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.

A getah virus-like-particle vaccine provides complete protection from viremia and arthritis in wild-type mice.

Getah virus (GETV) is an emerging mosquito-borne virus with economic impact on the livestock industry in East Asia. In this study, we successfully produced GETV virus-like particles (VLPs) in insect cells using the baculovirus expression vector system. We show that the GETV envelope glycoproteins were successfully expressed at the surface of the insect cell and were glycosylated. VLPs were isolated from the culture fluid as enveloped particles of 60-80 nm in diameter. Two 1 µg vaccinations with this GETV VLP vaccine, without adjuvant, generated neutralizing antibody responses and protected wild-type C57/BL6 mice against GETV viremia and arthritic disease. The GETV VLP vaccine may find application as a horse and/or pig vaccine in the future.

Scarless Baculovirus Genome Editing Using Lambda-Red Recombineering in E. coli.

Baculoviruses are widely used for their potential as biological pesticide and as platform for the production of recombinant proteins and gene therapy vectors. The Baculovirus Expression Vector System (BEVS) is used for high level of expression of (multiple) proteins in insect cells. Baculovirus recombinants can be quickly constructed by transposition of the gene(s) of interest into a so-called bacmid, which is a baculovirus infectious clone maintained as single-copy, bacterial artificial chromosome in Escherichia coli. A two-step homologous recombineering technique using the lambda-red system in E. coli allows for scarless editing of the bacmid with PCR products based on sequence homology. In the first step, a selection cassette with 50 bp homology arms, typically generated by PCR, is inserted into the designated locus. In the second step, the selection cassette is removed based on a negative selection marker, such as SacB or rpsL. This lambda-red recombineering technique can be used for multiple gene editing purposes, including (large) deletions, insertions, and even single point mutations. Moreover, since there are no remnants of the editing process, successive modifications of the same bacmid are possible. This chapter provides detailed instructions to design and perform two-step homologous recombineering of baculovirus bacmid DNA in E. coli. We present two case studies demonstrating the utility of this technique for creating a deletion mutant of the chitinase and cathepsin genes and for introducing a single point mutation in the baculovirus gene gp41. This scarless genome editing approach can facilitate functional studies of baculovirus genes and improve the production of recombinant proteins using the BEVS.

Safety concern of recombination between self-amplifying mRNA vaccines and viruses is mitigated in vivo.

Self-amplifying mRNA (SAM) vaccines can be rapidly deployed in the event of disease outbreaks. A legitimate safety concern is the potential for recombination between alphavirus-based SAM vaccines and circulating viruses. This theoretical risk needs to be assessed in the regulatory process for SAM vaccine approval. Herein, we undertake extensive in vitro and in vivo assessments to explore recombination between SAM vaccine and a wide selection of alphaviruses and a coronavirus. SAM vaccines were found to effectively limit alphavirus co-infection through superinfection exclusion, although some co-replication was still possible. Using sensitive cell-based assays, replication-competent alphavirus chimeras were generated in vitro as a result of rare, but reproducible, RNA recombination events. The chimeras displayed no increased fitness in cell culture. Viable alphavirus chimeras were not detected in vivo in C57BL/6J, Rag1-/- and Ifnar-/- mice, in which high levels of SAM vaccine and alphavirus co-replicated in the same tissue. Furthermore, recombination between a SAM-spike vaccine and a swine coronavirus was not observed. In conclusion we state that although the ability of SAM vaccines to recombine with alphaviruses might be viewed as an environmental safety concern, several key factors substantially mitigate against in vivo emergence of chimeric viruses from SAM vaccine recipients.

The potential role of the Asian bush mosquito Aedes japonicus as spillover vector for West Nile virus in the Netherlands.

