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Summary Dengue virus (DENV) is an emerging threat causing an estimated 390 million infections per year. Dengvaxia, the only licensed vaccine, may not be adequately safe in young and seronegative patients; hence, development of a safer, more effective vaccine is of great public health interest. Virus‐like particles (VLPs) are a safe and very efficient vaccine strategy, and DENV VLPs have been produced in various expression systems. Here, we describe the production of DENV VLPs in Nicotiana benthamiana using transient expression. The co‐expression of DENV structural proteins (SP) and a truncated version of the non‐structural proteins (NSPs), lacking NS5 that contains the RNA‐dependent RNA polymerase, led to the assembly of DENV VLPs in plants. These VLPs were comparable in appearance and size to VLPs produced in mammalian cells. Contrary to data from other expression systems, expression of the protein complex prM‐E was not successful, and strategies used in other expression systems to improve the VLP yield did not result in increased yields in plants but, rather, increased purification difficulties. Immunogenicity assays in BALB/c mice revealed that plant‐made DENV1‐SP + NSP VLPs led to a higher antibody response in mice compared with DENV‐E domain III displayed inside bluetongue virus core‐like particles and a DENV‐E domain III subunit. These results are consistent with the idea that VLPs could be the optimal approach to creating candidate vaccines against enveloped viruses.

Lassa fever remains the most imported viral haemorrhagic fever in Europe and is responsible for 5000 deaths per year throughout Western Africa. There is no vaccine and treatment is often ineffective. We have developed a vaccine based on modified Vaccinia Ankara expressing the nucleoprotein from Lassa virus (MVALassaNP). This study investigated the immunogenicity (in mice) and efficacy (in guinea pigs) of the MVALassaNP vaccine as a prime/boost or single vaccination regime. ELISA and ELISpot assays confirmed humoral and T-cell immunity following both a prime and prime/boost vaccination, with the prime/boost regime producing a statistically increased response compared to a prime only vaccine (P

Summary During the past decade several notable viruses have suddenly emerged from obscurity or anonymity to become serious global health threats, provoking concern regarding their sustained epidemic transmission in immunologically naive human populations. With each new threat comes the call for rapid vaccine development. Indeed, vaccines are considered a critical component of disease prevention for emerging viral infections because, in many cases, other medical options are limited or non-existent, or that infections result in such a rapid clinical deterioration that the effectiveness of therapeutics is limited. While classic approaches to vaccine development are still amenable to emerging viruses, the application of molecular techniques in virology has profoundly influenced our understanding of virus biology, and vaccination methods based on replicating, attenuated and non-replicating virus vector approaches have become useful vaccine platforms. Together with a growing understanding of viral disease emergence, a range of vaccine strategies and international commitment to underpin development, vaccine intervention for new and emerging viruses may become a possibility.

In the UK, research on hazard group 4 (HG4) pathogens requires specialised Containment Level 4 (CL4) facilities. These differ from Biosafety Level 4 (BSL4) conditions in that work is conducted in class III microbiological safety cabinets for primary containment instead of using positive pressure suits. This presents unique challenges associated with the physical restrictions of working in a limited space, and prohibits the use of many techniques and specialist equipment. In consequence, detailed studies on the biology of HG4 pathogens and in particular their immunological relationships with the host are understudied in the UK; for example, the majority of immunological assays with which the immune system is interrogated require specialist equipment that is unsuitable for CL4. Multiplexing to simultaneously measure multiple analytes is increasingly being used in immunological studies. This assay is attractive for CL4 work because it reduces the time spent in the laboratory whilst maximising the use of valuable sample volume. The Luminex microsphere approach allows for the determination of many cytokines and chemokines, however, the detection system uses fixed aligned lasers and integrated computer systems which are unsuitable for use at CL4. Therefore, we have developed an approach in which the Luminex assay is conducted within the CL4 laboratory and a formalin-fixation stage is introduced to allow for analysis to be undertaken outside of containment. Quality control preparations allow the assay characteristics to be monitored and analysis of assay performance to be evaluated. Our data demonstrate that Luminex is an applicable tool for use at CL4 and that assays can be run reliably to generate reproducible standardised data across different plates and individual experiments.

Respiratory viruses such as coronaviruses represent major ongoing global threats, causing epidemics and pandemics with huge economic burden. Rapid spread of virus through populations poses an enormous challenge for outbreak control. Like all respiratory viruses, the most recent novel human coronavirus SARS-CoV-2, initiates infection in the upper respiratory tract (URT). Infected individuals are often asymptomatic, yet highly infectious and readily transmit virus. A therapy that restricts initial replication in the URT has the potential to prevent progression of severe lower respiratory tract disease as well as limiting person-to-person transmission.We show that prophylactic intra-nasal administration of the TLR2/6 agonist INNA-051 in a SARS-CoV-2 ferret infection model effectively reduces levels of viral RNA in the nose and throat. The results of our study support clinical development of a therapy based on prophylactic TLR2/6 innate immune activation in the URT to reduce SARS-CoV-2 transmission and provide protection against COVID-19.

