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The zoonotic emerging Rift Valley fever virus (RVFV) causes sporadic disease in livestock and humans throughout Africa and the Saudi Arabian peninsula. Infection of people with RVFV can occur through mosquito bite or mucosal exposure during butchering or milking of infected livestock. Disease typically presents as a self-limiting fever; however, in rare cases, hepatitis, encephalitis and ocular disease may occur. Recent studies have illuminated the neuropathogenic mechanisms of RVFV in a rat aerosol infection model. Neurological disease in rats is characterized by breakdown of the blood–brain barrier late in infection, infiltration of leukocytes to the central nervous system (CNS) and massive viral replication in the brain. However, the route of RVFV entry into the CNS after inhalational exposure remains unknown. Here, we visualized the entire nasal olfactory route from snout to brain after RVFV infection using RNA in situ hybridization and immunofluorescence microscopy. We found widespread RVFV-infected cells within the olfactory epithelium, across the cribriform plate, and in the glomerular region of the olfactory bulb within 2 days of infection. These results indicate that the olfactory tract is a major route of infection of the brain after inhalational exposure. A better understanding of potential neuroinvasion pathways can support the design of more effective therapeutic regiments for the treatment of neurological disease caused by RVFV.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged at the end of 2019 and by mid-June 2020 the virus had spread to at least 215 countries, caused more than 8 000 000 confirmed infections and over 450 000 deaths, and overwhelmed healthcare systems worldwide. Like severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low-passage (P) strains of SARS-CoV-2 (Wash1 : P4 and Munich : P1) were cultured twice in Vero E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1 : P6 and minor variants in the Munich : P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated that the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies, we investigated the development of neutralizing antibody titres in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

Arenaviruses are a significant cause of hemorrhagic fever, an often-fatal disease for which there is no approved antiviral therapy. Lassa fever in particular generates high morbidity and mortality in West Africa, where the disease is endemic, and a recent outbreak in Nigeria was larger and more geographically diverse than usual. We are developing LHF-535, a small-molecule viral entry inhibitor that targets the arenavirus envelope glycoprotein, as a therapeutic candidate for Lassa fever and other hemorrhagic fevers of arenavirus origin. Using a lentiviral pseudotype infectivity assay, we determined that LHF-535 had sub-nanomolar potency against the viral envelope glycoproteins from all Lassa virus lineages, with the exception of the glycoprotein from the LP strain from lineage I, which was 100-fold less sensitive than that of other strains. This reduced sensitivity was mediated by a unique amino acid substitution, V434I, in the transmembrane domain of the envelope glycoprotein GP2 subunit. This position corresponds to the attenuation determinant of Candid#1, a live-attenuated Junín virus vaccine strain used to prevent Argentine hemorrhagic fever. Using a virus-yield reduction assay, we determined that LHF-535 potently inhibited Junín virus, but not Candid#1, and the Candid#1 attenuation determinant, F427I, regulated this difference in sensitivity. We also demonstrated that a daily oral dose of LHF-535 at 10 mg/kg protected mice from a lethal dose of Tacaribe virus. Serial passage of Tacaribe virus in LHF-535-treated Vero cells yielded viruses that were resistant to LHF-535, and the majority of drug-resistant viruses exhibited attenuated pathogenesis. These findings provide a framework for the clinical development of LHF-535 as a broad-spectrum inhibitor of arenavirus entry and provide an important context for monitoring the emergence of drug-resistant viruses., Author summary Lassa fever is a viral hemorrhagic fever disease that is transmitted to humans primarily through contact with the urine or feces of infected rodents. The disease is endemic in West Africa, and an unusually large outbreak occurred in Nigeria in early 2018. The case fatality rate was 25% among confirmed cases, underscoring the need for an effective antiviral therapy. Here, we evaluated the small-molecule drug LHF-535, which targets the arenavirus envelope glycoprotein, for broad-spectrum activity against Lassa viruses of different lineages and related arenaviruses that cause hemorrhagic fever diseases in South America. We also selected for LHF-535-resistant viruses and characterized their genotype and phenotype. Using a combination of surrogate systems and wild-type viruses, we determined that all tested Lassa virus strains and New World hemorrhagic fever arenaviruses were sensitive to LHF-535. Sensitivity to the drug was modulated by specific amino acid changes in the viral envelope glycoprotein, and the majority of emerging drug-resistant viruses were attenuated for virulence. Similarly, the live-attenuated vaccine strain for Argentine hemorrhagic fever was also resistant to LHF-535. These findings indicate that LHF-535 targets a viral virulence determinant, the mutation of which may result in the emergence of drug-resistant viruses, but with reduced capacity for virulence.