BACKGROUND: In recent years the Asian bush mosquito Aedes japonicus has invaded Europe, including the Netherlands. This species is a known vector for a range of arboviruses, possibly including West Nile virus (WNV). As WNV emerged in the Netherlands in 2020, it is important to investigate the vectorial capacity of mosquito species present in the Netherlands to estimate the risk of future outbreaks and further spread of the virus. Therefore, this study evaluates the potential role of Ae. japonicus in WNV transmission and spillover from birds to dead-end hosts in the Netherlands. METHODS: We conducted human landing collections in allotment gardens (Lelystad, the Netherlands) in June, August and September 2021 to study the diurnal and seasonal host-seeking behaviour of Ae. japonicus. Furthermore, their host preference in relation to birds using live chicken-baited traps was investigated. Vector competence of field-collected Ae. japonicus mosquitoes for two isolates of WNV at two different temperatures was determined. Based on the data generated from these studies, we developed a Susceptible-Exposed-Infectious-Recovered (SEIR) model to calculate the risk of WNV spillover from birds to humans via Ae. japonicus, under the condition that the virus is introduced and circulates in an enzootic cycle in a given area. RESULTS: Our results show that Ae. japonicus mosquitoes are actively host seeking throughout the day, with peaks in activity in the morning and evening. Their abundance in August was higher than in June and September. For the host-preference experiment, we documented a small number of mosquitoes feeding on birds: only six blood-fed females were caught over 4 full days of sampling. Finally, our vector competence experiments with Ae. japonicus compared to its natural vector Culex pipiens showed a higher infection and transmission rate when infected with a local, Dutch, WNV isolate compared to a Greek isolate of the virus. Interestingly, we also found a small number of infected Cx. pipiens males with virus-positive leg and saliva samples. CONCLUSIONS: Combining the field and laboratory derived data, our model predicts that Ae. japonicus could act as a spillover vector for WNV and could be responsible for a high initial invasion risk of WNV when present in large numbers.

Subgenomic flavivirus RNA as key target for live-attenuated vaccine development.

Live-attenuated flavivirus vaccines confer long-term protection against disease, but the design of attenuated flaviviruses does not follow a general approach. The non-coding, subgenomic flavivirus RNA (sfRNA) is produced by all flaviviruses and is an essential factor in viral pathogenesis and transmission. We argue that modulating sfRNA expression is a promising, universal strategy to finetune flavivirus attenuation for developing effective flavivirus vaccines of the future.

Differential susceptibility of geographically distinct Ixodes ricinus populations to tick-borne encephalitis virus and louping ill virus.

Tick-borne encephalitis virus (TBEV) is an emerging pathogen in the Netherlands. Multiple divergent viral strains are circulating and the focal distribution of TBEV remains poorly understood. This may, however, be explained by differences in the susceptibility of tick populations for specific viruses and viral strains, and by viral strains having higher infection success in their local tick population. We investigated this hypothesis by exposing Dutch Ixodes ricinus ticks to two different TBEV strains: TBEV-NL from the Netherlands and TBEV-Neudoerfl from Austria. In addition, we exposed ticks to louping Ill virus (LIV), which is endemic to large parts of the United Kingdom and Ireland, but has not been reported in the Netherlands. Ticks were collected from two locations in the Netherlands: one location without evidence of TBEV circulation and one location endemic for the TBEV-NL strain. Ticks were infected in a biosafety level 3 laboratory using an artificial membrane feeding system. Ticks collected from the region without evidence of TBEV circulation had lower infection rates for TBEV-NL as compared to TBEV-Neudoerfl. Vice versa, ticks collected from the TBEV-NL endemic region had higher infection rates for TBEV-NL compared to TBEV-Neudoerfl. In addition, LIV infection rates were much lower in Dutch ticks compared to TBEV, which may explain why LIV is not present in the Netherlands. Our findings show that ticks from two distinct geographical populations differ in their susceptibility to TBEV strains, which could be the result of differences in the genetic background of the tick populations.

Evaluation of bird-adapted self-amplifying mRNA vaccine formulations in chickens.