Type I interferon receptor knockout mice (strain A129) were assessed as a disease model of hantavirus infection. A range of infection routes (intramuscular, intraperitoneal and intranasal) were assessed using minimally passaged Seoul virus (strain Humber). Dissemination of virus to the spleen, kidney and lung was observed at 5 days after intramuscular and intraperitoneal challenge, which was resolved by day 14. In contrast, intranasal challenge of A129 mice demonstrated virus tropism to the lung, which was maintained to day 14 post-challenge. These data support the use of the A129 mouse model for future infection studies and thein vivoevaluation of interventions.

Crimean-Congo haemorrhagic fever virus (CCHFV) is a deadly human pathogen of the utmost seriousness being highly lethal causing devastating disease symptoms that result in intense and prolonged suffering to those infected. During the past 40 years, this virus has repeatedly caused sporadic outbreaks responsible for relatively low numbers of human casualties, but with an alarming fatality rate of up to 80% in clinically infected patients. CCHFV is transmitted to humans by Hyalomma ticks and contact with the blood of viremic livestock, additionally cases of human-to-human transmission are not uncommon in nosocomial settings. The incidence of CCHF closely matches the geographical range of permissive ticks, which are widespread throughout Africa, Asia, the Middle East and Europe. As such, CCHFV is the most widespread tick-borne virus on earth. It is a concern that recent data shows the geographic distribution of Hyalomma ticks is expanding. Migratory birds are also disseminating Hyalomma ticks into more northerly parts of Europe thus potentially exposing naïve human populations to CCHFV. The virus has been imported into the UK on two occasions in the last five years with the first fatal case being confirmed in 2012. A licensed vaccine to CCHF is not available. In this review, we discuss the background and complications surrounding this limitation and examine the current status and recent advances in the development of vaccines against CCHFV.

The presence of tick-borne encephalitis virus (TBEV) was detected in a questing tick pool in southern England in September 2019. Hitherto, TBEV had only been detected in a limited area in eastern England. This southern English viral genome sequence is distinct from TBEV-UK, being most similar to TBEV-NL. The new location of TBEV presence highlights that the diagnosis of tick-borne encephalitis should be considered in encephalitic patients in areas of the United Kingdom outside eastern England.

The presence of tick-borne encephalitis virus (TBEV) was detected in a questing tick pool in southern England in September 2019. Hitherto, TBEV had only been detected in a limited area in eastern England. This southern English viral genome sequence is distinct from TBEV-UK, being most similar to TBEV-NL. The new location of TBEV presence highlights that the diagnosis of tick-borne encephalitis should be considered in encephalitic patients in areas of the United Kingdom outside eastern England.

The United Kingdom (UK) has thus far been considered to be free from tick-borne encephalitis (TBE), yet in July 2019, a German infant developed serologically diagnosed TBE following a tick bite in southern England. This first report of a probable human case together with recent findings of TBE virus in ticks in foci in England suggest that TBE may be acquired in parts of England and should be considered in patients with aetiologically-unexplained neurological manifestations.

Crimean-Congo hemorrhagic fever virus is a risk group 4 pathogen, which mandates the use of maximum containment facilities, often termed biosafety level 4 or containment level 4 when working with infectious materials. Diagnostic and research work involving live viruses in such laboratories is time-consuming and inconvenient, resulting in delays. Herein, we show that serum neutralizing activity against the virus can be measured in low-containment laboratories using a pseudotyped virus.

The highly glycosylated glycoprotein spike of Ebola virus (EBOV-GP1,2) is the primary target of the humoral host response. Recombinant EBOV-GP ectodomain (EBOV-GP1,2ecto) expressed in mammalian cells was used to immunize sheep and elicited a robust immune response and produced high titers of high avidity polyclonal antibodies. Investigation of the neutralizing activity of the ovine antisera in vitro revealed that it neutralized EBOV. A pool of intact ovine immunoglobulin G, herein termed EBOTAb, was prepared from the antisera and used for an in vivo guinea pig study. When EBOTAb was delivered 6 hours after challenge, all animals survived without experiencing fever or other clinical manifestations. In a second series of guinea pig studies, the administration of EBOTAb dosing was delayed for 48 or 72 hours after challenge, resulting in 100% and 75% survival, respectively. These studies illustrate the usefulness of EBOTAb in protecting against EBOV-induced disease.

