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Splenocytes were depleted of erythrocytes by treatment with ACK lysis buffer. Sera from blood collected from the abdominal vena cava were isolated using BD Microtainers (Franklin Lakes,NJ), and decomplemented by heat-treating at 56°C for 30 minutes.
Two hundred nasal swabs provided by the Arkansas Department of Health were collected from March to July 2010 for influenza surveillance from patients showing respiratory disease symptoms. All patients exhibited influenza-like symptoms consisting of fever, chills, body aches, runny nose and/or nasal congestion. Patients had a median age of 56 years (ranging from <1 to 96 years of age); 47.5% were male and 52.5% female. No further information was available on the patients. Samples were collected in 3 ml of sterile viral transport medium, centrifuged, aliquoted and stored at −80°C until testing. All samples were screened using the CDC Human Influenza Virus Real Time RT-PCR Diagnostic Panel (cat# KT0096) and the CDC rRT-2009 A(H1N1) pdm Flu Panel (catalog #FLUSW04), by the Arkansas Department of Health. Only Influenza virus negative samples were selected for this study. All human samples were obtained by the Arkansas Department of Health according to institutional policies and Federal guidelines.
In comparison to non-viral infected individuals, viral-infected SARI ones had significantly lower rates of pneumonia (p=0.004) and admission to the ICU (p=0.000). Patients with influenza virus tended to have significantly different rates of admission to the ICU (p=0.045), and mechanical ventilation (p=0.001), in comparison to those with non-influenza infections. With regards to complications, viral-infected SARI patients had significant differences for developing respiratory failure (p=0.033), and acute respiratory distress syndrome; ARDS (p=0.011), in comparison to those without viral infections.
Overall mortality in SARI-positive patients was 24/1,075 (2.2%) and peaked at 1% in 2014. Overall, only 2(8%) were adults, while 22 (92%) were children. Among children, 18(75%) were aged <5 years. Overall, two-thirds (16/24) had comorbidities. All patients who died were admitted to the ICU and mechanically ventilated. Notably, all patients who died tested positive for a viral pathogen; twelve were positive for RSV, four for influenza virus, two for adenovirus, one for hMPV, one for PIV and four for mixed viral infections, respectively. Among those who died, there was a significant difference between those with (2.2%) and without (5%) viral detection (p = 0.005). Among individual viral pathogens, SARI patients with RSV and influenza had significant deaths (p= 0.045 and 0.006), in comparison to those with non-RSV and non-influenza viral infections. No mortality was reported for patients with atypical bacteria (Table 1).
Samples positive for human CoV by qRT-PCR but negative by RT-PCR, using the ORF1b primers, were passed in HRT-18 (human rectal tumor - ATCC cat# CCL-244), Vero (African green monkey kidney - ATCC cat# CCL-81) and/or MRC-5 (human fetal lung – ATCC cat# CCL-171) cells in an effort to isolate and further amplify CoV, if present. Briefly, cells were grown in supplemented Advanced-Minimum Essential Medium (Ad-MEM, Invitrogen), containing 1% antibiotic-antimycotic (Gibco) and 5% fetal bovine serum (FBS), or in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen), supplemented with 1% antibiotic-antimycotic and 10% FBS, for 2–5 days. Before infection, cells were rinsed and incubated with Minimum Essential Medium (MEM, Invitrogen) without FBS for 1–3 h at 37°C and 5% CO2. T25 cm2 flasks (Nunc, Inc.) were inoculated with 200–300 µL of filtered (0.22 µm) nasal swab fluids diluted 1:25 in MEM. After 1 hour of incubation at 37°C, the supernatant was removed and replaced with FBS free MEM containing 0.15 µg/ml of trypsin (Sigma cat# T1426). Cells were frozen when 50 to 80% cytopathic effects were evident or after 14 days of incubation. After a cycle of freezing and thawing, cell lysates were centrifuged for 15 min at 2,500 × g at 4°C, and the supernatants were stored for subsequent inoculation or used for viral RNA extraction. Samples were passaged until a RT-PCR band was detected, or for up to 10 blind passages in each cell line.
To enter the cat facility, the outer barrier and the inner barrier had to be passed (Figure 3). To pass the outer barrier, the following steps were necessary: Undressing of everyday clothes, change of shoes, hand wash with soap and disinfection with Desmanol pure alcoholic hand rub (Schülke, Schülke and Mayr AG, Zürich, Switzerland) and wearing of a single-use overall (Coverall PP, VWR International GmbH, Dietikon, Switzerland). The inner barrier consisted of the following steps: Change of shoes, undressing of the single-use overall, hand disinfection with Sterilium® Virugard (Paul Hartmann AG, Heidenheim, Germany), wearing of a clean cotton fabric overall, a clean pair of socks and a single-use surgical cap. To exit the cat facility, the inner barrier had to be passed firstly and the following steps were necessary: Disposal of the surgical cap to the bio hazard bin (Mauser, Benelux B.V., Oosterhout, The Netherlands), disposal of cotton overall and socks to the wash bin, hand wash with soap, hand disinfection with Sterilium® Virugard (Paul Hartmann AG), disinfection of shoes with Sterilium® Virugard (Paul Hartmann AG), showering of the body with soap, hair wash was only mandatory if the hair was in contact with the cats or anything else within the cat rooms, dressing of single-use overall and change of shoes. To pass the outer barrier, the following steps were necessary: Disposal of single-use overall to the normal bin, changing into everyday clothes and shoes.
