Animal models that accurately reflect COVID-19 are vital for understanding mechanisms of disease and advancing development of improved vaccines and therapeutics. Pigs are increasingly recognized as valuable models for human disease due to their genetic, anatomical, physiological, and immunological similarities to humans, and they present a more ethically viable alternative to non-human primates. However, pigs are not susceptible to SARS-CoV-2 infection which limits their utility as a model. To address this, we have developed transgenic pigs expressing human ACE2 that are susceptible to SARS-CoV-2 infection. Following challenge, clinical signs consistent with COVID-19, including fever, coughing and respiratory distress were observed, with virus replication detected in the nasal turbinates, trachea and lungs up to the study endpoint, seven days post-infection. Notably, examination of tissues revealed immunopathology in the lungs consistent with histological changes observed in fatal human COVID-19 cases. This study establishes human ACE2 transgenic pigs as a large animal model that accurately reflects many aspects of COVID-19 disease.

Animal Trypanosomosis (AT) is a significant disease affecting cattle across sub-Saharan Africa, Latin America, and Asia, posing a major threat to economic productivity and animal welfare. The absence of reliable diagnostic tests has led to an over-reliance on widespread pre-emptive drug treatments, which not only compromise animal health but also heighten the risk of drug resistance. The chronic nature of AT, characterized by cyclical low or undetectable parasite levels, and the necessity for field-applicable tests that can distinguish between active infection and prior exposure, present considerable challenges in developing effective diagnostics. In previous work, we identified a parasite-specific small RNA, 7SL-sRNA, which is detectable in the serum of infected cattle, even during the chronic stages of infection. However, existing methods for detecting sRNA require specialized equipment, making them unsuitable for field use. In this study, we have developed both a fluorescence-based and a lateral flow diagnostic test utilizing Cas10 technology for the detection of 7SL-sRNA from Trypanosoma congolense and Trypanosoma brucei. The fluorescence assay detects 10 - 100 fM T. congolense 7SL-sRNA and 1 pM T. brucei 7SL-sRNA, and the lateral flow assay showed a limit of detection of 1 - 10 pM for both species. Either assay can effectively identify active infections in cattle, including during chronic phases (with positive signals observed up to the experimental end point, 63 days post infection). This also highlights the effective use of Cas10 for small RNA detection, paving the way for a cost-effective, user-friendly, and field-deployable diagnostic test for AT, while establishing Cas10 technology for the detection of small RNAs in general.

COVID-19 continues to cause significant morbidity and mortality, with emerging strains rapidly spreading despite substantial immunity through vaccination and previous exposure. Animal models that accurately reflect COVID-19 are vital for testing mechanisms of disease, enabling development of improved vaccines and therapeutics. We have developed human ACE2 transgenic pigs that are highly susceptible to SARS-CoV-2 and display clinical signs, disease progression, and lung inflammation that faithfully replicate severe COVID-19 in humans.

African swine fever virus (ASFV) is a lethal animal pathogen that enters its host cells through endocytosis. So far, host factors specifically required for ASFV replication have been barely identified. In this study a genome-wide CRISPR/Cas9 knockout screen in porcine cells indicated that the genes RFXANK, RFXAP, SLA-DMA, SLA-DMB, and CIITA are important for productive ASFV infection. The proteins encoded by these genes belong to the major histocompatibility complex II (MHC II), or swine leucocyte antigen complex II (SLA II). RFXAP and CIITA are MHC II-specific transcription factors, whereas SLA-DMA/B are subunits of the non-classical MHC II molecule SLA-DM. Targeted knockout of either of these genes led to severe replication defects of different ASFV isolates, reflected by substantially reduced plating efficiency, cell-to-cell spread, progeny virus titers and viral DNA replication. Transgene-based reconstitution of SLA-DMA/B fully restored the replication capacity demonstrating that SLA-DM, which resides in late endosomes, plays a crucial role during early steps of ASFV infection.

