Kunjin virus replicons: an RNA-based, non-cytopathic viral vector system for protein production, vaccine and gene therapy applications

The application of viral vectors for gene expression and delivery is rapidly evolving, with several entering clinical trials. However, a number of issues, including safety, gene expression levels, cell selectivity and antivector immunity, are driving the search for new vector systems. A number of replicon-based vectors derived from positive-strand RNA viruses have recently been developed, and this paper reviews the current knowledge on the first flavivirus replicon system, which is based on the Australian flavivirus Kunjin NUN). Like most replicon systems, KUN replicons can be delivered as DNA, RNA or virus-like particles, they replicate their RNA in the cytoplasm and direct prolonged high-level gene expression. However, unlike most alphavirus replicon systems, KUN replicons are non-cytopathic, with transfected cells able to divide, allowing the establishment of cell lines stably expressing replicon RNA and heterologous genes. As vaccine vectors KUN replicons can induce potent, long-lived, protective, immunogen-specific CD8(+) T cell immunity, a feature potentially related to extended production of antigen and double-stranded RNA-induced 'danger signals'. The identification of KUN replicon mutants that induce increased levels of IFN-alpha/beta has also spawned investigation of KUN replicons for use in cancer gene therapy. The unique characteristics of KUN replicons may thus make them suitable for specific protein production, vaccine and gene therapy applications

Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells

Background: Chikungunya virus (CHIKV) is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively. Results: Expression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits. Conclusions: Chikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections

Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells

Background: Chikungunya virus (CHIKV) is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively. Results: Expression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits. Conclusions: Chikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections

Reverse genetics system for shuni virus, an emerging orthobunyavirus with zoonotic potential

The genus Orthobunyavirus (family Peribunyaviridae, order Bunyavirales) comprises over 170 named mosquito- and midge-borne viruses, several of which cause severe disease in animals or humans. Their three-segmented genomes enable reassortment with related viruses, which may result in novel viruses with altered host or tissue tropism and virulence. One such reassortant, Schmallenberg virus (SBV), emerged in north-western Europe in 2011. Shuni virus (SHUV) is an orthobunyavirus related to SBV that is associated with neurological disease in horses in southern Africa and recently caused an outbreak manifesting with neurological disease and birth defects among ruminants in Israel. The zoonotic potential of SHUV was recently underscored by its association with neurological disease in humans. We here report a reverse genetics system for SHUV and provide first evidence that the non-structural (NSs) protein of SHUV functions as an antagonist of host innate immune responses. We furthermore report the rescue of a reassortant containing the L and S segments of SBV and the M segment of SHUV. This novel reverse genetics system can now be used to study SHUV virulence and tropism, and to elucidate the molecular mechanisms that drive reassortment events.The Dutch Ministry of Agriculture, Nature and Food Quality of the Netherlands and the European Union’s Horizon 2020 research and innovation programme under LEAP-Agri grant agreement No 727715.http://www.mdpi.com/journal/viruseshj2020Medical Virolog

Recombinant Modified Vaccinia Virus Ankara Expressing Glycoprotein E2 of Chikungunya Virus Protects AG129 Mice against Lethal Challenge

Chikungunya virus (CHIKV) infection is characterized by rash, acute high fever, chills, headache, nausea, photophobia, vomiting, and severe polyarthralgia. There is evidence that arthralgia can persist for years and result in long-term discomfort. Neurologic disease with fatal outcome has been documented, although at low incidences. The CHIKV RNA genome encodes five structural proteins (C, E1, E2, E3 and 6K). The E1 spike protein drives the fusion process within the cytoplasm, while the E2 protein is believed to interact with cellular receptors and therefore most probably constitutes the target of neutralizing antibodies. We have constructed recombinant Modified Vaccinia Ankara (MVA) expressing E3E2, 6KE1, or the entire CHIKV envelope polyprotein cassette E3E26KE1. MVA is an appropriate platform because of its demonstrated clinical safety and its suitability for expression of various heterologous proteins. After completing the immunization scheme, animals were challenged with CHIV-S27. Immunization of AG129 mice with MVAs expressing E2 or E3E26KE1 elicited neutralizing antibodies in all animals and provided 100% protection against lethal disease. In contrast, 75% of the animals immunized with 6KE1 were protected against lethal infection. In conclusion, MVA expressing the glycoprotein E2 of CHIKV represents as an immunogenic and effective candidate vaccine against CHIKV infections

Complex dynamics of defective interfering baculoviruses during serial passage in insect cells

Defective interfering (DI) viruses are thought to cause oscillations in virus levels, known as the 'Von Magnus effect'. Interference by DI viruses has been proposed to underlie these dynamics, although experimental tests of this idea have not been forthcoming. For the baculoviruses, insect viruses commonly used for the expression of heterologous proteins in insect cells, the molecular mechanisms underlying DI generation have been investigated. However, the dynamics of baculovirus populations harboring DIs have not been studied in detail. In order to address this issue, we used quantitative real-time PCR to determine the levels of helper and DI viruses during 50 serial passages of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in Sf21 cells. Unexpectedly, the helper and DI viruses changed levels largely in phase, and oscillations were highly irregular, suggesting the presence of chaos. We therefore developed a simple mathematical model of baculovirus-DI dynamics. This theoretical model reproduced patterns qualitatively similar to the experimental data. Although we cannot exclude that experimental variation (noise) plays an important role in generating the observed patterns, the presence of chaos in the model dynamics was confirmed with the computation of the maximal Lyapunov exponent, and a Ruelle-Takens-Newhouse route to chaos was identified at decreasing production of DI viruses, using mutation as a control parameter. Our results contribute to a better understanding of the dynamics of DI baculoviruses, and suggest that changes in virus levels over passages may exhibit chaos.The authors thank Javier Carrera, Just Vlak and Lia Hemerik for helpful discussion. MPZ was supported by a Rubicon Grant from the Netherlands Organization for Scientific Research (NWO, www.nwo.nl) and a 'Juan de la Cierva' postdoctoral contract (JCI-2011-10379) from the Spanish 'Secretaria de Estado de Investigacion, Desarrollo e Innovacion'. 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Impact of Gut Bacteria on the Infection and Transmission of Pathogenic Arboviruses by Biting Midges and Mosquitoes