As part of on-going efforts to control hookworm infection, the “human hookworm vaccine initiative” has recognised blood feeding as a feasible therapeutic target for inducing immunity against hookworm infection. To this end, molecular approaches have been used to identify candidate targets, such as Necator americanus (Na) haemoglobinase aspartic protease-1 (APR-1), with immunogenicity profiled in canine and hamster models. We sought to accelerate the immune analysis of these identified therapeutic targets by developing an appropriate mouse model. Here we demonstrate that Nippostrongylus brasiliensis (Nb), a phylogenetically distant strongylid nematode of rodents, begins blood feeding early in its development and that immunisation with Na-APR-1 can block its growth and completion of its life cycle. Furthermore, we identify a new haem detoxification pathway in Nb required for blood feeding that can be blocked by drugs of the quinolone family, reducing both infection burden and the associated anaemia in rodents. Collectively, our findings show that haem metabolism has potential as a checkpoint for interrupting hookworm development in early stages of the hookworm life cycle and that the Nippostrongylus brasiliensis rodent model is relevant for identifying novel therapeutic targets against human hookworm., Author summary Hookworm infections (Necator americanus or Ancylostoma duodenale) represent a major neglected tropical disease affecting approximately 450 million people worldwide and causing morbidity due to their need to feed on host blood resulting in severe anemia. New chemotherapy and vaccines are needed to combat hookworm infections. Using a rodent parasite model, we describe a new haem detoxification pathway that is a metabolic checkpoint for parasite development, survival and reproduction. This provides a starting point for the development of novel therapies against such metazoan blood-feeders.

The equine herpesvirus type 1 (EHV-1) ORF1 and ORF71 genes have immune modulatory effects in vitro. Experimental infection of horses using virus mutants with multiple deletions including ORF1 and ORF71 showed promise as vaccine candidates against EHV-1. Here, the combined effects of ORF1 and ORF71 deletions from the neuropathogenic EHV-1 strain Ab4 on clinical disease and host immune response were further explored. Three groups of EHV-1 naive horses were experimentally infected with the ORF1/71 gene deletion mutant (Ab4ΔORF1/71), the parent Ab4 strain, or remained uninfected. In comparison to Ab4, horses infected with Ab4ΔORF1/71 did not show the initial high fever peak characteristic of EHV-1 infection. Ab4ΔORF1/71 infection had reduced nasal shedding (1/5 vs. 5/5) and, simultaneously, decreased intranasal interferon (IFN)-α, interleukin (IL)-10 and soluble CD14 secretion. However, Ab4 and Ab4ΔORF1/71 infection resulted in comparable viremia, suggesting these genes do not regulate the infection of the mononuclear cells and subsequent viremia. Intranasal and serum anti-EHV-1 antibodies to Ab4ΔORF1/71 developed slightly slower than those to Ab4. However, beyond day 12 post infection (d12pi) serum antibodies in both virus-infected groups were similar and remained increased until the end of the study (d114pi). EHV-1 immunoglobulin (Ig) G isotype responses were dominated by short-lasting IgG1 and long-lasting IgG4/7 antibodies. The IgG4/7 response closely resembled the total EHV-1 specific antibody response. Ex vivo re-stimulation of PBMC with Ab4 resulted in IFN-γ and IL-10 secretion by cells from both infected groups within two weeks pi. Flow cytometric analysis showed that IFN-γ producing EHV-1-specific T-cells were mainly CD8+/IFN-γ+ and detectable from d32pi on. Peripheral blood IFN-γ+ T-cell percentages were similar in both infected groups, albeit at low frequency (~0.1%). In summary, the Ab4ΔORF1/71 gene deletion mutant is less virulent but induced antibody responses and cellular immunity similar to the parent Ab4 strain.