Synonymous recoding of RNA virus genomes is a promising approach for generating attenuated viruses to use as vaccines. Problematically, recoding typically hinders virus growth, but this may be rectified using CpG dinucleotide enrichment. CpGs are recognised by cellular zinc-finger antiviral protein (ZAP), and so in principle, removing ZAP sensing from a virus propagation system will reverse attenuation of a CpG-enriched virus, enabling high titre yield of a vaccine virus. We tested this using a vaccine strain of influenza A virus (IAV) engineered for increased CpG content in genome segment 1. Virus attenuation was mediated by the short isoform of ZAP, correlated with the number of CpGs added, and was enacted via turnover of viral transcripts. The CpG-enriched virus was strongly attenuated in mice, yet conveyed protection from a potentially lethal challenge dose of wildtype virus. Importantly for vaccine development, CpG-enriched viruses were genetically stable during serial passage. Unexpectedly, in both MDCK cells and embryonated hens' eggs that are used to propagate live attenuated influenza vaccines, the ZAP-sensitive virus was fully replication competent. Thus, ZAP-sensitive CpG enriched viruses that are defective in human systems can yield high titre in vaccine propagation systems, providing a realistic, economically viable platform to augment existing live attenuated vaccines.

Spillover events of avian influenza A viruses (IAVs) to humans could represent the first step in a future pandemic 1 . Several factors that limit the transmission and replication of avian IAVs in mammals have been identified. There are several gaps in our understanding to predict which virus lineages are more likely to cross the species barrier and cause disease in humans 1 . Here, we identified human BTN3A3 (butyrophilin subfamily 3 member A3) 2 as a potent inhibitor of avian IAVs but not human IAVs. We determined that BTN3A3 is expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts primarily at the early stages of the virus life cycle by inhibiting avian IAV RNA replication. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F or, rarely, 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, avian IAV serotypes, such as H7 and H9, that spilled over into humans also evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure 3 . Thus, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.

Background Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. Results We explored how porcine pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages. Porcine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages and were productively infected by pig pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, porcine PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied either in parental stem cells or directly in the macrophages by lentiviral vector transduction. Conclusions We show that porcine PSCdMs exhibit key macrophage characteristics, including infection by a range of commercially relevant pig pathogens. In addition, genetic engineering of PSCs and PSCdMs affords new opportunities for functional analysis of macrophage biology in an important livestock species. PSCs and differentiated derivatives should therefore represent a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in pigs, and also have wider applications in livestock.

Animal trypanosomiasis (AT) is a significant livestock disease, affecting millions of animals across Sub-Saharan Africa, Central and South America, and Asia, and is caused by the protozoan parasites Trypanosoma brucei, Trypanosoma vivax, and Trypanosoma congolense, with the largest economic impact in cattle. There is over-reliance on presumptive chemotherapy due to inadequate existing diagnostic tests, highlighting the need for improved AT diagnostics. A small RNA species, the 7SL sRNA, is excreted/secreted by trypanosomes in infected animals, and has been previously shown to reliably diagnose active infection. We sought to explore key properties of 7SL sRNA RT-qPCR assays; namely, assessing the potential for cross-reaction with the widespread and benign Trypanosoma theileri, directly comparing assay performance against currently available diagnostic methods, quantitatively assessing specificity and sensitivity, and assessing the rate of decay of 7SL sRNA post-treatment. Results showed that the 7SL sRNA RT-qPCR assays specific for T. brucei, T. vivax, and T. congolense performed better than microscopy and DNA PCR in detecting infection. The 7SL sRNA signal was undetectable or significantly reduced by 96-h post treatment; at 1 × curative dose there was no detectable signal in 5/5 cattle infected with T. congolense, and in 3/5 cattle infected with T. vivax, with the signal being reduced 14,630-fold in the remaining two T. vivax cattle. Additionally, the assays did not cross-react with T. theileri. Finally, by using a large panel of validated infected and uninfected samples, the species-specific assays are shown to be highly sensitive and specific by receiver operating characteristic (ROC) analysis, with 100% sensitivity (95% CI, 96.44–100%) and 100% specificity (95% CI, 96.53–100%), 96.73% (95% CI, 95.54–99.96%) and 99.19% specificity (95% CI, 92.58–99.60%), and 93.42% (95% CI, 85.51–97.16% %) and 82.43% specificity (95% CI, 72.23–89.44% %) for the T brucei, T. congolense and T. vivax assays, respectively, under the conditions used. These findings indicate that the 7SL sRNA has many attributes that would be required for a potential diagnostic marker of AT: no cross-reaction with T. theileri, high specificity and sensitivity, early infection detection, continued signal even in the absence of detectable parasitaemia in blood, and clear discrimination between infected and treated animals.