Ebola virus (EBOV) is highly pathogenic, with a predisposition to cause outbreaks in human populations accompanied by significant mortality. Owing to the lack of approved therapies, screening programmes of potentially efficacious drugs have been undertaken. One of these studies has demonstrated the possible utility of chloroquine against EBOV using pseudotyped assays. In mouse models of EBOV disease there are conflicting reports of the therapeutic effects of chloroquine. There are currently no reports of its efficacy using the larger and more stringent guinea pig model of infection. In this study we have shown that replication of live EBOV is impaired by chloroquine in vitro. However, no protective effects were observed in vivo when EBOV-infected guinea pigs were treated with chloroquine. These results advocate that chloroquine should not be considered as a treatment strategy for EBOV.

Ebola virus is responsible for causing severe hemorrhagic fevers, with case fatality rates of up to 90%. Currently, no antiviral or vaccine is licensed against Ebola virus. A phosphatidylserine-targeting antibody (PGN401, bavituximab) has previously been shown to have broad-spectrum antiviral activity. Here, we demonstrate that PGN401 specifically binds to Ebola virus and recognizes infected cells. Our study provides the first evidence of phosphatidylserine-targeting antibody reactivity against Ebola virus.

Despite sporadic outbreaks of Ebola virus (EBOV) over the last 4 decades and the recent public health emergency in West Africa, there are still no approved vaccines or therapeutics for the treatment of acute EBOV disease (EVD). In response to the 2014 outbreak, an ovine immunoglobulin therapy was developed, termed EBOTAb. After promising results in the guinea pig model of EBOV infection, EBOTAb was tested in the cynomolgus macaque non-human primate model of lethal EBOV infection. To ensure stringent therapeutic testing conditions to replicate likely clinical usage, EBOTAb was first delivered 1, 2 or 3 days post-challenge with a lethal dose of EBOV. Results showed a protective effect of EBOTAb given post-exposurally, with survival rates decreasing with increasing time after challenge. Viremia results demonstrated that EBOTAb resulted in a decreased circulation of EBOV in the bloodstream. Additionally, assay of liver enzymes and histology analysis of local tissues identified differences between EBOTAb-treated and untreated groups. The results presented demonstrate that EBOTAb conferred protection against EBOV when given post-exposure and should be explored and developed further as a potential intervention strategy for future outbreaks, which are likely to occur.

Zika virus (ZIKV) is phylogenetically divided into two lineages comprising African (ZIKVAF) and Asian (ZIKVAS) genotypes. In the type-I interferon receptor deficient mouse model, ZIKVAF causes severe disease with all mice meeting humane endpoints with doses as low as 10 plaque-forming units (pfu) whereas a much milder infection is seen after challenge with ZIKVAS, including with doses as high as 106 pfu. Using this mouse model, the elucidation of cytokine, chemokine, growth factor and acute phase protein responses over the course of infection were studied to determine whether these analytes contributed to the stark difference in clinical outcome. Results demonstrated some significant differences, with the ZIKVAF infection being associated with increases in a higher number of biomarkers than ZIKVAS. When low (10 pfu) and high (106 pfu) challenge doses were compared, animals given the lower virus inoculum showed a wider range of responses, indicating a different disease progression compared to those challenged with high doses. These results aid with elucidating the different outcomes with the two lineages of ZIKV and with future work to assess pathogenicity of virus infection.

Viral pathogenesis in the infected cell is a balance between antiviral responses and subversion of host-cell processes. Many viral proteins specifically interact with host-cell proteins to promote virus biology. Understanding these interactions can lead to knowledge gains about infection and provide potential targets for antiviral therapy. One such virus is Ebola, which has profound consequences for human health and causes viral hemorrhagic fever where case fatality rates can approach 90%. The Ebola virus VP24 protein plays a critical role in the evasion of the host immune response and is likely to interact with multiple cellular proteins. To map these interactions and better understand the potential functions of VP24, label-free quantitative proteomics was used to identify cellular proteins that had a high probability of forming the VP24 cellular interactome. Several known interactions were confirmed, thus placing confidence in the technique, but new interactions were also discovered including one with ATP1A1, which is involved in osmoregulation and cell signaling. Disrupting the activity of ATP1A1 in Ebola-virus-infected cells with a small molecule inhibitor resulted in a decrease in progeny virus, thus illustrating how quantitative proteomics can be used to identify potential therapeutic targets.

