Host Range Restriction Research Articles

Enforced specificity of an animal symbiosis.

Insect diversification has been catalyzed by widespread specialization on novel hosts - a process underlying exceptional radiations of phytophagous beetles, lepidopterans, parasitoid wasps, and inordinate lineages of symbionts, predators and other trophic specialists. The strict fidelity of many such interspecies associations is posited to hinge on sensory tuning to host-derived cues, a model supported by studies of neural function in host-specific model species. Here, we investigated the sensory basis of symbiotic interactions between a myrmecophile rove beetle and its single, natural host ant species. We show that host cues trigger analogous behaviors in both ant and symbiont. Cuticular hydrocarbons - the ant's nestmate recognition pheromones - elicit partner recognition by the beetle and execution of ant grooming behavior, integrating the beetle into the colony via chemical mimicry. The beetle also follows host trail pheromones, permitting inter-colony dispersal. Remarkably, the rove beetle also performs its symbiotic behaviors with ant species separated by ~95 million years, and shows minimal preference for its natural host over non-host ants. Experimentally validated agent-based modeling supports a scenario in which specificity is enforced by physiological constraints on symbiont dispersal, and negative fitness interactions with alternative hosts, rather than via sensory tuning. Enforced specificity may be a pervasive mechanism of host range restriction of specialists embedded within host niches. Chance realization of latent compatibilities with alternative hosts may facilitate host switching, enabling deep-time persistence of obligately symbiotic lineages.

Chimeric Viruses Enable Study of Antibody Responses to Human Rotaviruses in Mice.

The leading cause of gastroenteritis in children under the age of five is rotavirus infection, accounting for 37% of diarrhoeal deaths in infants and young children globally. Oral rotavirus vaccines have been widely incorporated into national immunisation programs, but whilst these vaccines have excellent efficacy in high-income countries, they protect less than 50% of vaccinated individuals in low- and middle-income countries. In order to facilitate the development of improved vaccine strategies, a greater understanding of the immune response to existing vaccines is urgently needed. However, the use of mouse models to study immune responses to human rotavirus strains is currently limited as rotaviruses are highly species-specific and replication of human rotaviruses is minimal in mice. To enable characterisation of immune responses to human rotavirus in mice, we have generated chimeric viruses that combat the issue of rotavirus host range restriction. Using reverse genetics, the rotavirus outer capsid proteins (VP4 and VP7) from either human or murine rotavirus strains were encoded in a murine rotavirus backbone. Neonatal mice were infected with chimeric viruses and monitored daily for development of diarrhoea. Stool samples were collected to quantify viral shedding, and antibody responses were comprehensively evaluated. We demonstrated that chimeric rotaviruses were able to efficiently replicate in mice. Moreover, the chimeric rotavirus containing human rotavirus outer capsid proteins elicited a robust antibody response to human rotavirus antigens, whilst the control chimeric murine rotavirus did not. This chimeric human rotavirus therefore provides a new strategy for studying human-rotavirus-specific immunity to the outer capsid, and could be used to investigate factors causing variability in rotavirus vaccine efficacy. This small animal platform therefore has the potential to test the efficacy of new vaccines and antibody-based therapeutics.

Shared clusters between phylogenetic trees for genomic segments of Rotavirus A with distinct genotype constellations

Rotavirus A (RVA) possesses a genome of 11-segmented, double-stranded RNAs, each of which is classified into genotypes. A variety of genotype constellations have been generated through reassortment, but reassortment does not appear to occur randomly. Here genomic sequences for 281 RVA strains with distinct genotype constellations retrieved from the International Nucleotide Sequence Database were analyzed to identify shared clusters between phylogenetic trees for genomic segments. The largest numbers of shared clusters were observed between the genomic segments encoding viral protein 1 (VP1) and VP2 as well as VP2 and VP3, suggesting that not only VP1 but also VP3 may interact with VP2 inside the core. Larger numbers were also associated with the segment encoding non-structural protein 5/6 (NSP5/6), which may be a hub for bundling genomic segments. Although VP7 and VP4 segments were associated with smaller numbers, reassortments between these segments as well as VP7 and VP6 segments appeared to be restricted due to interactions in constructing the virion. For VP4 segment, the number with NSP1 segment was significantly greater than those with other segments, possibly reflecting that specificities in receptor binding and interferon antagonism may define host range restriction. Overall, no cluster was shared by all genomic segments, supporting that RVA constituted a single species.