Cairo University Hospital (CUH) is a 5100-beds tertiary referral teaching hospital. Inclusion criteria consisted of hospitalized adults (defined as age ≥ 18 years old), as well as pediatric patients (age < 18 years old), with the diagnosis of SARI, who provided a respiratory sample, from February 2010 to February 2014. Due to an annual review by dedicated investigators and updates to WHO guidelines, the case definition for SARI has evolved over the study period. Before February 2010, as a global-surveillance case definition of SARI did not exist, the definition for SARI was adapted from the WHO protocol on rapid response for persons ≥5 years old. Whereas, for children <5 years old, SARI definition was adapted from the program for Integrated Management of Childhood Illness. After March 2011, the global standards and tools for influenza surveillance developed by the WHO were adopted. As of January 2014, the WHO surveillance case definitions for SARI was implemented as follows, acute respiratory infection with history of fever or measured fever of ≥ 38 C°; and cough; with onset within the last 10 days; and requiring hospitalization. An enrollment form was used to collect data from enrolled eligible patients including patient demographics, medical history, clinical signs and symptoms, comorbidities, reported influenza vaccine status, recent travel history, treatment, clinical course, and outcome. Patients with incomplete medical records were excluded.
In the efforts to develop a universal influenza vaccine, various platforms for immunization to conserved antigens have been studied. Replication incompetent adenovirus vectors are promising, since their strong induction of innate immune responses provides a built-in adjuvant, and the antigen-specific B and T cell responses they induce are sustained for a long time. Animal adenoviruses have the potential advantage that humans have no prior exposure to them. For that reason chimpanzee adenoviruses have recently begun to be explored for use as vaccine vectors in humans, where they showed good safety and excellent immunogenicity,,. Furthermore, in tests of Ad5 and four chimpanzee adenovirus vectors, prior immunization with a GFP-expressing construct blocked subsequent responses to the transgene product only for homologous vector; cross-blocking was minimal.
In this study, we tested a new vector based on bonobo virus PanAd3. Both Ad5 and bonobo virus PanAd3 belong to the adenovirus species C. Species B (for example Ad35) has been shown in other studies to be less immunogenic than species C. Since they are so closely related, Ad5 and PanAd3 may share certain structural features providing powerful internal adjuvant effects and thus higher immunogenicity. Despite the structural similarity, human sera containing high anti-Ad5 neutralization titers (>1000) show no or marginal neutralization capacity on PanAd3. Moreover, in a screening study, PanAd3 was shown to be a potent vector in mice and in primates. We show here that a single administration of PanAd3-NPM1 influenza vaccine given i.n. provided highly effective protection against lethal challenge with mouse-adapted A/FM, with greatly reduced morbidity and mortality compared to controls. Protection was comparable to that in previously published studies of Ad5 expressing conserved influenza virus antigens for the same challenge virus and dose,.
The PanAd3 vaccine induced both antibody and T cell responses to NP. As mentioned earlier, the T cell response to NP is well-known to contribute to protection,–, and recent studies have reported that antibodies to NP can also contribute to protection.
The fusion protein of NP with M1 expressed by the PanAd3 vaccine has the advantage of including another major target of human immunity. Using multiple target antigens may invoke different immune mechanisms, reduce the likelihood of generating escape mutants, and provide a larger range of epitopes that may be suitable for different MHC types. Although M1 is not expected to play much of a role in protection in mice, it is a prominent target of T cell immunity in humans, and might contribute to the performance of the PanAd3-NPM1 vaccine in humans.
The results presented here support the use of the PanAd3 vector as a vaccine candidate that is highly effective at inducing T cell and antibody immunity, while at the same time having the advantage that it is not neutralized by human sera. Thus PanAd3, when used to express conserved influenza virus antigens, has promise as a “universal” influenza vaccine candidate.
The four connected cat rooms (Figure 3) were dusted daily using a broom, but no disinfection took the place of either the floor or any other material in the cat rooms, including food and water bowls. The bowls were only rinsed with soap after daily use. Obvious stains on the floor (e.g., vomit or urine) were cleaned with water and paper tissues. The cat feces and urine were removed daily from the two litter trays and if necessary new cat litter, Aspen 6 wood shavings (Le Comptoir Des Sciures, Meyzieu, France), was added. Prior to use, the cat litter was autoclaved to prevent the import of pathogens. The cat beds were cleaned with a small brush to remove hair, and, if obviously stained, brought for washing at 60 °C and autoclaving. No additional cleaning or disinfection was performed in-between the two FCV challenges.