Each year, millions of poultry succumb to highly pathogenic avian influenza A virus (AIV) and infectious bursal disease virus (IBDV) infections. Conventional vaccines based on inactivated or live-attenuated viruses are useful tools for disease prevention and control, yet, they often fall short in terms of safety, efficacy, and development times. Therefore, versatile vaccine platforms are crucial to protect poultry from emerging viral pathogens. Self-amplifying (replicon) RNA vaccines offer a well-defined and scalable option for the protection of both animals and humans. The best-studied replicon platform, based on the Venezuelan equine encephalitis virus (VEEV; family Togaviridae) TC-83 vaccine strain, however, displays limited efficacy in poultry, warranting the exploration of alternative, avian-adapted, replicon platforms. In this study, we engineered two Tembusu virus (TMUV; family Flaviviridae) replicons encoding varying capsid gene lengths and compared these to the benchmark VEEV replicon in vitro. The TMUV replicon system exhibited a robust and prolonged transgene expression compared to the VEEV replicon system in both avian and mammalian cells. Moreover, the TMUV replicon induced a lesser cytopathic effect compared to the VEEV replicon RNA in vitro. DNA-launched versions of the TMUV and VEEV replicons (DREP) were also developed. The replicons successfully expressed the AIV haemagglutinin (HA) glycoproteins and the IBDV capsid protein (pVP2). To assess the immune responses elicited by the TMUV replicon system in chickens, a prime-boost vaccination trial was conducted using lipid nanoparticle (LNP)-formulated replicon RNA and DREP encoding the viral (glyco)proteins of AIV or IBDV. Both TMUV and VEEV replicon RNAs were unable to induce a humoral response against AIV. However, TMUV replicon RNA induced IBDV-specific seroconversion in vaccinated chickens, in contrast to VEEV replicon RNA, which showed no significant humoral response. In both AIV and IBDV immunization studies, VEEV DREP generated the highest (neutralizing) antibody responses, which underscores the potential for self-amplifying mRNA vaccine technology to combat emerging poultry diseases.

Infectious clone of a contemporary Tembusu virus and replicons expressing reporter genes or heterologous antigens from poultry viruses.

The novel mosquito-borne Tembusu virus (TMUV, family Flaviviridae) was discovered as the cause of a severe outbreak of egg-drop syndrome affecting ducks in Southeast Asia in 2010. TMUV infection can also lead to high mortality in various additional avian species such as geese, pigeons, and chickens. This study describes the construction of an infectious cDNA clone of a contemporary duck-isolate (TMUV WU2016). The virus recovered after transfection of BHK-21 cells shows enhanced virus replication compared to the mosquito-derived MM1775 strain. Next, the WU2016 cDNA clone was modified to create a SP6 promoter-driven, self-amplifying mRNA (replicon) capable of expressing a range of different reporter genes (Renilla luciferase, mScarlet, mCherry, and GFP) and viral (glyco)proteins of avian influenza virus (AIV; family Orthomyxoviridae), infectious bursal disease virus (IDBV; family Bunyaviridae) and infectious bronchitis virus (IBV; family Coronaviridae). The current study demonstrates the flexibility of the TMUV replicon system, to produce different heterologous proteins over an extended period of time and its potential use as a platform technology for novel poultry vaccines.

Infection of wild-caught wood mice (Apodemus sylvaticus) and yellow-necked mice (A. flavicollis) with tick-borne encephalitis virus.

The distribution of tick-borne encephalitis virus (TBEV) is expanding to Western European countries, including the Netherlands, but the contribution of different rodent species to the transmission of TBEV is poorly understood. We investigated whether two species of wild rodents native to the Netherlands, the wood mouse Apodemus sylvaticus and the yellow-necked mouse Apodemus flavicollis, differ in their relative susceptibility to experimental infection with TBEV. Wild-caught individuals were inoculated subcutaneously with the classical European subtype of TBEV (Neudoerfl) or with TBEV-NL, a genetically divergent TBEV strain from the Netherlands. Mice were euthanised and necropsied between 3 and 21 days post-inoculation. None of the mice showed clinical signs or died during the experimental period. Nevertheless, TBEV RNA was detected up to 21 days in the blood of both mouse species and TBEV was also isolated from the brain of some mice. Moreover, no differences in infection rates between virus strains and mouse species were found in blood, spleen, or liver samples. Our results suggest that the wood mouse and the yellow-necked mouse may equally contribute to the transmission cycle of TBEV in the Netherlands. Future experimental infection studies that include feeding ticks will help elucidate the relative importance of viraemic transmission in the epidemiology of TBEV.

The virome of the invasive Asian bush mosquito Aedes japonicus in Europe.

