- Volume 1, Issue 10, 2019

Tick borne fever (TBF) caused by Anaplasma phagocytophilum (Ap), and louping ill caused by the flavivirus louping ill virus (LIV) are the two most economically important vector-borne diseases in UK sheep populations. Both pathogens are transmitted by the tick Ixodes ricinus, which also harbours protozoan parasites and Borrelia spp. spirochetes. I. ricinus ticks may be co-infected with multiple microorganisms and potentially transmit more than one pathogen to hosts during blood feeding.

Ap infection is not limited to sheep, causing pasture fever in cattle and granulocytic anaplasmosis in horses, dogs and humans. There is no vaccine available for TBF, and disease control relies on tick control and antibiotic treatment. LIV mainly infects ruminants, but can also infect other livestock including horses, pigs, alpacas and llamas. Since 2016, the LIV vaccine has been unavailable and there is no alternative prophylactic treatment for livestock. Both Ap and LIV are zoonotic diseases with occasional human cases reported in the UK.

Importantly, Ap infection leads to host immunosuppression and consequently increases vulnerability to secondary infections. Both Ap and LIV have been studied as single infections in tick and mammalian cells. However the dynamics and implications of co-infections within the arthropod vector or mammalian host have, to date, not been fully explored.

Using embryo-derived Ixodes spp. cell lines infected with Langat virus (a BSL-2 model for LIV) and Ap, we examined the effect of co-infection on viral RNA replication by qRT-PCR and bacterial growth by qPCR. The results of this study will be presented and discussed.

Japanese Encephalitis virus (JEV) is a zoonotic flavivirus that represents the most significant etiology of childhood viral neurological infections throughout the Asia. During the last 20 years, JEV genotype dominance has shifted from genotype III (GIII) to genotype I (GI). To date, the exact mechanism of this displacement is still not known. Culex (Cx.) mosquitoes are the most common species in China and play an essential role in maintaining JEV enzootic transmission cycle. In this study, we used Cx. pipiens mosquitoes from China as an in vivo mosquito model to explore if mosquitoes played a potential role in JEV genotype shift. We exposed female Cx. pipiens mosquitoes orally to either GI or GIII JEV strains. Midgut, whole mosquitoes, secondary organs, and salivary glands of JEV-infected mosquitoes were collected at 7 and 14 days of post infection (dpi) and subjected to measure the infection rate, replication kinetics, dissemination rate and transmission potential of the infected JEV strains in Cx. pipiens mosquitoes by 50% tissue culture infective dose assay. We found that Cx. pipiens mosquito was competent vector for both GI and GIII JEV infection, with similar infection rates and growth kinetics. After the establishment of infection, Cx. pipiens mosquitoes disseminated both JEV genotypes to secondary organs at similar rates of dissemination. A few GI-infected mosquito salivary glands (16.2%) were positive for GI virus, whereas GIII virus was undetectable in GIII-infected mosquito salivary glands at 7 dpi. However, 29.4% (5/17) and 36.3% (8/22) were positive for GI- and GIII-infected mosquito salivary glands at 14 dpi, respectively, showing an increase in JEV positive rate. No statistical difference in the transmission rate between GI- and GIII-infected mosquitoes was detected. Our experiment data demonstrated that GI and GIII viruses have similar infectivity in Cx. pipiens mosquitoes, suggesting that Cx. pipiens mosquitoes from China may not play a critical role in JEV genotype shift. Although the current data were obtained solely from Cx. pipiens mosquitoes, it is likely that the conclusion drawn could be extrapolated to the role of mosquitoes in JEV genotype shift.