Background Undifferentiated febrile illness (UFI) is one of the most common reasons for people seeking healthcare in low-income countries. While illness and death due to specific infections such as malaria are often well-quantified, others are frequently uncounted and their impact underappreciated. A number of high consequence infectious diseases, including Ebola virus, are endemic or epidemic in the Federal Republic of Sudan which has experienced at least 12 UFI outbreaks, frequently associated with haemorrhage and high case fatality rates (CFR), since 2012. One of these occurred in Darfur in 2015/2016 with 594 cases and 108 deaths (CFR 18.2%). The aetiology of these outbreaks remains unknown. Methodology/Principal findings We report a retrospective cohort study of the 2015/2016 Darfur outbreak, using a subset of 65 of 263 outbreak samples received by the National Public Health Laboratory which met selection criteria of sufficient sample volume and epidemiological data. Clinical features included fever (95.8%), bleeding (95.7%), headache (51.6%) and arthralgia (42.2%). No epidemiological patterns indicative of person-to-person transmission or health-worker cases were reported. Samples were tested at the Public Health England Rare and Imported Pathogens Laboratory using a bespoke panel of likely pathogens including haemorrhagic fever viruses, arboviruses and Rickettsia, Leptospira and Borrelia spp. Seven (11%) were positive for Crimean-Congo haemorrhagic fever virus (CCHFV) by real-time reverse transcription PCR. The remaining samples tested negative on all assays. Conclusions/Significance CCHFV is an important cause of fever and haemorrhage in Darfur, but not the sole major source of UFI outbreaks in Sudan. Prospective studies are needed to explore other aetiologies, including novel pathogens. The presence of CCHFV has critical infection, prevention and control as well as clinical implications for future response. Our study reinforces the need to boost surveillance, lab and investigative capacity to underpin effective response, and for local and international health security., Author summary The Federal Republic of Sudan has had at least 12 outbreaks of febrile illness of unknown cause associated with symptoms of haemorrhage and high case fatality rates since 2012. Outbreaks without clear diagnosis are concerning, particularly in countries such as Sudan where a range of high consequence diseases, including viral haemorrhagic fevers, are endemic or epidemic, and local laboratory capacity is limited. We transferred historical samples stored in the National Public Health Authority from one of these outbreaks that occurred in Darfur 2015–2016 to the Public Health England Laboratory at Porton, UK, and tested them against a wide range of infectious diseases to try to identify the cause, and to help the Sudanese Federal Ministry of Health to develop and target their limited laboratory capacity. We found that Crimean-Congo Haemorrhagic Fever was an important cause but not the only source of cases in this outbreak. This has implications for prevention and control as well as for treating cases. Our study also highlighted the need for future studies to explore other possible causes, including new pathogens, and reinforced the need to boost surveillance, lab and investigative capacity for more timely and complete outbreak response.

Lujo virus is an emerging arenavirus circulating in Southern Africa. Although to date there has only been a single outbreak of the novel haemorrhagic disease resulting from human infection with this virus, the case-fatality rate of exposed individuals, including nosocomial transmission, was 80%. The ability to identify viral haemorrhagic fevers accurately, especially those capable of nosocomial transmission, is of critical importance. Timely identification of these diseases allow medical professionals to isolate patients and implement barrier nursing techniques in order to prevent onward transmission of the virus. While rapid diagnostic methods are published for most viral haemorrhagic fevers, at present there are no such virus specific protocols for Lujo haemorrhagic fever. This report details the first set of diagnostic molecular assays designed to identify Lujo viral RNA rapidly, and demonstrates the potential functionality of these assays for use in the clinical setting. Although these assays have been designed and validated against a solitary isolate of Lujo virus, this represents the entirety of strains detected to date, and offer quick, cheap and easy methods for use in diagnostic laboratories.

Sequential sampling from animals challenged with highly pathogenic organisms, such as haemorrhagic fever viruses, is required for many pharmaceutical studies. Using the guinea pig model of Ebola virus infection, a catheterized system was used which had the benefits of allowing repeated sampling of the same cohort of animals, and also a reduction in the use of sharps at high biological containment. Levels of a PS-targeting antibody (Bavituximab) were measured in Ebola-infected animals and uninfected controls. Data showed that the pharmacokinetics were similar in both groups, therefore Ebola virus infection did not have an observable effect on the half-life of the antibody.

Zika virus (ZIKV) is an emerging pathogenic flavivirus currently circulating in numerous countries in South America, the Caribbean, and the Western Pacific Region. Using an unbiased metagenomic sequencing approach, we report here the first complete genome sequence of ZIKV isolated from a clinical semen sample.