Cross-species transmission of avian influenza A viruses (IAVs) into humans could represent the first step of a future pandemic 1 . Multiple factors limiting the spillover and adaptation of avian IAVs in humans have been identified, but they are not sufficient to explain which virus lineages are more likely to cross the species barrier 1,2 . Here, we identified human BTN3A3 3 (butyrophilin subfamily 3 member A3) as a potent inhibitor of avian but not human IAVs. We determined that BTN3A3 is constitutively expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts at the early stages of virus replication by inhibiting avian IAV vRNA transcription. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F, or rarely 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, several serotypes of avian IAVs that spilled over into humans in recent decades evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure 4 . Importantly, we identified more than 150 avian IAV lineages with a BTN3A3-resistant genotype. In conclusion, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.

Simian immunodeficiency virus (SIV) insert-expressing, 68-1 rhesus cytomegalovirus (RhCMV/SIV) vectors elicit major histocompatibility complex E (MHC-E)- and MHC-II-restricted, SIV-specific CD8(+) T cell responses, but the basis of these unconventional responses and their contribution to demonstrated vaccine efficacy against SIV challenge in the rhesus monkeys (RMs) have not been characterized. We show that these unconventional responses resulted from a chance genetic rearrangement in 68-1 RhCMV that abrogated the function of eight distinct immunomodulatory gene products encoded in two RhCMV genomic regions (Rh157.5/Rh157.4 and Rh158-161), revealing three patterns of unconventional response inhibition. Differential repair of these genes with either RhCMV-derived or orthologous human CMV (HCMV)-derived sequences (UL128/UL130; UL146/UL147) leads to either of two distinct CD8(+) T cell response types-MHC-Ia-restricted only or a mix of MHC-II- and MHC-Ia-restricted CD8(+) T cells. Response magnitude and functional differentiation are similar to RhCMV 68-1, but neither alternative response type mediated protection against SIV challenge. These findings implicate MHC-E-restricted CD8(+) T cell responses as mediators of anti-SIV efficacy and indicate that translation of RhCMV/SIV vector efficacy to humans will likely require deletion of all genes that inhibit these responses from the HCMV/HIV vector.

An evolutionary arms race occurs between viruses and hosts. Hosts have developed an array of antiviral mechanisms aimed at inhibiting replication and spread of viruses, reducing their fitness, and ultimately minimising pathogenic effects. In turn, viruses have evolved sophisticated counter-measures that mediate evasion of host defence mechanisms. A key aspect of host defences is the ability to differentiate between self and non-self. Previous studies have demonstrated significant suppression of CpG and UpA dinucleotide frequencies in the coding regions of RNA and small DNA viruses. Artificially increasing these dinucleotide frequencies results in a substantial attenuation of virus replication, suggesting dinucleotide bias could facilitate recognition of non-self RNA. The interferon-inducible gene, zinc finger antiviral protein (ZAP) is the host factor responsible for sensing CpG dinucleotides in viral RNA and restricting RNA viruses through direct binding and degradation of the target RNA. Herpesviruses are large DNA viruses that comprise three subfamilies, alpha, beta and gamma, which display divergent CpG dinucleotide patterns within their genomes. ZAP has recently been shown to act as a host restriction factor against human cytomegalovirus (HCMV), a beta-herpesvirus, which in turn evades ZAP detection by suppressing CpG levels in the major immediate-early transcript IE1, one of the first genes expressed by the virus. While suppression of CpG dinucleotides allows evasion of ZAP targeting, synonymous changes in nucleotide composition that cause genome biases, such as low GC content, can cause inefficient gene expression, especially in unspliced transcripts. To maintain compact genomes, the majority of herpesvirus transcripts are unspliced. Here we discuss how the conflicting pressures of ZAP evasion, the need to maintain compact genomes through the use of unspliced transcripts and maintaining efficient gene expression may have shaped the evolution of herpesvirus genomes, leading to characteristic CpG dinucleotide patterns.

