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Significance It is important to understand antiviral mechanisms in potential new arbovirus vectors, such as Anopheles mosquitoes, in order to assess risks associated with arbovirus spread. Using an arbovirus naturally transmitted by Anopheles , we find that important immune mechanisms involved in the first bottleneck to Anopheles infection, the midgut, have distinct effects on arbovirus or malaria. This result is, to our knowledge, the first concrete evidence of protection tradeoffs for different human pathogens in a human disease vector, and it suggests that design of genetically immune-modified mosquitoes could result in unexpected outcomes. These results also indicate that different mosquito tissues display distinct antiviral protection that probably imposes divergent selection pressures upon viral replication during different stages of the infection. Arboviruses are transmitted by mosquitoes and other arthropods to humans and animals. The risk associated with these viruses is increasing worldwide, including new emergence in Europe and the Americas. Anopheline mosquitoes are vectors of human malaria but are believed to transmit one known arbovirus, o’nyong-nyong virus, whereas Aedes mosquitoes transmit many. Anopheles interactions with viruses have been little studied, and the initial antiviral response in the midgut has not been examined. Here, we determine the antiviral immune pathways of the Anopheles gambiae midgut, the initial site of viral infection after an infective blood meal. We compare them with the responses of the post-midgut systemic compartment, which is the site of the subsequent disseminated viral infection. Normal viral infection of the midgut requires bacterial flora and is inhibited by the activities of immune deficiency (Imd), JAK/STAT, and Leu-rich repeat immune factors. We show that the exogenous siRNA pathway, thought of as the canonical mosquito antiviral pathway, plays no detectable role in antiviral defense in the midgut but only protects later in the systemic compartment. These results alter the prevailing antiviral paradigm by describing distinct protective mechanisms in different body compartments and infection stages. Importantly, the presence of the midgut bacterial flora is required for full viral infectivity to Anopheles , in contrast to malaria infection, where the presence of the midgut bacterial flora is required for protection against infection. Thus, the enteric flora controls a reciprocal protection tradeoff in the vector for resistance to different human pathogens.

There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic. Vaccines for SARS-COV-2 are needed in the ongoing pandemic. Here the authors characterize a vaccine candidate that presents the receptor-binding domain (RBD) of SARS-CoV-2 spike protein on a synthetic VLP platform using SpyTag/SpyCatcher technology and show immunogenicity of a prime-boost regimen in mice and pigs.

SARS Coronavirus 2 (SARS-CoV-2) emerged in late 2019, leading to the Coronavirus Disease 2019 (COVID-19) pandemic that continues to cause significant global mortality in human populations. Given its sequence similarity to SARS-CoV, as well as related coronaviruses circulating in bats, SARS-CoV-2 is thought to have originated in Chiroptera species in China. However, whether the virus spread directly to humans or through an intermediate host is currently unclear, as is the potential for this virus to infect companion animals, livestock, and wildlife that could act as viral reservoirs. Using a combination of surrogate entry assays and live virus, we demonstrate that, in addition to human angiotensin-converting enzyme 2 (ACE2), the Spike glycoprotein of SARS-CoV-2 has a broad host tropism for mammalian ACE2 receptors, despite divergence in the amino acids at the Spike receptor binding site on these proteins. Of the 22 different hosts we investigated, ACE2 proteins from dog, cat, and cattle were the most permissive to SARS-CoV-2, while bat and bird ACE2 proteins were the least efficiently used receptors. The absence of a significant tropism for any of the 3 genetically distinct bat ACE2 proteins we examined indicates that SARS-CoV-2 receptor usage likely shifted during zoonotic transmission from bats into people, possibly in an intermediate reservoir. Comparison of SARS-CoV-2 receptor usage to the related coronaviruses SARS-CoV and RaTG13 identified distinct tropisms, with the 2 human viruses being more closely aligned. Finally, using bioinformatics, structural data, and targeted mutagenesis, we identified amino acid residues within the Spike–ACE2 interface, which may have played a pivotal role in the emergence of SARS-CoV-2 in humans. The apparently broad tropism of SARS-CoV-2 at the point of viral entry confirms the potential risk of infection to a wide range of companion animals, livestock, and wildlife.