The Asian bush mosquito Aedes japonicus is rapidly invading North America and Europe. Due to its potential to transmit multiple pathogenic arthropod-borne (arbo)viruses including Zika virus, West Nile virus, and chikungunya virus, it is important to understand the biology of this vector mosquito in more detail. In addition to arboviruses, mosquitoes can also carry insect-specific viruses that are receiving increasing attention due to their potential effects on host physiology and arbovirus transmission. In this study, we characterized the collection of viruses, referred to as the virome, circulating in Ae. japonicus populations in the Netherlands and France. Applying a small RNA-based metagenomic approach to Ae. japonicus, we uncovered a distinct group of viruses present in samples from both the Netherlands and France. These included one known virus, Ae. japonicus narnavirus 1 (AejapNV1), and three new virus species that we named Ae. japonicus totivirus 1 (AejapTV1), Ae. japonicus anphevirus 1 (AejapAV1) and Ae. japonicus bunyavirus 1 (AejapBV1). We also discovered sequences that were presumably derived from two additional novel viruses: Ae. japonicus bunyavirus 2 (AejapBV2) and Ae. japonicus rhabdovirus 1 (AejapRV1). All six viruses induced strong RNA interference responses, including the production of twenty-one nucleotide-sized small interfering RNAs, a signature of active replication in the host. Notably, AejapBV1 and AejapBV2 belong to different viral families; however, no RNA-dependent RNA polymerase sequence has been found for AejapBV2. Intriguingly, our small RNA-based approach identified an ∼1-kb long ambigrammatic RNA that is associated with AejapNV1 as a secondary segment but showed no similarity to any sequence in public databases. We confirmed the presence of AejapNV1 primary and secondary segments, AejapTV1, AejapAV1, and AejapBV1 by reverse transcriptase polymerase chain reaction (PCR) in wild-caught Ae. japonicus mosquitoes. AejapNV1 and AejapTV1 were found at high prevalence (87-100 per cent) in adult females, adult males, and larvae. Using a small RNA-based, sequence-independent metagenomic strategy, we uncovered a conserved and prevalent virome among Ae. japonicus mosquito populations. The high prevalence of AejapNV1 and AejapTV1 across all tested mosquito life stages suggests that these viruses are intimately associated with Ae. japonicus.

Transcriptomic Profiling Reveals Intense Host-Pathogen Dispute Compromising Homeostasis during Acute Rift Valley Fever Virus Infection.

Rift Valley fever virus (RVFV) (family Phenuiviridae) can cause severe disease, and outbreaks of this mosquito-borne pathogen pose a significant threat to public and animal health. Yet many molecular aspects of RVFV pathogenesis remain incompletely understood. Natural RVFV infections are acute, characterized by a rapid onset of peak viremia during the first days post-infection, followed by a rapid decline. Although in vitro studies identified a major role of interferon (IFN) responses in counteracting the infection, a comprehensive overview of the specific host factors that play a role in RVFV pathogenesis in vivo is still lacking. Here, the host in vivo transcriptional profiles in the liver and spleen tissues of lambs exposed to RVFV are studied using RNA sequencing (RNA-seq) technology. We validate that IFN-mediated pathways are robustly activated in response to infection. We also link the observed hepatocellular necrosis with severely compromised organ function, which is reflected as a marked downregulation of multiple metabolic enzymes essential for homeostasis. Furthermore, we associate the elevated basal expression of LRP1 in the liver with RVFV tissue tropism. Collectively, the results of this study deepen the knowledge of the in vivo host response during RVFV infection and reveal new insights into the gene regulation networks underlying pathogenesis in a natural host. IMPORTANCE Rift Valley fever virus (RVFV) is a mosquito-transmitted pathogen capable of causing severe disease in animals and humans. Outbreaks of RVFV pose a significant threat to public health and can result in substantial economic losses. Little is known about the molecular basis of RVFV pathogenesis in vivo, particularly in its natural hosts. We employed RNA-seq technology to investigate genome-wide host responses in the liver and spleen of lambs during acute RVFV infection. We show that RVFV infection drastically decreases the expression of metabolic enzymes, which impairs normal liver function. Moreover, we highlight that basal expression levels of the host factor LRP1 may be a determinant of RVFV tissue tropism. This study links the typical pathological phenotype induced by RVFV infection with tissue-specific gene expression profiles, thereby improving our understanding of RVFV pathogenesis.

Rise of the RNA machines - self-amplification in mRNA vaccine design.

mRNA vaccines have won the race for early COVID-19 vaccine approval, yet improvements are necessary to retain this leading role in combating infectious diseases. A next generation of self-amplifying mRNAs, also known as replicons, form an ideal vaccine platform. Replicons induce potent humoral and cellular responses with few adverse effects upon a minimal, single-dose immunization. Delivery of replicons is achieved with virus-like replicon particles (VRPs), or in nonviral vehicles such as liposomes or lipid nanoparticles. Here, we discuss innovative advances, including multivalent, mucosal, and therapeutic replicon vaccines, and highlight novelties in replicon design. As soon as essential safety evaluations have been resolved, this promising vaccine concept can transform into a widely applied clinical platform technology taking center stage in pandemic preparedness.