Exploring the role of symbiotic modifier peptidases in the legume - rhizobium symbiosis.

Legumes can establish a mutual association with soil-derived nitrogen-fixing bacteria called 'rhizobia' forming lateral root organs called root nodules. Rhizobia inside the root nodules get transformed into 'bacteroids' that can fix atmospheric nitrogen to ammonia for host plants in return for nutrients and shelter. A substantial 200million tons of nitrogen is fixed annually through biological nitrogen fixation. Consequently, the symbiotic mechanism of nitrogen fixation is utilized worldwide for sustainable agriculture and plays a crucial role in the Earth's ecosystem. The development of effective nitrogen-fixing symbiosis between legumes and rhizobia is very specialized and requires coordinated signaling. A plethora of plant-derived nodule-specific cysteine-rich (NCR or NCR-like) peptides get actively involved in this complex and tightly regulated signaling process of symbiosis between some legumes of the IRLC (Inverted Repeat-Lacking Clade) and Dalbergioid clades and nitrogen-fixing rhizobia. Recent progress has been made in identifying two such peptidases that actively prevent bacterial differentiation, leading to symbiotic incompatibility. In this review, we outlined the functions of NCRs and two nitrogen-fixing blocking peptidases: HrrP (host range restriction peptidase) and SapA (symbiosis-associated peptidase A). SapA was identified through an overexpression screen from the Sinorhizobium meliloti 1021 core genome, whereas HrrP is inherited extra-chromosomally. Interestingly, both peptidases affect the symbiotic outcome by degrading the NCR peptides generated from the host plants. These NCR-degrading peptidases can shed light on symbiotic incompatibility, helping to elucidate the reasons behind the inefficiency of nitrogen fixation observed in certain groups of rhizobia with specific legumes.

Equine Rotavirus A under the One Health Lens: Potential Impacts on Public Health.

Group A rotaviruses are a well-known cause of viral gastroenteritis in infants and children, as well as in many mammalian species and birds, affecting them at a young age. This group of viruses has a double-stranded, segmented RNA genome with high genetic diversity linked to point mutations, recombination, and, importantly, reassortment. While initial molecular investigations undertaken in the 1900s suggested host range restriction among group A rotaviruses based on the fact that different gene segments were distributed among different animal species, recent molecular surveillance and genome constellation genotyping studies conducted by the Rotavirus Classification Working Group (RCWG) have shown that animal rotaviruses serve as a source of diversification of human rotavirus A, highlighting their zoonotic potential. Rotaviruses occurring in various animal species have been linked with contributing genetic material to human rotaviruses, including horses, with the most recent identification of equine-like G3 rotavirus A infecting children. The goal of this article is to review relevant information related to rotavirus structure/genomic organization, epidemiology (with a focus on human and equine rotavirus A), evolution, inter-species transmission, and the potential zoonotic role of equine and other animal rotaviruses. Diagnostics, surveillance and the current status of human and livestock vaccines against RVA are also reviewed.

Comparative phylogenomics and phylotranscriptomics provide insights into the genetic complexity of nitrogen-fixing root-nodule symbiosis