The antiviral properties of iminosugars have been reported previously in vitro and in small animal models against Ebola virus (EBOV); however, their effects have not been tested in larger animal models such as guinea pigs. We tested the iminosugars N-butyl-deoxynojirimycin (NB-DNJ) and N-(9-methoxynonyl)-1deoxynojirimycin (MON-DNJ) for safety in uninfected animals, and for antiviral efficacy in animals infected with a lethal dose of guinea pig adapted EBOV. 1850 mg/kg/day NB-DNJ and 120 mg/kg/day MON-DNJ administered intravenously, three times daily, caused no adverse effects and were well tolerated. A pilot study treating infected animals three times within an 8 hour period was promising with 1 of 4 infected NB-DNJ treated animals surviving and the remaining three showing improved clinical signs. MON-DNJ showed no protective effects when EBOV-infected guinea pigs were treated. On histopathological examination, animals treated with NB-DNJ had reduced lesion severity in liver and spleen. However, a second study, in which NB-DNJ was administered at equally-spaced 8 hour intervals, could not confirm drug-associated benefits. Neither was any antiviral effect of iminosugars detected in an EBOV glycoprotein pseudotyped virus assay. Overall, this study provides evidence that NB-DNJ and MON-DNJ do not protect guinea pigs from a lethal EBOV-infection at the dose levels and regimens tested. However, the one surviving animal and signs of improvements in three animals of the NB-DNJ treated cohort could indicate that NB-DNJ at these levels may have a marginal beneficial effect. Future work could be focused on the development of more potent iminosugars.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a major cause of tick-borne viral hemorrhagic disease in the world. Despite of its importance as a deadly pathogen, there is currently no licensed vaccine against CCHF disease. The attachment glycoprotein of CCHFV (Gn) is a potentially important target for protective antiviral immune responses. To characterize the expression of recombinant CCHFV Gn in an insect-cell-based system, we developed a gene expression system expressing the full-length coding sequence under a polyhedron promoter in Sf9 cells using recombinant baculovirus. Recombinant Gn was purified by affinity chromatography, and the immunoreactivity of the protein was evaluated using sera from patients with confirmed CCHF infection. Codon-optimized Gn was successfully expressed, and the product had the expected molecular weight for CCHFV Gn glycoprotein of 37 kDa. In time course studies, the optimum expression of Gn occurred between 36 and 48 hours postinfection. The immunoreactivity of the recombinant protein in Western blot assay against human sera was positive and was similar to the results obtained with the anti-V5 tag antibody. Additionally, mice were subjected to subcutaneous injection with recombinant Gn, and the cellular and humoral immune response was monitored. The results showed that recombinant Gn protein was highly immunogenic and could elicit high titers of antigen-specific antibodies. Induction of the inflammatory cytokine interferon-gamma and the regulatory cytokine IL-10 was also detected. In conclusion, a recombinant baculovirus harboring CCHFV Gn was constructed and expressed in Sf9 host cells for the first time, and it was demonstrated that this approach is a suitable expression system for producing immunogenic CCHFV Gn protein without any biosafety concerns.

The Nairovirus genus of the Bunyaviridae family contains serious human and animal pathogens classified within multiple serogroups and species. Of these serogroups, the Crimean-Congo hemorrhagic fever virus (CCHFV) serogroup comprises sole members CCHFV and Hazara virus (HAZV). CCHFV is an emerging zoonotic virus that causes often-fatal hemorrhagic fever in infected humans for which preventative or therapeutic strategies are not available. In contrast, HAZV is nonpathogenic to humans and thus represents an excellent model to study aspects of CCHFV biology under conditions of more-accessible biological containment. The three RNA segments that form the nairovirus genome are encapsidated by the viral nucleocapsid protein (N) to form ribonucleoprotein (RNP) complexes that are substrates for RNA synthesis and packaging into virus particles. We used quantitative proteomics to identify cellular interaction partners of CCHFV N and identified robust interactions with cellular chaperones. These interactions were validated using immunological methods, and the specific interaction between native CCHFV N and cellular chaperones of the HSP70 family was confirmed during live CCHFV infection. Using infectious HAZV, we showed for the first time that the nairovirus N-HSP70 association was maintained within both infected cells and virus particles, where N is assembled as RNPs. Reduction of active HSP70 levels in cells by the use of small-molecule inhibitors significantly reduced HAZV titers, and a model for chaperone function in the context of high genetic variability is proposed. These results suggest that chaperones of the HSP70 family are required for nairovirus replication and thus represent a genetically stable cellular therapeutic target for preventing nairovirus-mediated disease. IMPORTANCE Nairoviruses compose a group of human and animal viruses that are transmitted by ticks and associated with serious or fatal disease. One member is Crimean-Congo hemorrhagic fever virus (CCHFV), which is responsible for fatal human disease and is recognized as an emerging threat within Europe in response to climate change. No preventative or therapeutic strategies against nairovirus-mediated disease are currently available. Here we show that the N protein of CCHFV and the related Hazara virus interact with a cellular protein, HSP70, during both the intracellular and extracellular stages of the virus life cycle. The use of inhibitors that block HSP70 function reduces virus titers by up to 1,000-fold, suggesting that this interaction is important within the context of the nairovirus life cycle and may represent a potent target for antinairovirus therapies against which the virus cannot easily develop resistance.

Crimean-Congo hemorrhagic fever (CCHF) is a virulent tick-borne disease with a case fatality rate ranging from 10–50% for tick-borne transmission, and up to 80% for nosocomial transmission. Human cases have been reported in over 30 countries across Europe, Asia, and Africa. It appears to be spreading to new areas with several countries reporting their first human cases of CCHF disease within the past 10 years. We report a novel real-time RT-PCR assay designed to amplify a conserved region of the CCHF virus S segment. It is capable of detecting strains from all 7 groups of CCHF, including the AP92 strain that until recently represented a lineage of strains that were not associated with human disease. The limit of detection of the assay is 5 copies of target RNA, and the assay shows no cross-reactivity with other viruses from within the same genus, or with viruses causing similar human disease.