Herpesviruses are large DNA viruses, which encode up to 300 different proteins including enzymes enabling efficient replication. Nevertheless, they depend on a multitude of host cell proteins for successful propagation. To uncover cellular host factors important for replication of pseudorabies virus (PrV), an alphaherpesvirus of swine, we performed an unbiased genome-wide CRISPR/Cas9 forward screen. To this end, a porcine CRISPR-knockout sgRNA library (SsCRISPRko.v1) targeting 20,598 genes was generated and used to transduce porcine kidney cells. Cells were then infected with either wildtype PrV (PrV-Ka) or a PrV mutant (PrV-gD–Pass) lacking the receptor-binding protein gD, which regained infectivity after serial passaging in cell culture. While no cells survived infection with PrV-Ka, resistant cell colonies were observed after infection with PrV-gD–Pass. In these cells, sphingomyelin synthase 1 (SMS1) was identified as the top hit candidate. Infection efficiency was reduced by up to 90% for PrV-gD–Pass in rabbit RK13-sgms1KO cells compared to wildtype cells accompanied by lower viral progeny titers. Exogenous expression of SMS1 partly reverted the entry defect of PrV-gD–Pass. In contrast, infectivity of PrV-Ka was reduced by 50% on the knockout cells, which could not be restored by exogenous expression of SMS1. These data suggest that SMS1 plays a pivotal role for PrV infection, when the gD-mediated entry pathway is blocked.

Viral infections activate the powerful interferon (IFN) response that induces the expression of several hundred IFN stimulated genes (ISGs). The principal role of this extensive response is to create an unfavourable environment for virus replication and to limit spread; however, untangling the biological consequences of this large response is complicated. In addition to a seemingly high degree of redundancy, several ISGs are usually required in combination to limit infection as individual ISGs often have low to moderate antiviral activity. Furthermore, what ISG or combination of ISGs are antiviral for a given virus is usually not known. For these reasons, and since the function(s) of many ISGs remains unexplored, genome-wide approaches are well placed to investigate what aspects of this response result in an appropriate, virus-specific phenotype. This review discusses the advances screening approaches have provided for the study of host defence mechanisms, including clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9), ISG expression libraries and RNA interference (RNAi) technologies.

Cytomegalovirus (CMV) causes clinically important diseases in immune compromised and immune immature individuals. Based largely on work in the mouse model of murine (M)CMV, there is a consensus that myeloid cells are important for disseminating CMV from the site of infection. In theory, such dissemination should expose CMV to cell-mediated immunity and thus necessitate evasion of T cells and NK cells. However, this hypothesis remains untested. We constructed a recombinant MCMV encoding target sites for the hematopoietic specific miRNA miR-142-3p in the essential viral gene IE3. This virus disseminated poorly to the salivary gland following intranasal or footpad infections but not following intraperitoneal infection in C57BL/6 mice, demonstrating that dissemination by hematopoietic cells is essential for specific routes of infection. Remarkably, depletion of NK cells or T cells restored dissemination of this virus in C57BL/6 mice after intranasal infection, while dissemination occurred normally in BALB/c mice, which lack strong NK cell control of MCMV. These data show that cell-mediated immunity is responsible for restricting MCMV to hematopoietic cell-mediated dissemination. Infected hematopoietic cells avoided cell-mediated immunity via three immune evasion genes that modulate class I MHC and NKG2D ligands (m04, m06 and m152). MCMV lacking these 3 genes spread poorly to the salivary gland unless NK cells were depleted, but also failed to replicate persistently in either the nasal mucosa or salivary gland unless CD8+ T cells were depleted. Surprisingly, CD8+ T cells primed after intranasal infection required CD4+ T cell help to expand and become functional. Together, our data suggest that MCMV can use both hematopoietic cell-dependent and -independent means of dissemination after intranasal infection and that cell mediated immune responses restrict dissemination to infected hematopoietic cells, which are protected from NK cells during dissemination by viral immune evasion. In contrast, viral replication within mucosal tissues depends on evasion of T cells.