Plant root-nodule symbiosis (RNS) with mutualistic nitrogen-fixing bacteria is restricted to a single clade of angiosperms, the Nitrogen-Fixing Nodulation Clade (NFNC), and is best understood in the legume family. Nodulating species share many commonalities, explained either by divergence from a common ancestor over 100 million years ago or by convergence following independent origins over that same time period. Regardless, comparative analyses of diverse nodulation syndromes can provide insights into constraints on nodulation—what must be acquired or cannot be lost for a functional symbiosis—and the latitude for variation in the symbiosis. However, much remains to be learned about nodulation, especially outside of legumes. Here, we employed a large-scale phylogenomic analysis across 88 species, complemented by 151 RNA-seq libraries, to elucidate the evolution of RNS. Our phylogenomic analyses further emphasize the uniqueness of the transcription factor NIN as a master regulator of nodulation and identify key mutations that affect its function across the NFNC. Comparative transcriptomic assessment revealed nodule-specific upregulated genes across diverse nodulating plants, while also identifying nodule-specific and nitrogen-response genes. Approximately 70% of symbiosis-related genes are highly conserved in the four representative species, whereas defense-related and host-range restriction genes tend to be lineage specific. Our study also identified over 900 000 conserved non-coding elements (CNEs), over 300 000 of which are unique to sampled NFNC species. NFNC-specific CNEs are enriched with the active H3K9ac mark and are correlated with accessible chromatin regions, thus representing a pool of candidate regulatory elements for genes involved in RNS. Collectively, our results provide novel insights into the evolution of nodulation and lay a foundation for engineering of RNS traits in agriculturally important crops.

Global Infection Rate of Rotavirus C during 1980-2022 and Analysis of Critical Factors in the Host Range Restriction of Virus VP4.

Information on rotavirus C (RVC) infection is lacking, partly because the prevalence of RVC among humans and animals worldwide is undefined. Data on the characteristics of the P genotype among RVC strains are also required. We performed systematic searches on the infection rates of RVC since 1980 based on the literature and gene sequences of the PubMed and GenBank databases. A phylogenetic tree of VP4 genes was constructed to evaluate the distribution of the P genotype of RVC from various hosts. The specific mutation motifs in VP8* with P [2]/P [4]/P [5] specificity were analyzed to elucidate their roles in host range restriction. The rate of RVC infection in humans has fallen from 3% before 2009 to 1%, whereas in animals it has risen from 10% to 25%. The P genotype of RVC showed strict host species specificity, and current human RVC infections are exclusively caused by genotype P [2]. In the VP8* hemagglutinin domain of the P [4]/P [5] genotype of swine RVC, specific insertion or deletion were found relative to the human P [2] genotype, and these motifs are a possible critical factor for host range restriction. Our findings highlight the need for further epidemiological surveillance, preventive strategies, and elucidation of the factors involved in the specific host range restriction of RVC-circulating strains.

The Role of the VP4 Attachment Protein in Rotavirus Host Range Restriction in an In Vivo Suckling Mouse Model.

The basis for rotavirus (RV) host range restriction (HRR) is not fully understood but is likely multigenic. RV genes encoding VP3, VP4, NSP1, NSP2, NSP3, and NSP4 have been associated with HRR in various studies. With the exception of NSP1, little is known about the relative contribution of the other RV genes to HRR. VP4 has been linked to HRR because it functions as the RV cell attachment protein, but its actual role in HRR has not been fully assessed. We generated a collection of recombinant RVs (rRVs) in an isogenic murine-like RV genetic background, harboring either heterologous or homologous VP4 genes from simian, bovine, porcine, human, and murine RV strains, and characterized these rRVs in vitro and in vivo. We found that a murine-like rRV encoding a simian VP4 was shed, spread to uninoculated littermates, and induced diarrhea comparably to rRV harboring a murine VP4. However, rRVs carrying VP4s from both bovine and porcine RVs had reduced diarrhea, but no change in fecal shedding was observed. Both diarrhea and shedding were reduced when VP4 originated from a human RV strain. rRVs harboring VP4s from human or bovine RVs did not transmit to uninoculated littermates. We also generated two rRVs harboring reciprocal chimeric murine or bovine VP4. Both chimeras replicated and caused disease as efficiently as the parental strain with a fully murine VP4. These data suggest that the genetic origin of VP4 partially modulates HRR in the suckling mouse and that both the VP8* and VP5* domains independently contribute to pathogenesis and transmission. IMPORTANCE Human group A rotaviruses (RVs) remain the most important cause of severe acute gastroenteritis among infants and young children worldwide despite the introduction of several safe and effective live attenuated vaccines. The lack of knowledge regarding fundamental aspects of RV biology, such as the genetic basis of host range restriction (HRR), has made it difficult to predictively and efficiently design improved, next-generation live attenuated rotavirus vaccines. Here, we engineered a collection of VP4 monoreassortant RVs to systematically explore the role of VP4 in replication, pathogenicity, and spread, as measures of HRR, in a suckling mouse model. The genetic and mechanistic bases of HRR have substantial clinical relevance given that this restriction forms the basis of attenuation for several replication-competent human RV vaccines. In addition, a better understanding of RV pathogenesis and the determinants of RV spread is likely to enhance our ability to improve antiviral drug and therapy development.