Free-ranging spur-thighed tortoises Testudo graeca, captured in different habitat types of Northern Tunisia from March to April 2017, were examined for tick infestation: 134/147 (91%) were infested. The overall infestation intensity and abundance was 8.5 and 7.8, respectively. From these tortoises, 1174 ticks were collected, of which 10% (n = 120) taken from 18 randomly-selected tortoises were identified at the species level; the remaining ticks were examined for the presence of Crimean-Congo haemorrhagic fever virus (CCHFv) by real time RT-PCR. Only adult Hyalomma aegyptium were found, suggesting a high degree of host specificity to tortoises. No CCHFv was detected in ticks. Considering the absence of CCHFv in Hyalomma aegyptium infesting its main host, the spur-thighed tortoise, this tick species is unlikely to play a major role in the epidemiology of CCHF. Therefore, more studies are needed to investigate the circulation of this arbovirus between livestock and other tick species from North Africa.

Zika virus (ZIKV) is a mosquito-borne pathogen which has recently spread beyond Africa and into Pacific and South American regions. Despite first being detected in 1947, very little information is known about the virus and its spread has been associated with increases in Guillain-Barre syndrome and microcephaly. There are currently no known vaccines or antivirals against ZIKV infection. Progress in assessing interventions will require the development of animal models to test efficacies; however, there are only limited reports on in vivo studies. The only susceptible murine models have involved intracerebral inoculations or juvenile animals, which do not replicate natural infection. Our report has studied the effect of ZIKV infection in type-I interferon receptor deficient (A129) mice and the parent strain (129Sv/Ev) after subcutaneous challenge in the lower leg to mimic a mosquito bite. A129 mice developed severe symptoms with viral RNA being detected widespread in the blood, brain, spleen, liver and ovaries. Histological changes were also striking in these animals. 129Sv/Ev mice developed no clinical symptoms or histological changes, despite viral RNA being detectable in the blood, spleen and ovaries, albeit at lower levels to those seen in A129 mice. Our results identify A129 mice as being highly susceptible to ZIKV and thus a suitable small animal model for the testing of vaccines and antivirals which are urgently required.

International audience; Several outbreaks of human West Nile virus (WNV) infections were reported in Tunisia during the last two decades. Serological studies on humans as well as on equine showed intensive circulation of WNV in Tunisia. However, no virus screening of mosquitoes for WNV has been performed in Tunisia. In the present study, we collected mosquito samples from Central Tunisia to be examined for the presence of flaviviruses. A total of 102 Culex pipiens mosquitoes were collected in September 2014 from Central Tunisia. Mosquitoes were pooled according to the collection site, date and sex with a maximum of 5 specimens per pool and tested for the presence of flaviviruses by conventional reverse transcription heminested PCR and by a specific West Nile virus real time reverse transcription PCR. Of a total of 21 pools tested, 7 were positive for WNV and no other flavivirus could be evidenced in mosquito pools. In addition, WNV was isolated on Vero cells. Phylogenetic analysis showed that recent Tunisian WNV strains belong to lineage 1 WNV and are closely related to the Tunisian strain 1997 (PAH 001). This is the first detection and isolation of WNV from mosquitoes in Tunisia. Some areas of Tunisia are at high risk for human WNV infections. WNV is likely to cause future sporadic and foreseeable outbreaks. Therefore, it is of major epidemiological importance to set up an entomological surveillance as an early alert system. Timely detection of WNV should prompt vector control to prevent future outbreaks. In addition, education of people to protect themselves from mosquito bites is of major epidemiological importance as preventive measure against WNV infection.

Background: Hazara virus (HAZV) is a member of the Bunyaviridae family of segmented negative stranded RNA viruses, and shares the same serogroup as Crimean-Congo haemorrhagic fever virus (CCHFV). CCHFV is responsible for fatal human disease with a mortality rate approaching 30 %, which has an increased recent incidence within southern Europe. There are no preventative or therapeutic treatments for CCHFV-mediated disease, and thus CCHFV is classified as a hazard group 4 pathogen. In contrast HAZV is not associated with serious human disease, although infection of interferon receptor knockout mice with either CCHFV or HAZV results in similar disease progression. To characterise further similarities between HAZV and CCHFV, and support the use of HAZV as a model for CCHFV infection, we investigated the structure of the HAZV nucleocapsid protein (N) and compared it to CCHFV N. N performs an essential role in the viral life cycle by encapsidating the viral RNA genome, and thus, N represents a potential therapeutic target. Results: We present the purification, crystallisation and crystal structure of HAZV N at 2.7 Å resolution. HAZV N was expressed as an N-terminal glutathione S-transferase (GST) fusion protein then purified using glutathione affinity chromatography followed by ion-exchange chromatography. HAZV N crystallised in the P212121 space group with unit cell parameters a = 64.99, b = 76.10, and c = 449.28 Å. HAZV N consists of a globular domain formed mostly of alpha helices derived from both the N- and C-termini, and an arm domain comprising two long alpha helices. HAZV N has a similar overall structure to CCHFV N, with their globular domains superposing with an RMSD = 0.70 Å, over 368 alpha carbons that share 59 % sequence identity. Four HAZV N monomers crystallised in the asymmetric unit, and their head-to-tail assembly reveals a potential interaction site between monomers. Conclusions: The crystal structure of HAZV N reveals a close similarity to CCHFV N, supporting the use of HAZV as a model for CCHFV. Structural similarity between the N proteins should facilitate study of the CCHFV and HAZV replication cycles without the necessity of working under containment level 4 (CL-4) conditions.