Japanese Encephalitis virus (JEV) is a zoonotic flavivirus that represents the most significant etiology of childhood viral neurological infections throughout the Asia. During the last 20 years, JEV genotype dominance has shifted from genotype III (GIII) to genotype I (GI). To date, the exact mechanism of this displacement is still not known. Culex (Cx.) mosquitoes are the most common species in China and play an essential role in maintaining JEV enzootic transmission cycle. In this study, we used Cx. pipiens mosquitoes from China as an in vivo mosquito model to explore if mosquitoes played a potential role in JEV genotype shift. We exposed female Cx. pipiens mosquitoes orally to either GI or GIII JEV strains. Midgut, whole mosquitoes, secondary organs, and salivary glands of JEV-infected mosquitoes were collected at 7 and 14 days of post infection (dpi) and subjected to measure the infection rate, replication kinetics, dissemination rate and transmission potential of the infected JEV strains in Cx. pipiens mosquitoes by 50% tissue culture infective dose assay. We found that Cx. pipiens mosquito was competent vector for both GI and GIII JEV infection, with similar infection rates and growth kinetics. After the establishment of infection, Cx. pipiens mosquitoes disseminated both JEV genotypes to secondary organs at similar rates of dissemination. A few GI-infected mosquito salivary glands (16.2%) were positive for GI virus, whereas GIII virus was undetectable in GIII-infected mosquito salivary glands at 7 dpi. However, 29.4% (5/17) and 36.3% (8/22) were positive for GI- and GIII-infected mosquito salivary glands at 14 dpi, respectively, showing an increase in JEV positive rate. No statistical difference in the transmission rate between GI- and GIII-infected mosquitoes was detected. Our experiment data demonstrated that GI and GIII viruses have similar infectivity in Cx. pipiens mosquitoes, suggesting that Cx. pipiens mosquitoes from China may not play a critical role in JEV genotype shift. Although the current data were obtained solely from Cx. pipiens mosquitoes, it is likely that the conclusion drawn could be extrapolated to the role of mosquitoes in JEV genotype shift.

Mosquitoes harbor a diversity of viruses and are responsible for several mosquito-borne viral diseases of humans and animals, thereby leading to major public health concerns and significant economic losses across the globe. The viral metagenomics offers a great opportunity for bulk analysis of viral genomes retrieved directly from environmental samples. In this study, we performed a viral metagenomic analysis of five pools of mosquitoes belonging to Aedes, Anopheles and Culex species, collected from different pig farms in the vicinity of Shanghai, China to explore the viral community carried by mosquitoes. The resulting metagenomic data revealed that viral community in the mosquitoes was highly diverse and varied in abundance among pig farms, which comprised of more than 48 viral taxonomic families, specific to vertebrates, invertebrates, plants, fungi, bacteria, and protozoa. The read sequences related to animal viruses included parvoviruses, anelloviruses, circoviruses, flavivirus, rhabdovirus, and seadornaviruses, which might be taken by mosquitoes from viremic animal hosts during blood feeding. Notably, sample G1 contained the most abundant sequence related to Banna virus, which is of public health interest because it causes encephalitis in humans. Furthermore, non-classified viruses also shared considerable virus sequences in all the samples, presumably belonging to unexplored virus category. Overall, the present study provides a comprehensive knowledge of diverse viral populations carried by mosquitoes at pig farms, which is a potential source of diseases for mammals including humans and animals. These viral metagenomic data are valuable for assessment of emerging and re-emerging viral epidemics.

Mosquitoes harbor a diversity of viruses and are responsible for several mosquito-borne viral diseases of humans and animals, thereby leading to major public health concerns and significant economic losses across the globe. The viral metagenomics offers a great opportunity for bulk analysis of viral genomes retrieved directly from environmental samples. In this study, we performed a viral metagenomic analysis of five pools of mosquitoes belonging to Aedes, Anopheles and Culex species, collected from different pig farms in the vicinity of Shanghai, China to explore the viral community carried by mosquitoes. The resulting metagenomic data revealed that viral community in the mosquitoes was highly diverse and varied in abundance among pig farms, which comprised of more than 48 viral taxonomic families, specific to vertebrates, invertebrates, plants, fungi, bacteria, and protozoa. The read sequences related to animal viruses included parvoviruses, anelloviruses, circoviruses, flavivirus, rhabdovirus, and seadornaviruses, which might be taken by mosquitoes from viremic animal hosts during blood feeding. Notably, sample G1 contained the most abundant sequence related to Banna virus, which is of public health interest because it causes encephalitis in humans. Furthermore, non-classified viruses also shared considerable virus sequences in all the samples, presumably belonging to unexplored virus category. Overall, the present study provides a comprehensive knowledge of diverse viral populations carried by mosquitoes at pig farms, which is a potential source of diseases for mammals including humans and animals. These viral metagenomic data are valuable for assessment of emerging and re-emerging viral epidemics.

The expression of Japanese encephalitis virus (JEV) envelope gene in green cos leaf was a project undertaken at the John Innes Centre during 6 September to 20 October 2018 and is one of the projects of the research training program at the John Innes Centre during long vacation every year since 2010 until now.