Intranasal immunization with avian paramyxovirus type 3 expressing SARS-CoV-2 spike protein protects hamsters against SARS-CoV-2

Current vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are administered parenterally and appear to be more protective in the lower versus the upper respiratory tract. Vaccines are needed that directly stimulate immunity in the respiratory tract, as well as systemic immunity. We used avian paramyxovirus type 3 (APMV3) as an intranasal vaccine vector to express the SARS-CoV-2 spike (S) protein. A lack of pre-existing immunity in humans and attenuation by host-range restriction make APMV3 a vector of interest. The SARS-CoV-2 S protein was stabilized in its prefusion conformation by six proline substitutions (S-6P) rather than the two that are used in most vaccine candidates, providing increased stability. APMV3 expressing S-6P (APMV3/S-6P) replicated to high titers in embryonated chicken eggs and was genetically stable, whereas APMV3 expressing non-stabilized S or S-2P were unstable. In hamsters, a single intranasal dose of APMV3/S-6P induced strong serum IgG and IgA responses to the S protein and its receptor-binding domain, and strong serum neutralizing antibody responses to SARS-CoV-2 isolate WA1/2020 (lineage A). Sera from APMV3/S-6P-immunized hamsters also efficiently neutralized Alpha and Beta variants of concern. Immunized hamsters challenged with WA1/2020 did not exhibit the weight loss and lung inflammation observed in empty-vector-immunized controls; SARS-CoV-2 replication in the upper and lower respiratory tract of immunized animals was low or undetectable compared to the substantial replication in controls. Thus, a single intranasal dose of APMV3/S-6P was highly immunogenic and protective against SARS-CoV-2 challenge, suggesting that APMV3/S-6P is suitable for clinical development.

Re-Examining Rotavirus Innate Immune Evasion: Potential Applications of the Reverse Genetics System.

ABSTRACTRotaviruses represent one of the most successful pathogens in the world, with high infectivity and efficient transmission between the young of many animal species, including humans. To overcome host defenses, rotaviruses have evolved a plethora of strategies to effectively evade the innate immune response, establish initial infection in the small intestine, produce progeny, and shed into the environment. Previously, studying the roles and relative contributions of specific rotaviral factors in innate immune evasion had been challenging without a plasmid-only reverse genetics system. Although still in its infancy, current reverse genetics technology will help address important research questions regarding rotavirus innate immune evasion, host range restriction, and viral pathogenesis. In this review, we summarize the current knowledge about the antiviral host innate immune defense mechanisms, countermeasures of rotavirus-encoded factors, and strategies to better understand these interactions using the rotavirus reverse genetics system.

Negotiating mutualism: A locus for exploitation by rhizobia has a broad effect size distribution and context-dependent effects on legume hosts.