Recent avian and swine-origin influenza virus outbreaks illustrate the ongoing threat of influenza pandemics. We investigated immunogenicity and protective efficacy of a multi-antigen (MA) universal influenza DNA vaccine consisting of HA, M2, and NP antigens in cynomolgus macaques. Following challenge with a heterologous pandemic H1N1 strain, vaccinated animals exhibited significantly lower viral loads and more rapid viral clearance when compared to unvaccinated controls. The MA DNA vaccine induced robust serum and mucosal antibody responses but these high antibody titers were not broadly neutralizing. In contrast, the vaccine induced broadly-reactive NP specific T cell responses that cross-reacted with the challenge virus and inversely correlated with lower viral loads and inflammation. These results demonstrate that a MA DNA vaccine that induces strong cross-reactive T cell responses can, independent of neutralizing antibody, mediate significant cross-protection in a nonhuman primate model and further supports development as an effective approach to induce broad protection against circulating and emerging influenza strains.

The viruses circulating among Antarctic wildlife remain largely unknown. In an effort to identify viruses associated with Weddell seals (Leptonychotes weddellii) inhabiting the Ross Sea, vaginal and nasal swabs, and faecal samples were collected between November 2014 and February 2015. In addition, a Weddell seal kidney and South Polar skua (Stercorarius maccormicki) faeces were opportunistically sampled. Using high throughput sequencing, we identified and recovered 152 anellovirus genomes that share 63–70% genome-wide identities with other pinniped anelloviruses. Genome-wide pairwise comparisons coupled with phylogenetic analysis revealed two novel anellovirus species, tentatively named torque teno Leptonychotes weddellii virus (TTLwV) -1 and -2. TTLwV-1 (n = 133, genomes encompassing 40 genotypes) is highly recombinant, whereas TTLwV-2 (n = 19, genomes encompassing three genotypes) is relatively less recombinant. This study documents ubiquitous TTLwVs among Weddell seals in Antarctica with frequent co-infection by multiple genotypes, however, the role these anelloviruses play in seal health remains unknown.

Background Development of antiviral drugs that have broad-spectrum activity against a number of viral infections would be of significant benefit. Due to the evolution of resistance to currently licensed antiviral drugs, development of novel anti-influenza drugs is in progress, including Favipiravir (T-705), which is currently in human clinical trials. T-705 displays broad-spectrum in vitro activity against a number of viruses, including Rift Valley Fever virus (RVFV). RVF is an important neglected tropical disease that causes human, agricultural, and economic losses in endemic regions. RVF has the capacity to emerge in new locations and also presents a potential bioterrorism threat. In the current study, the in vivo efficacy of T-705 was evaluated in Wistar-Furth rats infected with the virulent ZH501 strain of RVFV by the aerosol route. Methodology/Principal Findings Wistar-Furth rats are highly susceptible to a rapidly lethal disease after parenteral or inhalational exposure to the pathogenic ZH501 strain of RVFV. In the current study, two experiments were performed: a dose-determination study and a delayed-treatment study. In both experiments, all untreated control rats succumbed to disease. Out of 72 total rats infected with RVFV and treated with T-705, only 6 succumbed to disease. The remaining 66 rats (92%) survived lethal infection with no significant weight loss or fever. The 6 treated rats that succumbed survived significantly longer before succumbing to encephalitic disease. Conclusions/Significance Currently, there are no licensed antiviral drugs for treating RVF. Here, T-705 showed remarkable efficacy in a highly lethal rat model of Rift Valley Fever, even when given up to 48 hours post-infection. This is the first study to show protection of rats infected with the pathogenic ZH501 strain of RVFV. Our data suggest that T-705 has potential to be a broad-spectrum antiviral drug., Author Summary Broad-spectrum antiviral drugs are preferred because they have the capacity to treat a range of viral illnesses rather than just one. Food and Drug Administration (FDA) approval of antiviral drugs to treat neglected tropical diseases is difficult to obtain due to ethical and logistical considerations when conducting human clinical trials. Rift Valley Fever (RVF) is an endemic tropical disease that causes human morbidity and mortality, as well as economic damage to the livestock industry. There are no licensed antiviral drugs to treat RVF. In this study, we found that a novel anti-influenza drug, Favipiravir (T-705), is able to prevent lethal RVF in rats, and therefore shows promise as a broad-spectrum antiviral treatment.