Tripartite interactions among insect vectors, midgut bacteria, and viruses may determine the ability of insects to transmit pathogenic arboviruses. Here, we investigated the impact of gut bacteria on the susceptibility of Culicoides nubeculosus and Culicoides sonorensis biting midges for Schmallenberg virus, and of Aedes aegypti mosquitoes for Zika and chikungunya viruses. Gut bacteria were manipulated by treating the adult insects with antibiotics. The gut bacterial communities were investigated using Illumina MiSeq sequencing of 16S rRNA, and susceptibility to arbovirus infection was tested by feeding insects with an infectious blood meal. Antibiotic treatment led to changes in gut bacteria for all insects. Interestingly, the gut bacterial composition of untreated Ae. aegypti and C. nubeculosus showed Asaia as the dominant genus, which was drastically reduced after antibiotic treatment. Furthermore, antibiotic treatment resulted in relatively more Delftia bacteria in both biting midge species, but not in mosquitoes. Antibiotic treatment and subsequent changes in gut bacterial communities were associated with a significant, 1.8-fold increased infection rate of C. nubeculosus with Schmallenberg virus, but not for C. sonorensis. We did not find any changes in infection rates for Ae. aegypti mosquitoes with Zika or chikungunya virus. We conclude that resident gut bacteria may dampen arbovirus transmission in biting midges, but not so in mosquitoes. Use of antimicrobial compounds at livestock farms might therefore have an unexpected contradictory effect on the health of animals, by increasing the transmission of viral pathogens by biting midges.</p

The invasive Asian bush mosquito Aedes japonicus found in the Netherlands can experimentally transmit Zika virus and Usutu virus

Background - The Asian bush mosquito Aedes japonicus is invading Europe and was first discovered in Lelystad, the Netherlands in 2013, where it has established a permanent population. In this study, we investigated the vector competence of Ae. japonicus from the Netherlands for the emerging Zika virus (ZIKV) and zoonotic Usutu virus (USUV). ZIKV causes severe congenital microcephaly and Guillain-Barré syndrome in humans. USUV is closely related to West Nile virus, has recently spread throughout Europe and is causing mass mortality of birds. USUV infection in humans can result in clinical manifestations ranging from mild disease to severe neurological impairments.Methodology/Principal findings - In our study, field-collected Ae. japonicus females received an infectious blood meal with ZIKV or USUV by droplet feeding. After 14 days at 28°C, 3% of the ZIKV-blood fed mosquitoes and 13% of the USUV-blood fed mosquitoes showed virus-positive saliva, indicating that Ae. japonicus can transmit both viruses. To investigate the effect of the mosquito midgut barrier on virus transmission, female mosquitoes were intrathoracically injected with ZIKV or USUV. Of the injected mosquitoes, 96% (ZIKV) and 88% (USUV) showed virus-positive saliva after 14 days at 28°C. This indicates that ZIKV and USUV can efficiently replicate in Ae. japonicus but that a strong midgut barrier is normally restricting virus dissemination. Small RNA deep sequencing of orally infected mosquitoes confirmed active replication of ZIKV and USUV, as demonstrated by potent small interfering RNA responses against both viruses. Additionally, de novo small RNA assembly revealed the presence of a novel narnavirus in Ae. japonicus.Conclusions/Significance - Given that Ae. japonicus can experimentally transmit arthropod-borne viruses (arboviruses) like ZIKV and USUV and is currently expanding its territories, we should consider this mosquito as a potential vector for arboviral diseases in Europ

Comparative Usutu and West Nile virus transmission potential by local Culex pipiens mosquitoes in north-western Europe

Originating from Africa, Usutu virus (USUV) first emerged in Europe in 2001. This mosquito-borne flavivirus caused high mortality rates in its bird reservoirs, which strongly resembled the introduction of West Nile virus (WNV) in 1999 in the United States. Mosquitoes infected with USUV incidentally transmit the virus to other vertebrates, including humans, which can result in neuroinvasive disease. USUV and WNV co-circulate in parts of southern Europe, but the distribution of USUV extends into central and northwestern Europe. In the field, both viruses have been detected in the northern house mosquito Culex pipiens, of which the potential for USUV transmission is unknown. To understand the transmission dynamics and assess the potential spread of USUV, we determined the vector competence of C. pipiens for USUV and compared it with the well characterized WNV. We show for the first time that northwestern European mosquitoes are highly effective vectors for USUV, with infection rates of 11% at 18. °C and 53% at 23. °C, which are comparable with values obtained for WNV. Interestingly, at a high temperature of 28. °C, mosquitoes became more effectively infected with USUV (90%) than with WNV (58%), which could be attributed to barriers in the mosquito midgut. Small RNA deep sequencing of infected mosquitoes showed for both viruses a strong bias for 21-nucleotide small interfering (si)RNAs, which map across the entire viral genome both on the sense and antisense strand. No evidence for viral PIWI-associated RNA (piRNA) was found, suggesting that the siRNA pathway is the major small RNA pathway that targets USUV and WNV infection in C. pipiens mosquitoes

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.info:eu-repo/semantics/publishedVersio