In mutualisms, variation at genes determining partner fitness provides the raw material upon which coevolutionary selection acts, setting the dynamics and pace of coevolution. However, we know little about variation in the effects of genes that underlie symbiotic fitness in natural mutualist populations. In some species of legumes that form root nodule symbioses with nitrogen‐fixing rhizobial bacteria, hosts secrete nodule‐specific cysteine‐rich (NCR) peptides that cause rhizobia to differentiate in the nodule environment. However, rhizobia can cleave NCR peptides through the expression of genes like the plasmid‐borne Host range restriction peptidase (hrrP), whose product degrades specific NCR peptides. Although hrrP activity can confer host exploitation by depressing host fitness and enhancing symbiont fitness, the effects of hrrP on symbiosis phenotypes depend strongly on the genotypes of the interacting partners. However, the effects of hrrP have yet to be characterised in a natural population context, so its contribution to variation in wild mutualist populations is unknown. To understand the distribution of effects of hrrP in wild rhizobia, we measured mutualism phenotypes conferred by hrrP in 12 wild Ensifer medicae strains. To evaluate context dependency of hrrP effects, we compared hrrP effects across two Medicago polymorpha host genotypes and across two experimental years for five E. medicae strains. We show for the first time in a natural population context that hrrP has a wide distribution of effect sizes for many mutualism traits, ranging from strongly positive to strongly negative. Furthermore, we show that hrrP effect size varies across host genotypes and experiment years, suggesting that researchers should be cautious about extrapolating the role of genes in natural populations from controlled laboratory studies of single genetic variants.

Molecular Mechanisms Underlying the Cellular Entry and Host Range Restriction of Lujo Virus.

ABSTRACTLike other human-pathogenic arenaviruses, Lujo virus (LUJV) is a causative agent of viral hemorrhagic fever in humans. LUJV infects humans with high mortality rates, but the susceptibilities of other animal species and the molecular determinants of its host specificity remain unknown. We found that mouse- and hamster-derived cell lines (NIH 3T3 and BHK, respectively) were less susceptible to a replication-incompetent recombinant vesicular stomatitis virus (Indiana) pseudotyped with the LUJV glycoprotein (GP) (VSVΔG*-LUJV/GP) than were human-derived cell lines (HEK293T and Huh7). To determine the cellular factors involved in the differential susceptibilities between the human and mouse cell lines, we focused on the CD63 molecule, which is required for pH-activated GP-mediated membrane fusion during LUJV entry into host cells. The exogenous introduction of human CD63, but not mouse or hamster CD63, into BHK cells significantly increased susceptibility to VSVΔG*-LUJV/GP. Using chimeric human-mouse CD63 proteins, we found that the amino acid residues at positions 141 to 150 in the large extracellular loop (LEL) region of CD63 were important for the cellular entry of VSVΔG*-LUJV/GP. By site-directed mutagenesis, we further determined that a phenylalanine at position 143 in human CD63 was the key residue for efficient membrane fusion and VSVΔG*-LUJV/GP infection. Our data suggest that the interaction of LUJV GP with the LEL region of CD63 is essential for cell susceptibility to LUJV, thus providing new insights into the molecular mechanisms underlying the cellular entry of LUJV and the host range restriction of this virus.

The host range restriction of bat-associated no-known-vector flaviviruses occurs post-entry

Most flaviviruses are transmitted horizontally between vertebrate hosts by haematophagous arthropods. Others exhibit host ranges restricted to vertebrates or arthropods. Vertebrate-specific flaviviruses are commonly referred to as no-known-vector (NKV) flaviviruses and can be separated into bat- and rodent-associated NKV flaviviruses. Rio Bravo virus (RBV) is one of eight recognized bat-associated NKV (B-NKV) flaviviruses. Studies designed to identify the genetic determinants that condition the host range restriction of B-NKV flaviviruses have never been performed. To investigate whether the host range restriction occurs at the level of attachment or entry, chimeric flaviviruses were created by inserting the pre-membrane and envelope protein genes of RBV into the genetic backbones of yellow fever virus (YFV) and Zika virus (ZIKV), two mosquito-borne flaviviruses associated with human disease. The chimeric viruses infected both vertebrate and mosquito cells. In vertebrate cells, all viruses produced similar mean peak titres, but the chimeric viruses grew more slowly than their parental viruses during early infection. In mosquito cells, the chimeric virus of YFV and RBV grew more slowly than YFV at early post-inoculation time points, but reached a similar mean peak titre. In contrast, the chimeric virus of ZIKV and RBV produced a mean peak titre that was approximately 10-fold lower than ZIKV. The chimeric virus of YFV and RBV produced an intermediate plaque phenotype, while the chimeric virus of ZIKV and RBV produced smaller plaques than both parental viruses. To conclude, we provide evidence that the structural glycoproteins of RBV permit entry into both mosquito and vertebrate cells, indicating that the host range restriction of B-NKV flaviviruses is mediated by a post-attachment/entry event.