Ovine pulmonary adenocarcinoma (OPA) is an infectious neoplastic lung disease of sheep caused by jaagsiekte sheep retrovirus. OPA is an important veterinary problem and is also a valuable large animal model for human lung adenocarcinoma. JSRV infects type 2 alveolar epithelial cells in the lung and induces the growth of tumors, but little is known about the molecular events that lead to the activation of oncogenic pathways in infected cells. MicroRNAs (miRNAs) are small RNA molecules of approximately 22 nucleotides with important roles in regulating gene expression in eukaryotes and with well-established roles in cancer. Here we used small-RNA sequencing to investigate the changes in miRNA expression that occur in JSRV-infected ovine lung. After filtering out low abundance miRNAs, we identified expression of 405 miRNAs, 32 of which were differentially expressed in JSRV-infected lung compared to mock-inoculated control lung. Highly upregulated miRNAs included miR-182, miR-183, miR-96 and miR-135b, which have also been associated with oncogenic changes in human lung cancer. Network analysis of genes potentially targeted by the deregulated miRNAs identified their involvement in pathways known to be dysregulated in OPA. We found no evidence to support the existence of miRNAs encoded by JSRV. This study provides the first information on miRNA expression in OPA and identifies a number of targets for future studies into the role of these molecules in the pathogenesis of this unique veterinary model for human lung adenocarcinoma.IMPORTANCE Ovine pulmonary adenocarcinoma is a neoplastic lung disease of sheep caused by jaagsiekte sheep retrovirus (JSRV). OPA is a significant welfare and economic concern for sheep producers and is a valuable large animal model for human lung adenocarcinoma. MicroRNAs are small RNA molecules of approximately 22 nucleotides with important functions in regulating gene expression in eukaryotes and with well-established roles in cancer. In this study, we examined the changes in microRNA expression that occur in the lung in response to JSRV infection. We identified differential expression of a number of host-encoded microRNAs in infected tissue, including microRNAs with roles in human cancer. We found no evidence that JSRV encodes a microRNA. This study provides new insights on the cellular response to JSRV infection in the ovine lung, which will inform future studies into the pathogenesis of OPA in sheep and its use as a model for human lung adenocarcinoma.

The genomes of RNA and small DNA viruses of vertebrates display significant suppression of CpG dinucleotide frequencies. Artificially increasing dinucleotide frequencies results in substantial attenuation of virus replication, suggesting that these compositional changes may facilitate recognition of non-self RNA sequences. Recently, the interferon inducible protein ZAP, was identified as the host factor responsible for sensing CpG in viral RNA, through direct binding and possibly downstream targeting for degradation. Using an arrayed interferon stimulated gene expression library screen, we identified ZAPS, and its associated factor TRIM25, as direct inhibitors of human cytomegalovirus (HCMV) replication. Exogenous expression of ZAPS and TRIM25 significantly reduced virus replication while knockdown resulted in increased virus replication. HCMV displays a strikingly heterogeneous pattern of CpG representation with a specific suppression of CpGs within the IE1 major immediate early transcript which is absent in subsequently expressed genes. We demonstrated that suppression of CpG dinucleotides in the IE1 gene allows evasion of inhibitory effects of ZAP. During HCMV infection, expression of ZAP and TRIM25 are rapidly reduced, removing pressure to suppress dinucleotide frequencies in viral genes expressed after the immediate early genes, while acute virus replication and high levels of ZAP are mutually exclusive. Finally, we show that TRIM25 regulates alternative splicing between the ZAP short and long isoforms during HCMV infection and interferon induction, with knockdown of TRIM25 resulting in decreased ZAPS and corresponding increased ZAPL expression. These results demonstrate for the first time that ZAP is a potent host restriction factor against large DNA viruses and that HCMV evades ZAP detection through suppression of CpG dinucleotides within the major immediate early transcripts. Furthermore, TRIM25 is required for efficient upregulation of the interferon inducible short isoform of ZAP through regulation of alternative splicing.

CpG dinucleotides are under-represented in the genomes of single-stranded RNA viruses, and SARS-CoV-2 is no exception to this. Artificial modification of CpG frequency is a valid approach for live attenuated vaccine development; if this is to be applied to SARS-CoV-2, we must first understand the role CpG motifs play in regulating SARS-CoV-2 replication. Accordingly, the CpG composition of the SARS-CoV-2 genome was characterised. CpG suppression among coronaviruses does not differ between virus genera but does vary with host species and primary replication site (a proxy for tissue tropism), supporting the hypothesis that viral CpG content may influence cross-species transmission. Although SARS-CoV-2 exhibits overall strong CpG suppression, this varies considerably across the genome, and the Envelope (E) open reading frame (ORF) and ORF10 demonstrate an absence of CpG suppression. Across the Coronaviridae, E genes display remarkably high variation in CpG composition, with those of SARS and SARS-CoV-2 having much higher CpG content than other coronaviruses isolated from humans. This is an ancestrally derived trait reflecting their bat origins. Conservation of CpG motifs in these regions suggests that they have a functionality which over-rides the need to suppress CpG; an observation relevant to future strategies towards a rationally attenuated SARS-CoV-2 vaccine.