To further investigate mechanisms of protective immunity that are induced by live, attenuated simian immunodeficiency virus (SIV), three macaques were infected with SIVmacGX2, a nef-disrupted molecular clone. In two of these animals, which expressed the MamuA*01 major histocompatibility complex class I allele, loss of functional activity against an SIV-Gag-encoded immunodominant cytotoxic T lymphocyte (CTL) epitope was observed following prolonged infection. Nonetheless, all three animals were resistant to challenge with an uncloned pool of wild-type SIVmac, whereas four naïve controls became infected. Tetramer staining revealed the rapid generation of CD8+ T-cell responses against gag- and tat-encoded immunodominant epitopes in MamuA*01+ challenge controls. The dynamics of these T-cell responses to the wild-type virus were similar to those observed following primary infection of the vaccine group with attenuated virus. In contrast, neither tetramer staining nor gamma interferon ELISpot assay revealed an immediate, systemic, anamnestic response in the wild-type-challenged, attenuated SIV-infected animals. Functional CTL capacity had not been lost in this group, as lytic activity was still evident 17 weeks after challenge. Both attenuated and wild-type viruses induced a disseminated CD8+ T-cell response, which was of a higher magnitude in lymphoid tissues than in the periphery. These results suggest that, at least as measured in the periphery, protection against wild-type infection that is induced by live, attenuated SIV is not dependent on a rechallenge-driven expansion of immunodominant epitope-specific CD8+ T cells and, therefore, pre-existing activity may be sufficient to prevent superinfection.

Crimean-Congo Haemorrhagic Fever (CCHF) is a severe tick-borne disease, endemic in many countries in Africa, the Middle East, Eastern Europe and Asia. Between 15–70% of reported cases are fatal. There is no approved vaccine available, and preclinical protection in vivo by an experimental vaccine has not been demonstrated previously. In the present study, the attenuated poxvirus vector, Modified Vaccinia virus Ankara, was used to develop a recombinant candidate vaccine expressing the CCHF virus glycoproteins. Cellular and humoral immunogenicity was confirmed in two mouse strains, including type I interferon receptor knockout mice, which are susceptible to CCHF disease. This vaccine protected all recipient animals from lethal disease in a challenge model adapted to represent infection via a tick bite. Histopathology and viral load analysis of protected animals confirmed that they had been exposed to challenge virus, even though they did not exhibit clinical signs. This is the first demonstration of efficacy of a CCHF vaccine.

Crimean-Congo Haemorrhagic Fever (CCHF) is a severe tick-borne disease, endemic in many countries in Africa, the Middle East, Eastern Europe and Asia. There is no approved vaccine currently available against CCHF. The most promising candidate, which has previously been shown to confer protection in the small animal model, is a modified Vaccinia Ankara virus vector expressing the CCHF viral glycoprotein (MVA-GP). It has been shown that MVA-GP induces both humoral and cellular immunogenicity. In the present study, sera and T-lymphocytes were passively and adoptively transferred into recipient mice prior to challenge with CCHF virus. Results demonstrated that mediators from both arms of the immune system were required to demonstrate protective effects against lethal challenge.

Crimean-Congo hemorrhagic fever (CCHF) is a virus transmitted predominantly by ticks. However, contact with infected body fluids or tissues can result in animal-to-human or human-to-human transmission. Numbers of CCHF cases appear to be increasing, especially in Europe. We reviewed cases admitted to a tertiary referral unit in Kosova with suspected CCHF in 2008 and 2009, and looked at a smaller number of specimens which were sent to the Health Protection Agency, Porton Down, U.K., in further detail. The clinical features of cases admitted with suspected CCHF infection were assessed in more detail, and these are the focus of this article. Between 2008 and 2009, the numbers of patients admitted for suspected CCHF infection increased. Of the samples received in Porton Down, CCHF virus was detected in urine samples, and these patients were found to have prolonged viremia. The detection of CCHF in urine, as well as the prolonged viremias seen, are important for clinicians to know, as they may have public health implications with regard to the risk of infection, as well as provide insights into the biology and pathophysiology of infection. Further studies are required regarding the pathogenesis of this virus.