Seasonal abundance and host-plant affinities of Longitarsus basutoensis and related flea beetles associated with the invasive Senecio madagascariensis in their native range in KwaZulu-Natal, South Africa

Senecio madagascariensis Poiret (Asteraceae) is a target for biological control in Australia and Hawaii, with several candidate insect agents recorded in KwaZulu-Natal, South Africa, the confirmed origin of the invasive populations. Although the root-feeding Longitarsus basutoensis Bechyné (Chrysomelidae: Galerucinae: Alticini) was prioritised for assessment, similar flea beetle taxa were also considered. We first investigated the seasonal abundance of L. basutoensis and related taxa on S. madagascariensis populations in the KwaZulu-Natal Midlands. Second, we assessed their host-plant affinities by surveying 17 non-target native Senecio species in the region. We used DNA CO1 barcoding to differentiate flea beetle taxa and their root-feeding larvae in both studies. Monthly sampling revealed three root-feeding taxa associated with S. madagascariensis, with L. basutoensis the most abundant. Longitarsus basutoensis was recorded throughout the year, with no clear patterns in seasonal abundance but significantly higher numbers at sites with deep, well drained soils that have high water holding capacity. Two unidentified flea beetle taxa, represented by larval specimens only, were recorded infrequently and in low numbers. The host-range study revealed 18 flea beetle taxa on 11 Senecio species, with L. basutoensis the most abundant species and recorded mostly on S. madagascariensis. Longitarsus basutoensis was also detected on four non-target Senecio species which, together with the results of preliminary host-specificity tests, indicated insufficient host-range restriction for countries like Australia that host a diverse Senecio flora. However, L basutoensis may have potential for release in Hawaii, due to a lack of native Senecio species on any of its islands.

Chimeric Zika viruses containing structural protein genes of insect-specific flaviviruses cannot replicate in vertebrate cells due to entry and post-translational restrictions

Long Pine Key virus (LPKV) and Lammi virus are insect-specific flaviviruses that phylogenetically affiliate with dual-host flaviviruses. The goal of this study was to provide insight into the genetic determinants that condition this host range restriction. Chimeras were initially created by replacing select regions of the Zika virus genome, including the premembrane and envelope protein (prM-E) genes, with the corresponding regions of the LPKV genome. Of the four chimeras produced, one (the prM-E swap) yielded virus that replicated in mosquito cells. Another chimeric virus with a mosquito replication-competent phenotype was created by inserting the prM-E genes of Lammi virus into a Zika virus genetic background. Vertebrate cells did not support the replication of either chimeric virus although trace to modest amounts of viral antigen were produced, consistent with suboptimal viral entry. These data suggest that dual-host affiliated insect-specific flaviviruses cannot replicate in vertebrate cells due to entry and post-translational restrictions.

Swine MicroRNAs ssc-miR-221-3p and ssc-miR-222 Restrict the Cross-Species Infection of Avian Influenza Virus.

Avian influenza virus (AIV) can cross species barriers to infect humans and other mammals. However, these species-cross transmissions are most often dead-end infections due to host restriction. Current research about host restriction focuses mainly on the barriers of cell membrane, nuclear envelope, and host proteins; whether microRNAs (miRNAs) play a role in host restriction is largely unknown. In this study, we used porcine alveolar macrophage (PAM) cells as a model to elucidate the role of miRNAs in host range restriction. During AIV infection, 40 dysregulation expressed miRNAs were selected in PAM cells. Among them, two Sus scrofa (ssc; swine) miRNAs, ssc-miR-221-3p and ssc-miR-222, could inhibit the infection and replication of AIV in PAM cells by directly targeting viral genome and inducing cell apoptosis via inhibiting the expression of anti-apoptotic protein HMBOX1. Avian but not swine influenza virus caused upregulated expressions of ssc-miR-221-3p and ssc-miR-222 in PAM cells. We further found that NF-κB P65 was more effectively phosphorylated upon AIV infection and that P65 functioned as a transcription activator to regulate the AIV-induced expression of miR-221-3p/222 Importantly, we found that ssc-miR-221-3p and ssc-miR-222 could also be specifically upregulated upon AIV infection in newborn pig tracheal epithelial (NPTr) cells and also exerted anti-AIV function. In summary, our study indicated that miRNAs act as a host barrier during cross-species infection of influenza A virus.IMPORTANCE The host range of an influenza A virus is determined by species-specific interactions between virus and host cell factors. Host miRNAs can regulate influenza A virus replication; however, the role of miRNAs in host species specificity is unclear. Here, we show that the induced expression of ssc-miR-221-3p and ssc-miR-222 in swine cells is modulated by NF-κB P65 phosphorylation in response to AIV infection but not swine influenza virus infection. ssc-miR-221-3p and ssc-miR-222 exerted antiviral function via targeting viral RNAs and causing apoptosis by inhibiting the expression of HMBOX1 in host cells. These findings uncover miRNAs as a host range restriction factor that limits cross-species infection of influenza A virus.