The summary of this research is as follows. The production of the envelope protein of the JEV in green cos by using the envelope gene expression by 8 epitopes (MEP: multiepitope) of Japanese encephalitis virus vaccine strain SA14-14-2 that provides the highest immune response against JEV. After incorporated the envelope gene into pEAQ-HT and pEAQ-HT-HBcAg-tEL vectors, we cloned genes in Escherichia coli and Agrobacterium tumefaciens, respectively, then expressed the envelope protein of JEV in green cos leaves. The result can be seen in photographs of green cos leaves by both visible light camera and ultraviolet light camera.

Further research should include the analysis and identification by chemiluminescence immunoassay and by the Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF). Further purification of the envelope protein expression of JEV in green cos that will be benefited for the production of the envelope protein of the JEV plant-based vaccine.

Key words: Japanese encephalitis virus, plant-based vaccine, chemiluminescence immunoassay, MALDI-TOF

Zika virus infection was recently linked to microcephaly and peripheral neuropathy (GBS) in Zika virus epidemic areas. Building on our previous work (Cumberworth et al, 2017) we investigated, in a time-course study, how the viral infection and the injury of cell processes of oligodendrocytes and neurons are related to each other in the same in vitro model.

We generated CNS myelinating cultures from a reporter mouse (Thy1-YFP) on the Ifnar1 -/-background. A proportion of neurons and their processes are positive for YFP in those cultures, which enabled us to visualise single neurons. Cultures were infected with the Brazilian Zika virus strain (PE243) at an MOI of 0.3 and cultured for up to 6 days post infection. We observed that the neuronal cell processes were affected as early as the appearance of the first clusters of infected glial cells.

To analyse the interrelation of neurons and myelin further, cultures were labeled with an antibody recognising proteolipid protein. We found that the myelin got injured as early as neuronal processes. These results suggest that the injury to neuronal processes might be a consequence of the infection of the primary target of Zika virus: oligodendroglia.

These data help us to understand disease pathogenesis of Zika virus infection of the CNS, and whether there is a time-window to intervene therapeutically. Furthermore, this gives an insight as to how viral infection of glial cells can affect neuronal processes such as axons.

Zika virus infection was recently linked to microcephaly and peripheral neuropathy (GBS) in Zika virus epidemic areas. Building on our previous work (Cumberworth et al, 2017) we investigated, in a time-course study, how the viral infection and the injury of cell processes of oligodendrocytes and neurons are related to each other in the same in vitro model.

We generated CNS myelinating cultures from a reporter mouse (Thy1-YFP) on the Ifnar1 -/-background. A proportion of neurons and their processes are positive for YFP in those cultures, which enabled us to visualise single neurons. Cultures were infected with the Brazilian Zika virus strain (PE243) at an MOI of 0.3 and cultured for up to 6 days post infection. We observed that the neuronal cell processes were affected as early as the appearance of the first clusters of infected glial cells.

To analyse the interrelation of neurons and myelin further, cultures were labeled with an antibody recognising proteolipid protein. We found that the myelin got injured as early as neuronal processes. These results suggest that the injury to neuronal processes might be a consequence of the infection of the primary target of Zika virus: oligodendroglia.

These data help us to understand disease pathogenesis of Zika virus infection of the CNS, and whether there is a time-window to intervene therapeutically. Furthermore, this gives an insight as to how viral infection of glial cells can affect neuronal processes such as axons.

Developmental vaccines against emerging pathogens face many hurdles including determining what protective level of serological responses must be generated. Knowledge of a likely protective titre is critical where human challenge studies are not possible.

We have used an anti-zika plasma pool from convalescent patients (candidate serological reference reagent NIBSC16/320-14), infused into cynomolgus macaques and Type-1 IFN deficient mice to determine likely protective neutralising titres.

Anti-zika plasma was administered to a group of 4 macaques (single concentration) and groups of 8 A129 mice (4 group titration series) 24 hours prior to sub-cutaneous challenge with Zika virus PRVABC59. Plasma/sera samples were collected at regular intervals to track peripheral viremia and anti-zika antibody responses. FFPE tissues were collected at termination for histological analysis.

Immediately prior to challenge, human IgG was detectable in all infused animals. Within macaques the NT50 at this time was 250. All macaques that received plasma were protected against zika virus infection as determined by plasma/tissue qRT-PCR and IgM responses.

Titration within A129 mice gave a neutralisation titre of 110, above which mice were generally protected against zika infection. However this was not absolute as a small number of mice with high neutralisation levels were infected.

