Tick-Borne Encephalitis Virus, United Kingdom.

During February 2018-January 2019, we conducted large-scale surveillance for the presence and prevalence of tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) in sentinel animals and ticks in the United Kingdom. Serum was collected from 1,309 deer culled across England and Scotland. Overall, 4% of samples were ELISA-positive for the TBEV serocomplex. A focus in the Thetford Forest area had the highest proportion (47.7%) of seropositive samples. Ticks collected from culled deer within seropositive regions were tested for viral RNA; 5 of 2,041 ticks tested positive by LIV/TBEV real-time reverse transcription PCR, all from within the Thetford Forest area. From 1 tick, we identified a full-length genomic sequence of TBEV. Thus, using deer as sentinels revealed a potential TBEV focus in the United Kingdom. This detection of TBEV genomic sequence in UK ticks has important public health implications, especially for undiagnosed encephalitis.

Hantavirus infection in type I interferon receptor-deficient (A129) mice.

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 the in vivo evaluation of interventions.

Lineage-dependent differences of Zika virus infection in a susceptible mouse model are associated with different profiles of cytokines, chemokines, growth factors and acute phase proteins.

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.

Use and reliability of multiplex bead-based assays (Luminex) at Containment Level 4.

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.

Detection of new endemic focus of tick-borne encephalitis virus (TBEV), Hampshire/Dorset border, England, September 2019.

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.

Heat Shock Protein 70 Family Members Interact with Crimean-Congo Hemorrhagic Fever Virus and Hazara Virus Nucleocapsid Proteins and Perform a Functional Role in the Nairovirus Replication Cycle.

UNLABELLED: 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.

Expression and characterization of codon-optimized Crimean-Congo hemorrhagic fever virus Gn glycoprotein in insect cells.

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.

Chloroquine inhibited Ebola virus replication in vitro but failed to protect against infection and disease in the in vivo guinea pig model.

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.

The crystal structure of the Hazara virus nucleocapsid protein.

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.

Development of humanised antibodies for Crimean-Congo Haemorrhagic fever virus: Comparison of hybridoma-based versus phage library techniques.

Humanised antibodies targeting Crimean-Congo Haemorrhagic virus (CCHFV) are needed for the development and standardisation of serological assays. These assays are needed to address a shortfall in available tests that meet regulatory diagnostic standards and to aid surveillance activities to extend knowledge on the distribution of CCHFV. To generate a humanised monoclonal antibody against CCHFV, we have compared two methods: the traditional mouse hybridoma approach with subsequent sequencing and humanisation of antibodies versus a non-animal alternative using a human combinatorial antibody library (HuCAL). Our results demonstrated that the mouse hybridoma followed by humanisation protocol gave higher affinity antibodies. Whilst not yet able to demonstrate the generation of equivalent humanised antibodies without the use of animals, sequencing data enables the subsequent production of recombinant antibodies, thus providing a reduction in future animal usage for this application. Ultimately, our report provides information on development of a humanised standardised control, which can form an important positive control component of serological assays against CCHFV.

Use of a Preclinical Natural Transmission Model to Study Antiviral Effects of a Carbohydrate-Binding Module Therapy against SARS-CoV-2 in Hamsters.

The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV-2) and its expansion to a worldwide pandemic resulted in efforts to assess and develop interventions to reduce the disease burden. Despite the introduction of vaccine programmes against SARS-CoV-2, global incidence levels in early 2022 remained high, demonstrating a need for the development of physiologically relevant models, which are essential for the identification of alternative antiviral strategies. The hamster model of SARS-CoV-2 infection has been widely adopted due to similarities with humans in terms of host cell entry mechanism (via ACE2), and aspects of symptomology and virus shedding. We have previously described a natural transmission hamster model that better represents the natural course of infection. In the present study, we have conducted further testing of the model using the first-in-class antiviral Neumifil, which has previously shown promise against SARS-CoV-2 after a direct intranasal challenge. Neumifil is an intranasally delivered carbohydrate-binding module (CBM) which reduces the binding of viruses to their cellular receptor. By targeting the host cell, Neumifil has the potential to provide broad protection against multiple pathogens and variants. This study demonstrates that using a combination of a prophylactic and therapeutic delivery of Neumifil significantly reduces the severity of clinical signs in animals infected via a natural route of transmission and indicates a reduction of viral loads in the upper respiratory tract. Further refinements of the model are required in order to ensure the adequate transmission of the virus. However, our results provide additional data to the evidence base of Neumifil efficacy against respiratory virus infection and demonstrate that the transmission model is a potentially valuable tool for testing antiviral compounds against SARS-CoV-2.