Rotavirus Reprograms Multiple Interferon Receptors and Restricts Their Intestinal Antiviral and Inflammatory Functions.

Rotaviruses (RV) cause acute severe diarrhea in the absence of substantial intestinal inflammation. They are also highly infectious in their homologous host species. The replication capacity of RV in the small bowel is substantially due to its ability to inhibit different types of interferons (IFNs). Here, we found that during RV infection in vitro, both virus-infected and uninfected bystander cells resist STAT1 phosphorylation and interferon regulatory factor 7 (IRF7) induction in response to exogenous interferon (IFN). Functionally, cellular transcription in response to stimulation with IFN, but not intracellular double-stranded RNA (dsRNA), was inhibited by RV. Further, IFNAR1 stimulation during RV infection significantly repressed a set of virus-induced transcripts. Regulation of IFN signaling in vivo was studied in suckling mice using the highly infectious murine EW RV strain. Kinetic studies indicated that sustained EW RV replication and IFN induction in the small intestine are accompanied by significant decreases in IFN-stimulated transcripts. Lipopolysaccharide (LPS)-mediated intestinal damage, driven by STAT1-induced inflammation, was also prevented in EW RV-infected mice. Remarkably, by ectopically stimulating either IFNAR1 or IFNGR1 in EW RV-infected mice, we could eliminate several intestinal antiviral and inflammatory transcriptional responses to RV. In contrast to infection with homologous RV, infection with a STAT1-sensitive heterologous RV strain induced IFN-stimulated transcripts, inflammatory cytokines, and intestinal expression of STAT1-pY701. Finally, RV strain-specific STAT1 regulation also likely determines the intestinal activation of multiple caspases. The simian RRV strain, but not murine EW RV, uniquely triggers the cleavage of both extrinsic and intrinsic caspases (caspases 8, 9, and 3) in a STAT1-mediated manner. Collectively, our findings reveal efficient reprograming of multiple IFN receptors toward a negative-feedback mode of signaling, accompanied by suppression of IFN-mediated antiviral, apoptotic, and inflammatory functions, during natural RV intestinal infection.IMPORTANCE Rotavirus is a highly infectious pathogen that causes severe diarrhea. Replication of RV in the small intestine is restricted to homologous host species, and host range restriction is substantially determined by the interferon response. In this study, we demonstrate that during infection, RV bystander cells resist exogenous IFN-mediated STAT1 signaling and transcription. In a suckling mouse model, ectopically stimulating different intestinal interferon receptors during RV infection eliminates several innate and inflammatory antiviral responses. Different intestinal inflammatory cytokines were also suppressed by homologous RV, as was intestinal damage in response to endotoxin. The ability of RV to suppress IFN-mediated receptors likely impacts intestinal cell homeostasis, as the cleavage of multiple intestinal caspases during RV infection is mediated by the IFN-STAT1 signaling pathway. Together, our results provide a mechanism underlying both the remarkable interferon resistance of homologous RV and its ability to prevent substantial inflammatory damage to the small bowel.