Homologs of the LuxR acyl-homoserine lactone (AHL) quorum-sensing signal receptor are prevalent in Proteobacteria isolated from roots of the Eastern cottonwood tree, Populus deltoides. Many of these isolates possess an orphan LuxR homolog, closely related to OryR from the rice pathogen Xanthomonas oryzae. OryR does not respond to AHL signals but, instead, responds to an unknown plant compound. We discovered an OryR homolog, PipR, in the cottonwood endophyte Pseudomonas sp. strain GM79. The genes adjacent to pipR encode a predicted ATP-binding cassette (ABC) peptide transporter and peptidases. We purified the putative peptidases, PipA and AapA, and confirmed their predicted activities. A transcriptional pipA-gfp reporter was responsive to PipR in the presence of plant leaf macerates, but it was not influenced by AHLs, similar to findings with OryR. We found that PipR also responded to protein hydrolysates to activate pipA-gfp expression. Among many peptides tested, the tripeptide Ser-His-Ser showed inducer activity but at relatively high concentrations. An ABC peptide transporter mutant failed to respond to leaf macerates, peptone, or Ser-His-Ser, while peptidase mutants expressed higher-than-wild-type levels of pipA-gfp in response to any of these signals. Our studies are consistent with a model where active transport of a peptidelike signal is required for the signal to interact with PipR, which then activates peptidase gene expression. The identification of a peptide ligand for PipR sets the stage to identify plant-derived signals for the OryR family of orphan LuxR proteins., IMPORTANCE We describe the transcription factor PipR from a Pseudomonas strain isolated as a cottonwood tree endophyte. PipR is a member of the LuxR family of transcriptional factors. LuxR family members are generally thought of as quorum-sensing signal receptors, but PipR is one of an emerging subfamily of LuxR family members that respond to compounds produced by plants. We found that PipR responds to a peptidelike compound, and we present a model for Pip system signal transduction. A better understanding of plant-responsive LuxR homologs and the compounds to which they respond is of general importance, as they occur in dozens of bacterial species that are associated with economically important plants and, as we report here, they also occur in members of certain root endophyte communities.