We report a unique outbreak of Rift Valley fever in the Eldamar area, Sudan, May-July 2019, that resulted in 1,129 case-patients and 19 (1.7%) deaths. Patients exhibited clinical signs including fever (100%), headache (79%), and bleeding (4%). Most (98%) patients also reported death and abortions among their livestock.

Most vertebrate and plant RNA and small DNA viruses suppress genomic CpG and UpA dinucleotide frequencies, apparently mimicking host mRNA composition. Artificially increasing CpG/UpA dinucleotides attenuates viruses through an entirely unknown mechanism. Using the echovirus 7 (E7) model in several cell types, we show that the restriction in E7 replication in mutants with increased CpG/UpA dinucleotides occurred immediately after viral entry, with incoming virions failing to form replication complexes. Sequences of CpG/UpA-high virus stocks showed no evidence of increased mutational errors that would render them replication defective, these viral RNAs were not differentially sequestered in cytoplasmic stress granules nor did they induce a systemic antiviral state. Importantly, restriction was not mediated through effects on translation efficiency since replicons with high CpG/UpA sequences inserted into a non-coding region were similarly replication defective. Host-cells thus possess intrinsic defence pathways that prevent replication of viruses with increased CpG/UpA frequencies independently of codon usage. Living things store their genetic material as molecules of DNA or a related chemical called RNA. Both DNA and RNA contain building blocks known as bases. There are several different types of bases and the specific order they appear in a DNA or RNA molecule encodes the genetic information. In RNA these bases are known as cytosine, guanine, adenine and uracil (or C, G, A and U for short). The order that bases appear in DNA and RNA can be highly biased. For example, in RNAs from animals with backbones (also known as vertebrates), cytosine followed by guanine and uracil followed by adenine occur less often than mathematics would predict. Viruses are particles that contain DNA or RNA surrounded by a coat made of proteins. They are unable to multiply by themselves and must therefore invade the cells of host organisms. Viruses that infect vertebrates mimic the base biases found in their host, a strategy that likely helps the virus’ genetic material to hide within host cells. Previous experiments have shown that viruses engineered to have more cytosines followed by guanines and uracils followed by adenines were easier to eliminate. However, it is not clear how this worked. Fros et al. investigated the ability of a virus called echovirus 7 to multiply inside the cells of humans and several other vertebrates. The experiments show that artificially increasing the number of cytosines followed by guanines and uracils followed by adenines in this virus reduced the ability of the virus to multiply immediately after the virus had entered the host cell. The location of the changes did not have any effect on how strongly the virus was inhibited. Furthermore, Fros et al. confirmed that these changes did not affect the ability of the virus’ genetic material to make the proteins it needs to multiply and make its coat. This suggests that the host specifically prevents the virus genetic material from being copied, solely based on the order of the bases in the viral genetic material. These findings provide evidence that human and other vertebrate cells contain factors that recognize and rapidly respond to foreign genetic material with biases in their genetic code that do not match their own. In the future, artificially increasing the frequency of specific orders of bases in viral genomes could be used to design more effective vaccines against diseases caused by viruses.

The risk of emergence and/or re-emergence of arthropod-borne viral (arboviral) infections is rapidly growing worldwide, particularly in Africa. The burden of arboviral infections and diseases is not well scrutinized because of the inefficient surveillance systems in endemic countries. Furthermore, the health systems are fully occupied by the burden of other co-existing febrile illnesses, especially malaria. In this review we summarize the epidemiology and risk factors associated with the major human arboviral diseases and highlight the gap in knowledge, research, and control in Sudan. Published data in English up to March 2019 were reviewed and are discussed to identify the risks and challenges for the control of arboviruses in the country. In addition, the lack of suitable diagnostic tools such as viral genome sequencing, and the urgent need for establishing a genomic database of the circulating viruses and potential sources of entry are discussed. Moreover, the research and healthcare gaps and global health threats are analyzed, and suggestions for developing strategic health policy for the prevention and control of arboviruses with focus on building the local diagnostic and research capacity and establishing an early warning surveillance system for the early detection and containment of arboviral epidemics are offered.

Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic arboviral disease that poses a great threat to global health in the Old World, and it is endemic in Europe, Asia, and Africa, including Sudan. In this retrospective study, we reviewed previous epidemiological reports about the major epidemics of CCHF throughout Sudan between 2010 and 2020. During these epidemics, the infection of humans with Crimean-Congo hemorrhagic fever virus (CCHFV), the causative agent of CCHF, was diagnosed using qRT-PCR. We have identified 88 cases of CCHF, including 13 fatalities reported during five epidemics that occurred in 2010, 2011, 2015, 2019, and 2020. The two epidemics in 2010 and 2011 were by far the largest, with 51 and 27 cases reported, respectively. The majority of cases (78%) were reported in the endemic region of Kordofan. Here, we document that the first emergence of CCHFV in the Darfur region, West Sudan, occurred in 2010. We were not able to investigate outbreak dynamics through phylogenetic analysis due to the limited diagnostic capacity and the lack of sequencing services in the country. These findings call for establishing a genomic-based integrated One Health surveillance and response system for the early preparedness, prevention, and control of CCHF in the country.

Vector arthropods control arbovirus replication and spread through antiviral innate immune responses including RNA interference (RNAi) pathways. Arbovirus infections have been shown to induce the exogenous small interfering RNA (siRNA) and Piwi-interacting RNA (piRNA) pathways, but direct antiviral activity by these host responses in mosquito cells has only been demonstrated against a limited number of positive-strand RNA arboviruses. For bunyaviruses in general, the relative contribution of small RNA pathways in antiviral defences is unknown. The genus Orthobunyavirus in the Bunyaviridae family harbours a diverse range of mosquito-, midge- and tick-borne arboviruses. We hypothesized that differences in the antiviral RNAi response in vector versus non-vector cells may exist and that could influence viral host range. Using Aedes aegypti-derived mosquito cells, mosquito-borne orthobunyaviruses and midge-borne orthobunyaviruses we showed that bunyavirus infection commonly induced the production of small RNAs and the effects of the small RNA pathways on individual viruses differ in specific vector-arbovirus interactions. These findings have important implications for our understanding of antiviral RNAi pathways and orthobunyavirus-vector interactions and tropism.

No vaccine is currently available for dengue virus (DENV), therefore control programmes usually focus on managing mosquito vector populations. Entomological surveys provide the most common means of characterising vector populations and predicting the risk of local dengue virus transmission. Despite Indonesia being a country strongly affected by DENV, only limited information is available on the local factors affecting DENV transmission and the suitability of available survey methods for assessing risk. We conducted entomological surveys in the Banyumas Regency (Central Java) where dengue cases occur on an annual basis. Four villages were sampled during the dry and rainy seasons: two villages where dengue was endemic, one where dengue cases occurred sporadically and one which was dengue-free. In addition to data for conventional larvae indices, we collected data on pupae indices, and collected adult mosquitoes for species identification in order to determine mosquito species composition and population density. Traditionally used larval indices (House indices, Container indices and Breteau indices) were found to be inadequate as indicators for DENV transmission risk. In contrast, species composition of adult mosquitoes revealed that competent vector species were dominant in dengue endemic and sporadic villages. Our data suggested that the utility of traditional larvae indices, which continue to be used in many dengue endemic countries, should be re-evaluated locally. The results highlight the need for validation of risk indicators and control strategies across DENV affected areas here and perhaps elsewhere in SE Asia.

Mosquitoes transmit several human- and animal-pathogenic alphaviruses (Togaviridae family). In alphavirus-infected mosquito cells two different types of virus-specific small RNAs are produced as part of the RNA interference response: short-interfering (si)RNAs and PIWI-interacting (pi)RNAs. The siRNA pathway is generally thought to be the main antiviral pathway. Although an antiviral activity has been suggested for the piRNA pathway its role in host defences is not clear. Knock down of key proteins of the piRNA pathway (Ago3 and Piwi5) in Aedes aegypti-derived cells reduced the production of alphavirus chikungunya virus (CHIKV)-specific piRNAs but had no effect on virus replication. In contrast, knock down of the siRNA pathway key protein Ago2 resulted in an increase in virus replication. Similar results were obtained when expression of Piwi4 was silenced. Knock down of the helicase Spindle-E (SpnE), an essential co-factor of the piRNA pathway in Drosophila melanogaster, resulted in increased virus replication indicating that SpnE acts as an antiviral against alphaviruses such as CHIKV and the related Semliki Forest virus (SFV). Surprisingly, this effect was found to be independent of the siRNA and piRNA pathways in Ae. aegypti cells and specific for alphaviruses. This suggests a small RNA-independent antiviral function for this protein in mosquitoes.