A protective NT50 of 250 has been identified for macaques and this further titrated in A129 mice to give a guide protective neutralisation titre of 110. The lack of protection in some mice with higher titres is currently unclear and studies are underway to compare their infection pathology with that of controls.

Developmental vaccines against emerging pathogens face many hurdles including determining what protective level of serological responses must be generated. Knowledge of a likely protective titre is critical where human challenge studies are not possible.

We have used an anti-zika plasma pool from convalescent patients (candidate serological reference reagent NIBSC16/320-14), infused into cynomolgus macaques and Type-1 IFN deficient mice to determine likely protective neutralising titres.

Anti-zika plasma was administered to a group of 4 macaques (single concentration) and groups of 8 A129 mice (4 group titration series) 24 hours prior to sub-cutaneous challenge with Zika virus PRVABC59. Plasma/sera samples were collected at regular intervals to track peripheral viremia and anti-zika antibody responses. FFPE tissues were collected at termination for histological analysis.

Immediately prior to challenge, human IgG was detectable in all infused animals. Within macaques the NT50 at this time was 250. All macaques that received plasma were protected against zika virus infection as determined by plasma/tissue qRT-PCR and IgM responses.

Titration within A129 mice gave a neutralisation titre of 110, above which mice were generally protected against zika infection. However this was not absolute as a small number of mice with high neutralisation levels were infected.

A protective NT50 of 250 has been identified for macaques and this further titrated in A129 mice to give a guide protective neutralisation titre of 110. The lack of protection in some mice with higher titres is currently unclear and studies are underway to compare their infection pathology with that of controls.

Arthropod borne diseases are ubiquitously discussed topic whose relevance increases with ongoing changing climate, which extends the area of their incidence and affects profoundly the size of vector population, as well as its reproductive capacity, the abundance and spread of reservoir hosts and other variables that are generally tightly correlated with the spread of zoonoses.

The life cycle of arthropod borne pathogens are tightly bound to the life cycle of their host as well as to their vector organism. Therefore, the description of the vector life cycle should elucidate some questions related to the vector-pathogen dynamics including the factors important for successful disease transmission.

Tick has complex life cycle and for its completion it requires feeding on several host organisms, which is abused by the pathogen for its spreading within reservoir and host organisms.

Thus, more thorough description of factors driving tick developmental processes controlling its life cycle will be instrumental in understanding the nature of I. ricinus and its pathogens interactions and may also shed light on the process of blood feeding as an integral event in tick development as well as in potential pathogen transmission.

In our study we perform transcriptional profiling of all life stages of I. ricinus and provide a list of genes associated with particular life stages of I. ricinus and hence extend our knowledge in pursuit of potential acaricidal strategies.

Arthropod borne diseases are ubiquitously discussed topic whose relevance increases with ongoing changing climate, which extends the area of their incidence and affects profoundly the size of vector population, as well as its reproductive capacity, the abundance and spread of reservoir hosts and other variables that are generally tightly correlated with the spread of zoonoses.

The life cycle of arthropod borne pathogens are tightly bound to the life cycle of their host as well as to their vector organism. Therefore, the description of the vector life cycle should elucidate some questions related to the vector-pathogen dynamics including the factors important for successful disease transmission.

Tick has complex life cycle and for its completion it requires feeding on several host organisms, which is abused by the pathogen for its spreading within reservoir and host organisms.

Thus, more thorough description of factors driving tick developmental processes controlling its life cycle will be instrumental in understanding the nature of I. ricinus and its pathogens interactions and may also shed light on the process of blood feeding as an integral event in tick development as well as in potential pathogen transmission.

In our study we perform transcriptional profiling of all life stages of I. ricinus and provide a list of genes associated with particular life stages of I. ricinus and hence extend our knowledge in pursuit of potential acaricidal strategies.

Lumpy skin disease (LSD) is a tropical neglected viral disease of cattle, characterised by numerous cutaneous lesions disseminated throughout the body. Historically endemic to the African continent, it has become a threat to Europe following the outbreaks of LSD in the Middle East and Eastern Europe.