Swine influenza presents a substantial disease burden for pig populations worldwide and poses a potential pandemic threat to humans. There is considerable diversity in both H1 and H3 influenza viruses circulating in swine due to the frequent introductions of viruses from humans and birds coupled with geographic segregation of global swine populations. Much of this diversity is characterized genetically but the antigenic diversity of these viruses is poorly understood. Critically, the antigenic diversity shapes the risk profile of swine influenza viruses in terms of their epizootic and pandemic potential. Here, using the most comprehensive set of swine influenza virus antigenic data compiled to date, we quantify the antigenic diversity of swine influenza viruses on a multi-continental scale. The substantial antigenic diversity of recently circulating viruses in different parts of the world adds complexity to the risk profiles for the movement of swine and the potential for swine-derived infections in humans. DOI: http://dx.doi.org/10.7554/eLife.12217.001, eLife digest Influenza viruses, commonly called flu, infect millions of people and animals every year and occasionally causes pandemics in humans. The immune system can neutralise flu viruses by recognising the proteins on the virus surface, generically referred to as antigens. These antigens change as flu viruses evolve to escape detection by the immune system. These changes tend to be relatively small such that exposure to one flu virus generates immunity that is still effective against other related flu viruses. However, over time, the accumulation of these small changes can result in larger differences such that prior infections no longer provide protection against the new virus. Influenza A viruses infect a wide variety of birds and mammals. Viruses can also transmit from one species to another, which may result in the introduction of viruses with antigens that are new to the recipient species and which have the potential to cause substantial outbreaks. Pig flu viruses have long been considered to be a potential risk for human pandemic viruses and were the source of the 2009 pandemic H1N1 virus. Importantly, humans often transmit flu viruses to pigs. Understanding the dynamics and consequences of this two-way transmission is important for designing effective strategies to detect and respond to new strains of flu. Influenza A viruses of the H1 and H3 subtypes circulate widely in pigs. However, it was poorly understood how closely related swine and human viruses circulating in different regions were to one another and how much the antigens varied between the different viruses. Lewis, Russell et al. have now analysed the antigenic variation of hundreds of H1 and H3 viruses from pigs on multiple continents. The antigenic diversity of recent swine flu viruses resembles the diversity of H1 and H3 viruses observed in humans over the last 40 years. A key factor driving the diversity of the H1 and H3 viruses in pigs is the frequent introduction of human viruses to pigs. In contrast, only one flu virus from a bird had contributed to the observed antigenic diversity in pigs in a substantial way. Once in pigs, human-derived flu viruses continue to evolve their antigens. This results in a tremendous diversity of flu viruses that can be transmitted to other pigs and also to humans. These flu viruses could pose a serious risk to public health because they are no longer similar to the current human flu strains. These findings have important implications not only for developing flu vaccines for pigs but also for informing the development of more-effective surveillance and disease-control strategies to prevent the spread of new flu variants. DOI: http://dx.doi.org/10.7554/eLife.12217.002

Objective Cationic proteins found in cervicovaginal secretions (CVS) are known to contribute to the early antiviral immune response against HIV-infection in vitro. We here aimed to define additional antiviral factors that are over-expressed in CVS from female sex workers at high risk of infection. Methods CVS were collected from Kenyan HIV-seronegative (n = 34) and HIV-seropositive (n = 12) female sex workers, and were compared with those from HIV-seronegative low-risk women (n = 12). The highly exposed seronegative (HESN) sex workers were further divided into those with less (n = 22) or more (n = 12) than three years of documented sex work. Cationic protein-depleted CVS were assessed for HIV-neutralizing activity by a PBMC-based HIV-neutralizing assay, and then characterized by proteomics. Results HIV neutralizing activity was detected in all unprocessed CVS, however only CVS from the female sex worker groups maintained its HIV neutralizing activity after cationic protein-depletion. Differentially abundant proteins were identified in the cationic protein-depleted secretions including 26, 42, and 11 in the HESN>3yr, HESN

Swine influenza causes concern for global veterinary and public health officials. In continuing two previous networks that initiated the surveillance of swine influenza viruses (SIVs) circulating in European pigs between 2001 and 2008, a third European Surveillance Network for Influenza in Pigs (ESNIP3, 2010-2013) aimed to expand widely the knowledge of the epidemiology of European SIVs. ESNIP3 stimulated programs of harmonized SIV surveillance in European countries and supported the coordination of appropriate diagnostic tools and subtyping methods. Thus, an extensive virological monitoring, mainly conducted through passive surveillance programs, resulted in the examination of more than 9 000 herds in 17 countries. Influenza A viruses were detected in 31% of herds examined from which 1887 viruses were preliminary characterized. The dominating subtypes were the three European enzootic SIVs: avian-like swine H1N1 (53.6%), human-like reassortant swine H1N2 (13%) and human-like reassortant swine H3N2 (9.1%), as well as pandemic A/H1N1 2009 (H1N1pdm) virus (10.3%). Viruses from these four lineages co-circulated in several countries but with very different relative levels of incidence. For instance, the H3N2 subtype was not detected at all in some geographic areas whereas it was still prevalent in other parts of Europe. Interestingly, H3N2-free areas were those that exhibited highest frequencies of circulating H1N2 viruses. H1N1pdm viruses were isolated at an increasing incidence in some countries from 2010 to 2013, indicating that this subtype has become established in the European pig population. Finally, 13.9% of the viruses represented reassortants between these four lineages, especially between previous enzootic SIVs and H1N1pdm. These novel viruses were detected at the same time in several countries, with increasing prevalence. Some of them might become established in pig herds, causing implications for zoonotic infections.