Hazara virus infection is lethal for adult type I interferon receptor-knockout mice and may act as a surrogate for infection with the human-pathogenic Crimean-Congo hemorrhagic fever 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.

Heterologous prime-boost-boost immunisation of Chinese cynomolgus macaques using DNA and recombinant poxvirus vectors expressing HIV-1 virus-like particles.

BACKGROUND: There is renewed interest in the development of poxvirus vector-based HIV vaccines due to the protective effect observed with repeated recombinant canarypox priming with gp120 boosting in the recent Thai placebo-controlled trial. This study sought to investigate whether a heterologous prime-boost-boost vaccine regimen in Chinese cynomolgus macaques with a DNA vaccine and recombinant poxviral vectors expressing HIV virus-like particles bearing envelopes derived from the most prevalent clades circulating in sub-Saharan Africa, focused the antibody response to shared neutralising epitopes. METHODS: Three Chinese cynomolgus macaques were immunised via intramuscular injections using a regimen composed of a prime with two DNA vaccines expressing clade A Env/clade B Gag followed by boosting with recombinant fowlpox virus expressing HIV-1 clade D Gag, Env and cholera toxin B subunit followed by the final boost with recombinant modified vaccinia virus Ankara expressing HIV-1 clade C Env, Gag and human complement protein C3d. We measured the macaque serum antibody responses by ELISA, enumerated T cell responses by IFN-γ ELISpot and assessed seroneutralisation of HIV-1 using the TZM-bl ß-galactosidase assay with primary isolates of HIV-1. RESULTS: This study shows that large and complex synthetic DNA sequences can be successfully cloned in a single step into two poxvirus vectors: MVA and FPV and the recombinant poxviruses could be grown to high titres. The vaccine candidates showed appropriate expression of recombinant proteins with the formation of authentic HIV virus-like particles seen on transmission electron microscopy. In addition the b12 epitope was shown to be held in common by the vaccine candidates using confocal immunofluorescent microscopy. The vaccine candidates were safely administered to Chinese cynomolgus macaques which elicited modest T cell responses at the end of the study but only one out of the three macaques elicited an HIV-specific antibody response. However, the antibodies did not neutralise primary isolates of HIV-1 or the V3-sensitive isolate SF162 using the TZM-bl ß-galactosidase assay. CONCLUSIONS: MVA and FP9 are ideal replication-deficient viral vectors for HIV-1 vaccines due to their excellent safety profile for use in humans. This study shows this novel prime-boost-boost regimen was poorly immunogenic in Chinese cynomolgus macaques.

Towards quantification of protective antibody responses by passive transfer of the 1st WHO International Standard for Ebola virus antibody in a guinea pig model.

Ebola virus (EBOV) represents a major concern to global health due to the unpredictable nature of outbreaks. Infection with EBOV can cause a severe viral haemorrhagic fever with no licensed vaccine or treatment, restricting work with live EBOV to Containment/Biosafety Level 4 facilities. Whilst the magnitude of recent outbreaks has provided an impetus for vaccine and antiviral development, establishing the efficacy of candidate vaccine materials relies on EBOV challenge models and advanced human trials should outbreaks occur and where logistics and funding allow. To address these hurdles in vaccine development, we investigated whether a recently established serological reference standard, the 1st WHO International Standard for Ebola virus antibody, could be used to provide a quantifiable correlate of immune protection in vivo. Dilutions of the International Standard were inoculated into naïve guinea pigs 24 h before challenge with a lethal dose of Ebola virus. Only subjects receiving the highest dose of the International Standard exhibited evidence of delayed progression. Due to it being a WHO established reagent and available globally upon request, this standard allows for effective comparisons of data between laboratories and may prove valuable to select the candidate vaccines that are most likely to confer humoral immune protection ensuring the most promising candidates progress into efficacy studies.