Novel approaches for the rapid development of rationally designed arbovirus vaccines.

Vector-borne diseases, including those transmitted by mosquitoes, account for more than 17% of infectious diseases worldwide. This number is expected to rise with an increased spread of vector mosquitoes and viruses due to climate change and man-made alterations to ecosystems. Among the most common, medically relevant mosquito-borne infections are those caused by arthropod-borne viruses (arboviruses), especially members of the genera Flavivirus and Alphavirus. Arbovirus infections can cause severe disease in humans, livestock and wildlife. Severe consequences from infections include congenital malformations as well as arthritogenic, haemorrhagic or neuroinvasive disease. Inactivated or live-attenuated vaccines (LAVs) are available for a small number of arboviruses; however there are no licensed vaccines for the majority of these infections. Here we discuss recent developments in pan-arbovirus LAV approaches, from site-directed attenuation strategies targeting conserved determinants of virulence to universal strategies that utilize genome-wide re-coding of viral genomes. In addition to these approaches, we discuss novel strategies targeting mosquito saliva proteins that play an important role in virus transmission and pathogenesis in vertebrate hosts. For rapid pre-clinical evaluations of novel arbovirus vaccine candidates, representative in vitro and in vivo experimental systems are required to assess the desired specific immune responses. Here we discuss promising models to study attenuation of neuroinvasion, neurovirulence and virus transmission, as well as antibody induction and potential for cross-reactivity. Investigating broadly applicable vaccination strategies to target the direct interface of the vertebrate host, the mosquito vector and the viral pathogen is a prime example of a One Health strategy to tackle human and animal diseases.

Competition between two Usutu virus isolates in cell culture and in the common house mosquito Culex pipiens.

Usutu virus (USUV) is a mosquito-borne flavivirus of African origin. Over the past decades, USUV has spread through Europe causing mass die-offs among multiple bird species. The natural transmission cycle of USUV involves Culex spp. mosquitoes as vectors and birds as amplifying hosts. Next to birds and mosquitoes, USUV has also been isolated from multiple mammalian species, including humans, which are considered dead-end hosts. USUV isolates are phylogenetically classified into an African and European branch, subdivided into eight genetic lineages (Africa 1, 2, and 3 and Europe 1, 2, 3, 4, and 5 lineages). Currently, multiple African and European lineages are co-circulating in Europe. Despite increased knowledge of the epidemiology and pathogenicity of the different lineages, the effects of co-infection and transmission efficacy of the co-circulating USUV strains remain unclear. In this study, we report a comparative study between two USUV isolates as follows: a Dutch isolate (USUV-NL, Africa lineage 3) and an Italian isolate (USUV-IT, Europe lineage 2). Upon co-infection, USUV-NL was consistently outcompeted by USUV-IT in mosquito, mammalian, and avian cell lines. In mosquito cells, the fitness advantage of USUV-IT was most prominently observed in comparison to the mammalian or avian cell lines. When Culex pipiens mosquitoes were orally infected with the different isolates, no overall differences in vector competence for USUV-IT and USUV-NL were observed. However, during the in vivo co-infection assay, it was observed that USUV-NL infectivity and transmission were negatively affected by USUV-IT but not vice versa.

Comparative Efficacy of Mayaro Virus-Like Particle Vaccines Produced in Insect or Mammalian Cells.

Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that causes often debilitating rheumatic disease in tropical Central and South America. There are currently no licensed vaccines or antiviral drugs available for MAYV disease. Here, we generated Mayaro virus-like particles (VLPs) using the scalable baculovirus-insect cell expression system. High-level secretion of MAYV VLPs in the culture fluid of Sf9 insect cells was achieved, and particles with a diameter of 64 to 70 nm were obtained after purification. We characterize a C57BL/6J adult wild-type mouse model of MAYV infection and disease and used this model to compare the immunogenicity of VLPs from insect cells with that of VLPs produced in mammalian cells. Mice received two intramuscular immunizations with 1 µg of nonadjuvanted MAYV VLPs. Potent neutralizing antibody responses were generated against the vaccine strain, BeH407, with comparable activity seen against a contemporary 2018 isolate from Brazil (BR-18), whereas neutralizing activity against chikungunya virus was marginal. Sequencing of BR-18 illustrated that this virus segregates with genotype D isolates, whereas MAYV BeH407 belongs to genotype L. The mammalian cell-derived VLPs induced higher mean neutralizing antibody titers than those produced in insect cells. Both VLP vaccines completely protected adult wild-type mice against viremia, myositis, tendonitis, and joint inflammation after MAYV challenge. IMPORTANCE Mayaro virus (MAYV) is associated with acute rheumatic disease that can be debilitating and can evolve into months of chronic arthralgia. MAYV is believed to have the potential to emerge as a tropical public health threat, especially if it develops the ability to be efficiently transmitted by urban mosquito vectors, such as Aedes aegypti and/or Aedes albopictus. Here, we describe a scalable virus-like particle vaccine against MAYV that induced neutralizing antibodies against a historical and a contemporary isolate of MAYV and protected mice against infection and disease, providing a potential new intervention for MAYV epidemic preparedness.

Real-time online monitoring of insect cell proliferation and baculovirus infection using digital differential holographic microscopy and machine learning.

Real-time, detailed online information on cell cultures is essential for understanding modern biopharmaceutical production processes. The determination of key parameters, such as cell density and viability, is usually based on the offline sampling of bioreactors. Gathering offline samples is invasive, has a low time resolution, and risks altering or contaminating the production process. In contrast, measuring process parameters online provides more safety for the process, has a high time resolution, and thus can aid in timely process control actions. We used online double differential digital holographic microscopy (D3HM) and machine learning to perform non-invasive online cell concentration and viability monitoring of insect cell cultures in bioreactors. The performance of D3HM and the machine learning model was tested for a selected variety of baculovirus constructs, products, and multiplicities of infection (MOI). The results show that with online holographic microscopy insect cell proliferation and baculovirus infection can be monitored effectively in real time with high resolution for a broad range of process parameters and baculovirus constructs. The high-resolution data generated by D3HM showed the exact moment of peak cell densities and temporary events caused by feeding. Furthermore, D3HM allowed us to obtain information on the state of the cell culture at the individual cell level. Combining this detailed, real-time information about cell cultures with methodical machine learning models can increase process understanding, aid in decision-making, and allow for timely process control actions during bioreactor production of recombinant proteins.

Incomplete bunyavirus particles can cooperatively support virus infection and spread.

Bunyaviruses lack a specific mechanism to ensure the incorporation of a complete set of genome segments into each virion, explaining the generation of incomplete virus particles lacking one or more genome segments. Such incomplete virus particles, which may represent the majority of particles produced, are generally considered to interfere with virus infection and spread. Using the three-segmented arthropod-borne Rift Valley fever virus as a model bunyavirus, we here show that two distinct incomplete virus particle populations unable to spread autonomously are able to efficiently complement each other in both mammalian and insect cells following co-infection. We further show that complementing incomplete virus particles can co-infect mosquitoes, resulting in the reconstitution of infectious virus that is able to disseminate to the mosquito salivary glands. Computational models of infection dynamics predict that incomplete virus particles can positively impact virus spread over a wide range of conditions, with the strongest effect at intermediate multiplicities of infection. Our findings suggest that incomplete particles may play a significant role in within-host spread and between-host transmission, reminiscent of the infection cycle of multipartite viruses.

Zika vector competence data reveals risks of outbreaks: the contribution of the European ZIKAlliance project.

First identified in 1947, Zika virus took roughly 70 years to cause a pandemic unusually associated with virus-induced brain damage in newborns. Zika virus is transmitted by mosquitoes, mainly Aedes aegypti, and secondarily, Aedes albopictus, both colonizing a large strip encompassing tropical and temperate regions. As part of the international project ZIKAlliance initiated in 2016, 50 mosquito populations from six species collected in 12 countries were experimentally infected with different Zika viruses. Here, we show that Ae. aegypti is mainly responsible for Zika virus transmission having the highest susceptibility to viral infections. Other species play a secondary role in transmission while Culex mosquitoes are largely non-susceptible. Zika strain is expected to significantly modulate transmission efficiency with African strains being more likely to cause an outbreak. As the distribution of Ae. aegypti will doubtless expand with climate change and without new marketed vaccines, all the ingredients are in place to relive a new pandemic of Zika.