International audience; Respiratory infections stemming from influenza viruses and the Severe Acute Respiratory Syndrome corona virus (SARS-CoV) represent a serious public health threat as emerging pandemics. Despite efforts to identify the critical interactions of these viruses with host machinery, the key regulatory events that lead to disease pathology remain poorly targeted with therapeutics. Here we implement an integrated network interrogation approach, in which proteome and transcriptome datasets from infection of both viruses in human lung epithelial cells are utilized to predict regulatory genes involved in the host response. We take advantage of a novel ``crowd-based'' approach to identify and combine ranking metrics that isolate genes/proteins likely related to the pathogenicity of SARS-CoV and influenza virus. Subsequently, a multivariate regression model is used to compare predicted lung epithelial regulatory influences with data derived from other respiratory virus infection models. We predicted a small set of regulatory factors with conserved behavior for consideration as important components of viral pathogenesis that might also serve as therapeutic targets for intervention. Our results demonstrate the utility of integrating diverse `omic datasets to predict and prioritize regulatory features conserved across multiple pathogen infection models.

Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide, are peptides designed to competitively inhibit the viral fusion protein gp41. With the emergence of drug resistance, there is an increased need for effective and unique alternatives within this class of antivirals. One such alternative is a class of cyclic, cationic, antimicrobial peptides known as θ-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the θ-defensin analog RC-101 is being developed as a microbicide due to its specific antiviral activity, lack of toxicity to cells and tissues, and safety in animals. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development. In previous studies, we identified a mutant, R5-tropic virus that had evolved partial resistance to RC-101 during in vitro selection. Here, we report that a secondary mutation in gp41 was found to restore replicative fitness, membrane fusion, and the rate of viral entry, which were compromised by an initial mutation providing partial RC-101 resistance. Interestingly, we show that RC-101 is effective against two enfuvirtide-resistant mutants, demonstrating the clinical importance of RC-101 as a unique fusion inhibitor. These findings both expand our understanding of HIV drug-resistance to diverse peptide fusion inhibitors and emphasize the significance of compensatory gp41 mutations.

BACKGROUNDThe Canadian National Antiviral Stockpile (NAS) contains treatment for 17.5% of Canadians. This assumes no concurrent intervention strategies and no wastage due to non-influenza respiratory infections. A dynamic model can provide a mechanism to consider complex scenarios to support decisions regarding the optimal NAS size under uncertainty.METHODSWe developed a dynamic model for pandemic influenza in Canada that is structured by age and risk to calculate the demand for antivirals to treat persons with pandemic influenza under a wide-range of scenarios that incorporated transmission dynamics, disease severity, and intervention strategies. The anticipated per capita number of acute respiratory infections due to viruses other than influenza was estimated for the full pandemic period from surveys based on criteria to identify potential respiratory infections.RESULTSOur results demonstrate that up to two thirds of the population could develop respiratory symptoms as a result of infection with a pandemic strain. In the case of perfect antiviral allocation, up to 39.8% of the population could request antiviral treatment. As transmission dynamics, severity and timing of the emergence of a novel influenza strain are unknown, the sensitivity analysis produced considerable variation in potential demand (median: 11%, IQR: 2-21%). If the next pandemic strain emerges in late spring or summer and a vaccine is available before the anticipated fall wave, the median prediction was reduced to 6% and IQR to 0.7-14%. Under the strategy of offering empirical treatment to all patients with influenza like symptoms who present for care, demand could increase to between 65 and 144%.CONCLUSIONSThe demand for antivirals during a pandemic is uncertain. Unless an accurate, timely and cost-effective test is available to identify influenza cases, demand for antivirals from persons infected with other respiratory viruses will be substantial and have a significant impact on the NAS.