As a first line of defence, the interferon-mediated innate immune response is pivotal to protect cells against invading pathogens. At the heart of it are hundreds of interferon-stimulated genes (ISGs) upregulated substantially shortly after viral infection. Some of these ISGs act indirectly to interfere with virus life cycle via regulation of interferon signalling pathways whereas other ISG can act directly to inhibit viral replication via interaction with viral components. Although many of the ISGs have been identified decades ago, only a handful of them have been characterized about their antiviral activity. Here, we used an arrayed expression lentivirus library of more than 400 ISGs to identify the ISGs with anti-HCMV activity. We performed parallel screens using wild type fibroblast cells and IRF3 KO fibroblast cells generated by CRISPR/Cas9 editing to identify ISGs more likely to directly inhibit HCMV, as opposed to activation of IFN signalling. The IRF3-independent ISGs identified in the screen include those that signal through IRF3 independent pathways such as IRF7 or known to inhibit HCMV directly such as IDO, RIPK2 and AIM2 validating the screening approach. Interestingly, we also identified novel IRF3-independent anti-HCMV ISGs, indicating they may play a role in directly inhibiting the virus.

Human Cytomegalovirus (HCMV) is a highly prevalent herpesvirus, persistently infecting between 30 and 100% of the population, depending on socio-economic status (Fields et al., 2013). HCMV remains an important clinical pathogen accounting for more than 60% of complications associated with solid organ transplant patients (Kotton, 2013; Kowalsky et al., 2013; Bruminhent and Razonable, 2014). It is also the leading cause of infectious congenital birth defects and has been linked to chronic inflammation and immune aging (Ballard et al., 1979; Griffith et al., 2016; Jergovic et al., 2019). There is currently no effective vaccine and HCMV antivirals have significant side effects. As current antivirals target viral genes, the virus can develop resistance, reducing drug efficacy. There is therefore an urgent need for new antiviral agents that are effective against HCMV, have better toxicity profiles and are less vulnerable to the emergence of resistant strains. Targeting of host factors that are critical to virus replication is a potential strategy for the development of novel antivirals that circumvent the development of viral resistance. Systematic high throughput approaches provide powerful methods for the identification of novel host-virus interactions. As well as contributing to our basic understanding of virus and cell biology, such studies provide potential targets for the development of novel antiviral agents. High-throughput studies, such as RNA sequencing, proteomics, and RNA interference screens, are useful tools to identify HCMV-induced global changes in host mRNA and protein expression levels and host factors important for virus replication. Here, we summarize new findings on HCMV lytic infection from high-throughput studies since 2014 and how screening approaches have evolved.

Equine trypanosomosis comprises different parasitic diseases caused by protozoa of the subgenusTrypanozoon:Trypanosoma equiperdum(causative agent of dourine),Trypanosoma brucei(nagana) andTrypanosoma evansi(surra). Due to the absence of a vaccine and the lack of efficacy of the few available drugs, these diseases represent a major health and economic problem for international equine trade. Development of affordable, sensitive and specific diagnostic tests is therefore crucial to ensure the control of these diseases. Recently, it has been shown that a small RNA derived from the 7SL gene (7SL-sRNA) is produced in high concentrations in sera of cattle infected withTrypanosoma congolense,Trypanosoma vivaxandTrypanosoma brucei. Our objective was to determine whether 7SL-sRNA could serve as a marker of active infection in equids experimentally infected withTrypanosoma equiperdumby analysing the sensitivity, specificity and stability of the 7SL-sRNA. Using a two-step RT-qPCR, we were able to detect the presence of 7SL-sRNA between 2 and 7 days post-infection, whereas seroconversion was detected by complement fixation test between 5 and 14 days post-infection. There was a rapid loss of 7SL-sRNA signal from the blood of infected animals one day post-trypanocide treatment. The 7SL-sRNA RT-qPCR allowed an early detection of a treatment failure revealed by glucocorticoid-induced immunosuppression. In addition, the 7SL-sRNA remains detectable in positive sera after 7 days of storage at either 4 degrees C, room temperature or 30 degrees C, suggesting that there is no need to refrigerate serum samples before analysis. Our findings demonstrate continual detection of 7SL-sRNA over an extended period of experimental infection, with signals detected more than six weeks after inoculation. The detection of a strong and consistent 7SL-sRNA signal even during subpatent parasitemia and the early detection of treatment failure highlight the very promising nature of this new diagnostic method.