LSD virus (LSDV) is a Capripoxvirus transmitted by insect vectors. Experimental and epidemiological studies have indicated a role for the stable fly (Stomoxys calcintrans) and the mosquito Aedes aegypti. Nevertheless the relative importance of these vector species and others is unclear. A study was designed to explore the risk of transmission of LSDV from cattle to different vector species including Aedes aegypti, Culex quinquefasciatus, Stomoxys calcitrans and Culicoides nubeculosus. Cattle was challenged with LSDV to produce a bovine experimental model used as a natural source of LSDV to the potential vectors. Cattle samples were taken to quantify LSDV in different tissues and characterise the disease. All insect species were allowed to feed on LSDV-challenged cattle at regular intervals and incubated for up to eight days. This data was then used to model the dynamics of LSDV infection and transmission. All four species were able to acquire and maintain LSDV for up to eight days post feeding, and the risk of transmission from bovine donor to insect was dependent on the severity of the disease. A model was then generated using ex-vivo skin lesions and infectious blood that will allow further studies of the role of these vectors.

Lumpy skin disease (LSD) is a tropical neglected viral disease of cattle, characterised by numerous cutaneous lesions disseminated throughout the body. Historically endemic to the African continent, it has become a threat to Europe following the outbreaks of LSD in the Middle East and Eastern Europe.

LSD virus (LSDV) is a Capripoxvirus transmitted by insect vectors. Experimental and epidemiological studies have indicated a role for the stable fly (Stomoxys calcintrans) and the mosquito Aedes aegypti. Nevertheless the relative importance of these vector species and others is unclear. A study was designed to explore the risk of transmission of LSDV from cattle to different vector species including Aedes aegypti, Culex quinquefasciatus, Stomoxys calcitrans and Culicoides nubeculosus. Cattle was challenged with LSDV to produce a bovine experimental model used as a natural source of LSDV to the potential vectors. Cattle samples were taken to quantify LSDV in different tissues and characterise the disease. All insect species were allowed to feed on LSDV-challenged cattle at regular intervals and incubated for up to eight days. This data was then used to model the dynamics of LSDV infection and transmission. All four species were able to acquire and maintain LSDV for up to eight days post feeding, and the risk of transmission from bovine donor to insect was dependent on the severity of the disease. A model was then generated using ex-vivo skin lesions and infectious blood that will allow further studies of the role of these vectors.

Arthropod-borne viruses are able to infect vertebrate and invertebrate hosts. One such arbovirus is Zika virus (family Flaviviridae) that is mainly transmitted to humans by Aedes mosquitoes causing febrile illness and congenital Zika syndrome in infants. An interplay between host and virus proteins enables ZIKV to manipulate its host's cellular machineries in order to facilitate infection and evade antiviral responses. A possible mechanism it utilises is the ubiquitin-proteasome pathway (UPP) where target proteins are ubiquitinated and subsequently degraded by the proteasome. Results of proteomics analysis of Ae. aegypti cell lines (AF5) stably expressing V5-tagged ZIKV capsid (C), anchored capsid (AC) or non-structural 3 (NS3) proteins revealed that these viral proteins interact with effector proteins of the UPP. One of these proteins is TER94 an AAA-ATPase that acts as a chaperone segregating ubiquitinated proteins to the proteasome complex. Knockdown experiments of TER94 or its human ortholog VCP using dsRNA or siRNAs showed reduced virus replication in AF5 or A549 cells. Using small molecule inhibitors of UPP proteins also diminished ZIKV replication. Inhibiting different stages of the pathway have identified critical steps during early stages of infection. The ubiquitination of lysine-rich ZIKV C and its interaction with TER94/VCP could be one of the many universal strategies that the virus employs when it switches between mosquito and human hosts. Understanding how ZIKV is able to infect both human and insect species could provide novel strategies for prevention and therapeutics.

Keywords: Zika virus, Aedes aegypti, ubiquitin-proteasome pathway, mosquito, virus-host interaction