Influenza A viruses cause acute respiratory disease in swine. Viruses with H1 hemagglutinin genes from the human seasonal lineage (-cluster) have been isolated from North American swine since 2003. The objective of this work was to study the pathogenesis and transmission of -cluster H1 influenza viruses in swine, comparing three isolates from different phylogenetic subclusters, geographic locations, and years of isolation. Two isolates from the 2 subcluster, A/sw/MN/07002083/07 H1N1 (MN07) and A/sw/IL/00685/05 H1N1 (IL05), and A/sw/TX/01976/08 H1N2 (TX08) from the 1 sub-cluster were evaluated. All isolates caused disease and were transmitted to contact pigs. Respiratory disease was apparent in pigs infected with MN07 and IL05 viruses; however, clinical signs and lung lesions were reduced in severity as compared to TX08. On day 5 following infection MN07-infected pigs had lower virus titers than the TX08 pigs, suggesting that although this H1N1 was successfully transmitted, it may not replicate as efficiently in the upper or lower respiratory tract. MN07 and IL05 H1N1 induced higher serum antibody titers than TX08. Greater serological cross-reactivity was observed for viruses from the same HA phylogenetic sub-cluster; however, antigenic differences between the sub-clusters may have implications for disease control strategies for pigs.

Background HIV exposed seronegative (HESN) women represent the population most in need of a prophylactic antiviral strategy. Mucosal cationic polypeptides can potentially be regulated for this purpose and we here aimed to determine their endogenous expression and HIV neutralizing activity in genital secretions of women at risk of HIV infection. Methodology/Principal Findings Cervicovaginal secretions (CVS) of Kenyan women in HIV-serodiscordant relationships (HESN, n = 164; HIV seropositive, n = 60) and low-risk controls (n = 72) were assessed for the cationic polypeptides HNP1–3, LL-37 and SLPI by ELISA and for HIV neutralizing activity by a PBMC-based assay using an HIV primary isolate. Median levels of HNP1–3 and LL-37 in CVS were similar across study groups. Neither HSV-2 serostatus, nor presence of bacterial vaginosis, correlated with levels of HNP1–3 or LL-37 in the HESN women. However, an association with their partner's viral load was observed. High viral load (>10,000 HIV RNA copies/ml plasma) correlated with higher levels of HNP1–3 and LL-37 (p = 0.04 and 0.03, respectively). SLPI was most abundant in the low-risk group and did not correlate with male partner's viral load in the HESN women. HIV neutralizing activity was found in CVS of all study groups. In experimental studies, selective depletion of cationic polypeptides from CVS rendered the remaining CVS fraction non-neutralizing, whereas the cationic polypeptide fraction retained the activity. Furthermore, recombinant HNP1–3 and LL-37 could induce neutralizing activity when added to CVS lacking intrinsic activity. Conclusions/Significance These findings show that CVS from HESN, low-risk, and HIV seropositive women contain HIV neutralizing activity. Although several innate immune proteins, including HNP1–3 and LL-37, contribute to this activity these molecules can also have inflammatory properties. This balance is influenced by hormonal and environmental factors and in the present HIV serodiscordant couple cohort study we show that a partner's viral load is associated with levels of such molecules.

Many pathogenic bacteria, fungi, and protozoa achieve chronic infection through an immune evasion strategy known as antigenic variation. In the human malaria parasite Plasmodium falciparum, this involves transcriptional switching among members of the var gene family, causing parasites with different antigenic and phenotypic characteristics to appear at different times within a population. Here we use a genome-wide approach to explore this process in vitro within a set of cloned parasite populations. Our analyses reveal a non-random, highly structured switch pathway where an initially dominant transcript switches via a set of switch-intermediates either to a new dominant transcript, or back to the original. We show that this specific pathway can arise through an evolutionary conflict in which the pathogen has to optimise between safeguarding its limited antigenic repertoire and remaining capable of establishing infections in non-naïve individuals. Our results thus demonstrate a crucial role for structured switching during the early phases of infections and provide a unifying theory of antigenic variation in P. falciparum malaria as a balanced process of parasite-intrinsic switching and immune-mediated selection., Author Summary The malaria parasite Plasmodium falciparum avoids recognition and clearance by the immune system by sequentially switching between members of the var multi-gene family which encode the immunodominant surface proteins PfEMP1. However, some mechanism must exist to prevent rapid exposure of the pathogen's entire antigenic repertoire as this would quickly terminate the infection. It has previously been shown that the immune system can play an important role in orchestrating the sequential display of variants once an infection is established; however this does not explain how repertoire exhaustion is avoided in the initial phases of infection before an immune response has been established. Here we show that P. falciparum has evolved a highly structured switching pattern to prevent repertoire exhaustion in the early stages of infection without compromising the ability to establish new infections among partially immune individuals.

