 through their interactions with the putative L domain motif of NS3, while CHMP4A is recruited to E. In addition, we demonstrate the antiviral mechanism of ISG15 and HERC5, which degrades ALIX and CHIMP4A, indirectly targets virus infection. In summary, we reveal host-dependency factors supporting flavivirus infection, but these factors may also be targeted by antiviral host effector mechanisms.

Innate Immune Antagonism of Mosquito-Borne Flaviviruses in Humans and Mosquitoes.

Mosquito-borne viruses of the Flavivirus genus (Flaviviridae family) pose an ongoing threat to global public health. For example, dengue, Japanese encephalitis, West Nile, yellow fever, and Zika viruses are transmitted by infected mosquitoes and cause severe and fatal diseases in humans. The means by which mosquito-borne flaviviruses establish persistent infection in mosquitoes and cause disease in humans are complex and depend upon a myriad of virus-host interactions, such as those of the innate immune system, which are the main focus of our review. This review also covers the different strategies utilized by mosquito-borne flaviviruses to antagonize the innate immune response in humans and mosquitoes. Given the lack of antiviral therapeutics for mosquito-borne flaviviruses, improving our understanding of these virus-immune interactions could lead to new antiviral therapies and strategies for developing refractory vectors incapable of transmitting these viruses, and can also provide insights into determinants of viral tropism that influence virus emergence into new species.

Rocio Virus: An Updated View on an Elusive Flavivirus.

Rocio virus (ROCV) is a mosquito-borne flavivirus and human pathogen. The virus is indigenous to Brazil and was first detected in 1975 in the Sao Paulo State, and over a period of two years was responsible for several epidemics of meningoencephalitis in coastal communities leading to over 100 deaths. The vast majority of ROCV infections are believed to be subclinical and clinical manifestations can range from uncomplicated fever to fatal meningoencephalitis. Birds are the natural reservoir and amplification hosts and ROCV is maintained in nature in a mosquito-bird-mosquito transmission cycle, primarily involving Psorophora ferox mosquitoes. While ROCV has remained mostly undetected since 1976, in 2011 it re-emerged in Goiás State causing a limited outbreak. Control of ROCV outbreaks depends on sustainable vector control measures and public education. To date there is no specific treatment or licensed vaccine available. Here we provide an overview of the ecology, transmission cycles, epidemiology, pathogenesis, and treatment options, aiming to improve our ability to understand, predict, and ideally avert further ROCV emergence.

Pleiotropic Odorant-Binding Proteins Promote Aedes aegypti Reproduction and Flavivirus Transmission.

Insect odorant-binding proteins (OBPs) are small soluble proteins that have been assigned roles in olfaction, but their other potential functions have not been extensively explored. Using CRISPR/Cas9-mediated disruption of Aedes aegypti Obp10 and Obp22, we demonstrate the pleiotropic contribution of these proteins to multiple processes that are essential for vectorial capacity. Mutant mosquitoes have impaired host-seeking and oviposition behavior, reproduction, and arbovirus transmission. Here, we show that Obp22 is linked to the male-determining sex locus (M) on chromosome 1 and is involved in male reproduction, likely by mediating the development of spermatozoa. Although OBP10 and OBP22 are not involved in flavivirus replication, abolition of these proteins significantly reduces transmission of dengue and Zika viruses through a mechanism affecting secretion of viral particles into the saliva. These results extend our current understanding of the role of insect OBPs in insect reproduction and transmission of human pathogens, making them essential determinants of vectorial capacity. IMPORTANCE Aedes aegypti is the major vector for many arthropod-borne viral diseases, such as dengue, Zika, and chikungunya viruses. Previous studies suggested that odorant-binding proteins (OBPs) may have diverse physiological functions beyond the olfactory system in mosquitoes; however, these hypothesized functions have not yet been demonstrated. Here, we have used CRISPR/Cas9-based genome editing to functionally delete (knock out) Obp10 and Obp22 in Aedes aegypti. We showed that disruption of Obp10 or Obp22 significantly impairs female and male reproductive capacity by adversely affecting blood feeding, oviposition, fecundity and fertility, and the development of spermatozoa. We also showed that disruption of Obp10 or Obp22 significantly reduces the transmission of dengue and Zika viruses through a mechanism affecting secretion of viral particles into the saliva. Thus, our study is not only significant in understanding the functions of OBPs in mosquito biology, but also shows that OBPs may represent potent flavivirus transmission-blocking targets. Our study is in this regard particularly timely and important from a translational and public health perspective.

Flavivirus Persistence in Wildlife Populations.

A substantial number of humans are at risk for infection by vector-borne flaviviruses, resulting in considerable morbidity and mortality worldwide. These viruses also infect wildlife at a considerable rate, persistently cycling between ticks/mosquitoes and small mammals and reptiles and non-human primates and humans. Substantially increasing evidence of viral persistence in wildlife continues to be reported. In addition to in humans, viral persistence has been shown to establish in mammalian, reptile, arachnid, and mosquito systems, as well as insect cell lines. Although a considerable amount of research has centered on the potential roles of defective virus particles, autophagy and/or apoptosis-induced evasion of the immune response, and the precise mechanism of these features in flavivirus persistence have yet to be elucidated. In this review, we present findings that aid in understanding how vector-borne flavivirus persistence is established in wildlife. Research studies to be discussed include determining the critical roles universal flavivirus non-structural proteins played in flaviviral persistence, the advancement of animal models of viral persistence, and studying host factors that allow vector-borne flavivirus replication without destructive effects on infected cells. These findings underscore the viral-host relationships in wildlife animals and could be used to elucidate the underlying mechanisms responsible for the establishment of viral persistence in these animals.