Arthropod-borne viruses are able to infect vertebrate and invertebrate hosts. One such arbovirus is Zika virus (family Flaviviridae) that is mainly transmitted to humans by Aedes mosquitoes causing febrile illness and congenital Zika syndrome in infants. An interplay between host and virus proteins enables ZIKV to manipulate its host's cellular machineries in order to facilitate infection and evade antiviral responses. A possible mechanism it utilises is the ubiquitin-proteasome pathway (UPP) where target proteins are ubiquitinated and subsequently degraded by the proteasome. Results of proteomics analysis of Ae. aegypti cell lines (AF5) stably expressing V5-tagged ZIKV capsid (C), anchored capsid (AC) or non-structural 3 (NS3) proteins revealed that these viral proteins interact with effector proteins of the UPP. One of these proteins is TER94 an AAA-ATPase that acts as a chaperone segregating ubiquitinated proteins to the proteasome complex. Knockdown experiments of TER94 or its human ortholog VCP using dsRNA or siRNAs showed reduced virus replication in AF5 or A549 cells. Using small molecule inhibitors of UPP proteins also diminished ZIKV replication. Inhibiting different stages of the pathway have identified critical steps during early stages of infection. The ubiquitination of lysine-rich ZIKV C and its interaction with TER94/VCP could be one of the many universal strategies that the virus employs when it switches between mosquito and human hosts. Understanding how ZIKV is able to infect both human and insect species could provide novel strategies for prevention and therapeutics.

Keywords: Zika virus, Aedes aegypti, ubiquitin-proteasome pathway, mosquito, virus-host interaction

St Abbs Head virus (SAHV), member of Phlebovirus genus (family Phenuiviridae, order Bunyavirales), belongs to the largest group of negative strand RNA viruses. All phleboviruses share a genome structure that comprises three segments of negative-sense or ambi-sense RNA. The viral genome is composed of the small (S), medium (M) and large (L) RNA segments. The S segment encodes the nucleocapsid (N) protein, the M segment encodes the precursor for the viral glycoproteins (Gn and Gc) and the L segment encodes the viral RNA-dependent RNA polymerase (RdRp). Some viruses within the genus also encode non-structural proteins within their S or M segments.

SAHV was isolated from a pool of Ixodes uriae ticks collected at a seabird colony in Berwickshire, Scotland in 1979. There were quite a few related bunyaviruses found in tick and bird samples on the East Coast of Scotland and England in the 70s and 80s. Recently we have sequenced a sample of SAHV using next generation sequencing technology. The results suggested that this virus is very closely related to the Uukuniemi phlebovirus (UUKV). To determine how similar are SAHV and UUKV, we compared virus growth in various mammalian, bird and tick cell lines.

St Abbs Head virus (SAHV), member of Phlebovirus genus (family Phenuiviridae, order Bunyavirales), belongs to the largest group of negative strand RNA viruses. All phleboviruses share a genome structure that comprises three segments of negative-sense or ambi-sense RNA. The viral genome is composed of the small (S), medium (M) and large (L) RNA segments. The S segment encodes the nucleocapsid (N) protein, the M segment encodes the precursor for the viral glycoproteins (Gn and Gc) and the L segment encodes the viral RNA-dependent RNA polymerase (RdRp). Some viruses within the genus also encode non-structural proteins within their S or M segments.

SAHV was isolated from a pool of Ixodes uriae ticks collected at a seabird colony in Berwickshire, Scotland in 1979. There were quite a few related bunyaviruses found in tick and bird samples on the East Coast of Scotland and England in the 70s and 80s. Recently we have sequenced a sample of SAHV using next generation sequencing technology. The results suggested that this virus is very closely related to the Uukuniemi phlebovirus (UUKV). To determine how similar are SAHV and UUKV, we compared virus growth in various mammalian, bird and tick cell lines.

Background. Some of the best characterized alphaviruses are chikungunya virus (CHIKV), Semliki Forest virus (SFV) and Sindbis virus (SINV). E1 is a class II fusion protein, containing 3 domains (DI, DII, DIII) folded essentially as β-sheet, plus a stem region connecting DIII to the transmembrane (TM) segment. The fusion loop (FL) is at the tip of the elongated DII. The X-ray structure of the SFV E1 post-fusion trimer, truncated of the stem region, displayed a tripod-shape with the DII legs open and the FLs away from each other, contrary to the class II viral fusion proteins from other viral genera, in which the FLs interact at the tip of the post-fusion trimer. Here, we set to identify if the stem plays a structural role in zippering together the E1 trimer to bring the fusion loops into contact.

Methods. We produced the recombinant ectodomains of E1 of CHIKV, SINV and SFV containing or not the stem, crystallized them and determined the X-ray structure.

Results. We observed that CHIKV and SINV display E1 in closed conformation, in contrast to SFV, which displays a tripod even with the full stem. We identified a sequence motif in the stem responsible for the conformational difference.

Conclusion. Our results point to potential mechanistic differences between alphavirus E1 in driving fusion. Further functional studies are ongoing to pin-point the significance of these new findings, and the reasons for the alternative post-fusion conformations.