Hazara virus (HAZV) is closely related to the Crimean–Congo hemorrhagic fever virus (CCHFV). HAZV has not been reported to cause human disease; work with infectious material can be carried out at containment level (CL)-2. By contrast, CCHFV causes a haemorrhagic fever in humans and requires CL-4 facilities. A disease model of HAZV infection in mice deficient in the type I interferon receptor is reported in this study. Dose–response effects were seen with higher doses, resulting in a shorter time to death and earlier detection of viral loads in organs. The lowest dose of 10 p.f.u. was still lethal in over 50 % of the mice. Histopathological findings were identified in the liver, spleen and lymph nodes, with changes similar to a recent mouse model of CCHFV infection. The findings demonstrate that inoculation of mice with HAZV may act as a useful surrogate model for the testing of antiviral agents against CCHFV.

Recombinant nucleoprotein from Crimean-Congo Haemorrhagic Fever (CCHF) virus was successfully derived from a baculovirus expression system and purified for use in a novel enzyme-linked immunosorbent assay (ELISA) diagnostic test. Comparable tests were used for detection of IgG and IgM antibodies, thus allowing efficient detection of both antibodies in parallel. The major benefits of the assay also included removing any requirement for polyclonal sera, thus eliminating variation in preparations and allowing standardisation between laboratories. The assay was successfully tested using a panel of positive sera supplied from samples identified as being positive in Turkey, Tajikistan and Kosovo and shown to be sensitive and specific. It is envisaged that this simple diagnostic ELISA for CCHF virus infection which removes the reliance on polyclonal antibody preparations, will be accessible to a wider range of laboratories enabling them to carry out routine diagnosis. This will improve the efficiency of diagnosis and subsequent management of infected patients.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease associated with a high case fatality rate and transmitted mainly by Hyalomma marginatum . The geographical distribution of H. marginatum covers most of the Western Mediterranean basin. We aimed to investigate whether CCHF virus (CCHFv) is circulating in Tunisia. Samples from unexplained acute febrile patients (n = 181) and a high risk group of humans, mainly slaughter workers (n = 38), were collected in the summer of 2014 and analyzed for exposure to CCHFv using serological tests and real-time RT-PCR. Ticks were collected from Northern and Southern Tunisia during May–June 2014 and examined for the presence of CCHFv by real-time RT-PCR. Of the 181 febrile patients, 5 showed only high titers of IgM suggesting a recent exposure to CCHFv. Among 38 slaughter workers, 2 had IgG anti-CCHFv responses yielding a seroprevalence of 5.2%. No CCHFv was detected in ticks and sera. Our results provide evidence of human exposure to CCHFv in Tunisia.

Cells isolated directly from tissues (primary cultures) have many applications in research and applications in the fields of toxicology, pharmacology, and virology. Where such preparations remain the only scientific option to achieve necessary results it is still possible to refine and reduce the use of animal and human sources of the tissue by cryopreservation of the primary cells for later use, this reduces the need for fresh tissues and enables improvements in standardization through the ability to provide the same cell preparation at different times and to different laboratories. The methods described provide options for adherent cultures as monolayers, harvested cell suspensions, and also lymphocytes isolated from peripheral blood.

In light of the recent outbreak of Ebola virus (EBOV) disease in West Africa, there have been renewed efforts to search for effective antiviral countermeasures. A range of compounds currently available with broad antimicrobial activity have been tested for activity against EBOV. Using live EBOV, eighteen candidate compounds were screened for antiviral activity in vitro. The compounds were selected on a rational basis because their mechanisms of action suggested that they had the potential to disrupt EBOV entry, replication or exit from cells or because they had displayed some antiviral activity against EBOV in previous tests. Nine compounds caused no reduction in viral replication despite cells remaining healthy, so they were excluded from further analysis (zidovudine; didanosine; stavudine; abacavir sulphate; entecavir; JB1a; Aimspro; celgosivir; and castanospermine). A second screen of the remaining compounds and the feasibility of appropriateness for in vivo testing removed six further compounds (ouabain; omeprazole; esomeprazole; Gleevec; D-LANA-14; and Tasigna). The three most promising compounds (17-DMAG; BGB324; and NCK-8) were further screened for in vivo activity in the guinea pig model of EBOV disease. Two of the compounds, BGB324 and NCK-8, showed some effect against lethal infection in vivo at the concentrations tested, which warrants further investigation. Further, these data add to the body of knowledge on the antiviral activities of multiple compounds against EBOV and indicate that the scientific community should invest more effort into the development of novel and specific antiviral compounds to treat Ebola virus disease.