Investigating the Cellular Transcriptomic Response Induced by the Makona Variant of Ebola Virus in Differentiated THP-1 Cells.

Recent studies have shown that transcriptomic analysis of blood samples taken from patients with acute Ebola virus disease (EVD) during the 2013-2016 West African outbreak was suggestive that a severe inflammatory response took place in acutely ill patients. The significant knowledge gained from studying the Makona variant, a cause of the largest known EVD outbreak, may be applicable to other species of ebolavirus, and other variants of the Ebola virus (EBOV) species. To investigate the ability of Makona to initiate an inflammatory response in human macrophages and characterise the host response in a similar manner to previously characterised EBOV variants, the human monocytic cell line THP-1 was differentiated into macrophage-like cells and infected with Makona. RNA-Seq and quantitative proteomics were used to identify and quantify host mRNA and protein abundance during infection. Data from infection with Reston virus (RESTV) were used as comparators to investigate changes that may be specific to, or enhanced in, Makona infection in relation to a less pathogenic species of ebolavirus.. This study found demonstrable induction of the inflammatory response, and increase in the activation state of THP-1 macrophages infected with Makona. NFκB and inflammation-associated transcripts displayed significant changes in abundance, reflective of what was observed in human patients during the 2013-2016 EBOV outbreak in West Africa, and demonstrated that transcriptomic changes found in Makona-infected cells were similar to that observed in Reston virus infection and that have been described in previous studies of other variants of EBOV.

The Development of a Novel Diagnostic Assay That Utilizes a Pseudotyped Vesicular Stomatitis Virus for the Detection of Neutralizing Activity against Crimean-Congo Hemorrhagic Fever Virus.

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.

A Protective Monoclonal Antibody Targets a Site of Vulnerability on the Surface of Rift Valley Fever Virus.

The Gn subcomponent of the Gn-Gc assembly that envelopes the human and animal pathogen, Rift Valley fever virus (RVFV), is a primary target of the neutralizing antibody response. To better understand the molecular basis for immune recognition, we raised a class of neutralizing monoclonal antibodies (nAbs) against RVFV Gn, which exhibited protective efficacy in a mouse infection model. Structural characterization revealed that these nAbs were directed to the membrane-distal domain of RVFV Gn and likely prevented virus entry into a host cell by blocking fusogenic rearrangements of the Gn-Gc lattice. Genome sequence analysis confirmed that this region of the RVFV Gn-Gc assembly was under selective pressure and constituted a site of vulnerability on the virion surface. These data provide a blueprint for the rational design of immunotherapeutics and vaccines capable of preventing RVFV infection and a model for understanding Ab-mediated neutralization of bunyaviruses more generally.

Development of vaccines against Crimean-Congo haemorrhagic fever virus.

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 40years, 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.

Lineage-dependent differences in the disease progression of Zika virus infection in type-I interferon receptor knockout (A129) mice.

Zika virus (ZIKV) falls into two lineages: African (ZIKVAF) and Asian (ZIKVAS). These lineages have not been tested comprehensively in parallel for disease progression using an animal model system. Here, using the established type-I interferon receptor knockout (A129) mouse model, it is first demonstrated that ZIKVAF causes lethal infection, with different kinetics of disease manifestations according to the challenge dose. Animals challenged with a low dose of 10 plaque-forming units (pfu) developed more neurological symptoms than those challenged with 5-log higher doses. By contrast, animals challenged with ZIKVAS displayed no clinical signs or mortality, even at doses of 106 pfu. However, viral RNA was detected in the tissues of animals infected with ZIKV strains from both lineages and similar histological changes were observed. The present study highlights strain specific virulence differences between the African and Asian lineages in a ZIKV mouse model.

Post-exposure treatment of non-human primates lethally infected with Ebola virus with EBOTAb, a purified ovine IgG product.

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.