We recently reported that HIV-1 infection can be inhibited by innate antimicrobial components of human seminal plasma (SP). Conversely, naturally occurring peptidic fragments from the SP-derived prostatic acid phosphatase ( PAP) have been reported to form amyloid fibrils called "SEVI'' and enhance HIV-1 infection in vitro. In order to understand the biological consequence of this proviral effect, we extended these studies in the presence of human SP. PAP-derived peptides were agitated to form SEVI and incubated in the presence or absence of SP. While PAP-derived peptides and SEVI alone were proviral, the presence of 1% SP ablated their proviral activity in several different anti-HIV-1 assays. The anti-HIV-1 activity of SP was concentration dependent and was reduced following filtration. Supraphysiological concentrations of PAP peptides and SEVI incubated with diluted SP were degraded within hours, with SP exhibiting proteolytic activity at dilutions as high as 1:200. Sub-physiological concentrations of two prominent proteases of SP, prostate-specific antigen (PSA) and matriptase, could degrade physiological and supraphysiological concentrations of PAP peptides and SEVI. While human SP is a complex biological fluid, containing both antiviral and proviral factors, our results suggest that PAP peptides and SEVI may be subject to naturally occurring proteolytic components capable of reducing their proviral activity.

Although overall pollinator populations have declined over the last couple of decades, the honey bee (Apis mellifera) malady, colony collapse disorder (CCD), has caused major concern in the agricultural community. Among honey bee pathogens, RNA viruses are emerging as a serious threat and are suspected as major contributors to CCD. Recent detection of these viral species in bumble bees suggests a possible wider environmental spread of these viruses with potential broader impact. It is therefore vital to study the ecology and epidemiology of these viruses in the hymenopteran pollinator community as a whole. We studied the viral distribution in honey bees, in their pollen loads, and in other non-Apis hymenopteran pollinators collected from flowering plants in Pennsylvania, New York, and Illinois in the United States. Viruses in the samples were detected using reverse transcriptase-PCR and confirmed by sequencing. For the first time, we report the molecular detection of picorna-like RNA viruses (deformed wing virus, sacbrood virus and black queen cell virus) in pollen pellets collected directly from forager bees. Pollen pellets from several uninfected forager bees were detected with virus, indicating that pollen itself may harbor viruses. The viruses in the pollen and honey stored in the hive were demonstrated to be infective, with the queen becoming infected and laying infected eggs after these virus-contaminated foods were given to virus-free colonies. These viruses were detected in eleven other non-Apis hymenopteran species, ranging from many solitary bees to bumble bees and wasps. This finding further expands the viral host range and implies a possible deeper impact on the health of our ecosystem. Phylogenetic analyses support that these viruses are disseminating freely among the pollinators via the flower pollen itself. Notably, in cases where honey bee apiaries affected by CCD harbored honey bees with Israeli Acute Paralysis virus (IAPV), nearby non-Apis hymenopteran pollinators also had IAPV, while those near apiaries without IAPV did not. In containment greenhouse experiments, IAPV moved from infected honey bees to bumble bees and from infected bumble bees to honey bees within a week, demonstrating that the viruses could be transmitted from one species to another. This study adds to our present understanding of virus epidemiology and may help explain bee disease patterns and pollinator population decline in general.