Niemann-Pick C1 Heterogeneity of Bat Cells Controls Filovirus Tropism

Fruit bats are suspected to be natural hosts of filoviruses, including Ebola virus (EBOV) and Marburg virus (MARV). Interestingly, however, previous studies suggest that these viruses have different tropisms depending on the bat species. Here, we show a molecular basis underlying the host-range restriction of filoviruses. We find that bat-derived cell lines FBKT1 and ZFBK13-76E show preferential susceptibility to EBOV and MARV, respectively, whereas the other bat cell lines tested are similarly infected with both viruses. In FBKT1 and ZFBK13-76E, unique amino acid (aa) sequences are found in the Niemann-Pick C1 (NPC1) protein, one of the cellular receptors interacting with the filovirus glycoprotein (GP). These aa residues, as well as a few aa differences between EBOV and MARV GPs, are crucial for the differential susceptibility to filoviruses. Taken together, our findings indicate that the heterogeneity of bat NPC1 orthologs is an important factor controlling filovirus species-specific host tropism.

Circulation of low pathogenic avian influenza (LPAI) viruses in wild birds and poultry in the Netherlands, 2006\u20132016

In this study, we explore the circulation of low pathogenic avian influenza (LPAI) viruses in wild birds and poultry in the Netherlands. Surveillance data collected between 2006 and 2016 was used to evaluate subtype diversity, spatiotemporal distribution and genetic relationships between wild bird and poultry viruses. We observed close species-dependent associations among hemagglutinin and neuraminidase subtypes. Not all subtypes detected in wild birds were found in poultry, suggesting transmission to poultry is selective and likely depends on viral factors that determine host range restriction. Subtypes commonly detected in poultry were in wild birds most frequently detected in mallards and geese. Different temporal patterns in virus prevalence were observed between wild bird species. Virus detections in domestic ducks coincided with the prevalence peak in wild ducks, whereas virus detections in other poultry types were made throughout the year. Genetic analysis of the surface genes demonstrated that most poultry viruses were related to locally circulating wild bird viruses, but no direct spatiotemporal link was observed. Results indicate prolonged undetected virus circulation and frequent reassortment events with local and newly introduced viruses within the wild bird population. Increased knowledge on LPAI virus circulation can be used to improve surveillance strategies.

SPI-1 is a missing host-range factor required for replication of the attenuated modified vaccinia Ankara (MVA) vaccine vector in human cells

Modified vaccinia virus Ankara (MVA) is the leading poxvirus vector for development of vaccines against diverse infectious diseases. This distinction is based on high expression of proteins and good immunogenicity despite an inability to assemble infectious progeny in human cells, which together promote efficacy and safety. Nevertheless, the basis for the host-range restriction is unknown despite past systematic attempts to identify the relevant missing viral gene(s). The search for host-range factors is exacerbated by the large number of deletions, truncations and mutations that occurred during the long passage history of MVA in chicken embryo fibroblasts. By whole genome sequencing of a panel of recombinant host-range extended (HRE) MVAs generated by marker rescue with 40 kbp segments of vaccinia virus DNA, we identified serine protease inhibitor 1 (SPI-1) as one of several candidate host-range factors present in those viruses that gained the ability to replicate in human cells. Electron microscopy revealed that the interruption of morphogenesis in human cells infected with MVA occurred at a similar stage as that of a vaccinia virus strain WR SPI-1 deletion mutant. Moreover, the introduction of the SPI-1 gene into the MVA genome led to more than a 2-log enhancement of virus spread in human diploid MRC-5 cells, whereas deletion of the gene diminished the spread of HRE viruses by similar extents. Furthermore, MRC-5 cells stably expressing SPI-1 also enhanced replication of MVA. A role for additional host range genes was suggested by the restoration of MVA replication to a lower level relative to HRE viruses, particularly in other human cell lines. Although multiple sequence alignments revealed genetic changes in addition to SPI-1 common to the HRE MVAs, no evidence for their host-range function was found by analysis thus far. Our finding that SPI-1 is host range factor for MVA should simplify use of high throughput RNAi or CRISPR/Cas single gene methods to identify additional viral and human restriction elements.