In contrast to the cat rooms, the rest of the area within the inner barrier (room for clinical examination and sample collection, corridors, etc.; Figure 3) was cleaned daily with hot water to remove solid dirt and stains and after removing all excess water with a floor squeegee, the floor was disinfected with 5% sodium bicarbonate (kaia.ch/SFT AG, Pratteln, Switzerland) dissolved in hot water as recommended against FCV. Afterwards the excessive water was again removed with a floor squeegee and the disinfection with IncidinTM Plus (Ecolab, Monheim am Rhein, Germany), containing the active agent glucoprotamin, was performed to prevent introduction of any additional pathogens from outside of the facility; glucoprotamin has only a limited virucidal effect, mostly against enveloped viruses. The IncidinTM Plus (Ecolab) was allowed to stay on the floors for at least 15 min before removing it using water and a floor squeegee. All waste that was generated within the inner barrier area was collected in biohazard bins (Mauser), sealed and removed for autoclaving to prevent FCV contamination outside the cat facility.
The used cotton overalls and the dirty cat beds were transported in a leak-proof container to the washing facility outside the cat facility, washed at 60 °C with regular washing powder, dried with a clothes drier, and autoclaved prior to reuse.
Multiplex PCR‐based NAATs have been increasingly used for syndromic diagnosis, due to their high throughput, high sensitivity, high specificity, cost‐effectiveness, and great clinical significance.10, 11, 12 The ResP assay is based on multiplex PCR amplification and capillary electrophoretic separation of PCR amplicons by length. This technique has been used for pathogen detection and subtype classification of pediatric acute lymphoblastic leukemia.13, 14 By comparing the results with a standard size marker of targeted pathogens, pathogens in samples can be separated and identified as expected.15 The subtypes of most viruses were not designed to be further distinguished by this assay, except for influenza virus A. The influenza virus A pdmH1N1 (2009) and H3N2 are the two subtypes which are most popular in China recently. Therefore, a patient whose specimen is positive for influenza virus A but negative for influenza virus A pdmH1N1 (2009) or H3N2 is probably infected by an uncommon influenza virus A, such as H7N9, H5N1, H5N6 avian influenza virus A16, 17, 18 and has to be immediately quarantined once it is confirmed. It should be noted that hospitals, not CDCs, are the first to reach such patients, so this assay helps hospitals identifying such high‐risk patients and make appropriate quarantine measurement in a timely manner to control further spread of avian influenza A virus.
This assay has previously been clinically applied to detection of respiratory pathogens in hospitalized children suffered with community‐acquired pneumonia (CAP)14 or lower respiratory tract infections.19 The assay was evaluated by comparing with Sanger sequencing, showing great performance with 100% positive prediction value (PPV) and 99.85% negative prediction value (NPV).20 To our knowledge, this is the first study evaluating the performance of the ResP in oropharyngeal swab specimens from outpatients with ARIs.
Our study showed almost perfect kappa statistics for the ResP on rhinovirus, adenovirus, influenza virus A pdmH1N1(2009), respiratory syncytial virus, and influenza virus B, suggesting that the performance of ResP on these viruses was as effective as pathogen‐specific PCRs. On human metapneumovirus, the kappa statistics were lower than 0.8, presumably due to the small number of positive cases. Overall, this assay demonstrated 86.5% PPV and 97.8% NPV. This work suggested that the performance of ResP was sufficient enough be used for respiratory pathogen identification in outpatients with flu‐like manifestations.
The major limitation of this study is the small number of human metapneumovirus, parainfluenza virus, Mycoplasma pneumoniae, boca virus, influenza virus A H3N2, coronavirus, and Chlamydia. Further investigation is needed to evaluate the performance of ResP on these pathogens.
In conclusion, the performance of ResP showed a high‐degree agreement with pathogen‐specific PCRs in oropharyngeal swabs from outpatients. The implementation of ResP may facilitate the diagnosis of respiratory infections in a variety of clinical scenarios.
Generally, virus-specific proteins have drawn attention for the treatment of viral infection as targets. However, the focus of antiviral approaches has recently started to move toward targeting host factors essential to virus multiplication. Hsp90, a molecular chaperone that regulates the function, turnover, and trafficking of several proteins including signaling and regulatory proteins, is one of the important host factors that play critical roles in the viral life cycle. Hsp90 inhibitors have been reported to inhibit Ebola virus (EBOV) replication, and cause degradation of the viral polymerase (Smith et al., 2010). However, the exact mechanism underlying the anti-EBOV activity of Hsp90 inhibitors remains unknown. In influenza virus infection, Hsp90 is required for viral genome replication. As Hsp90 associates with subunits of the influenza virus, inhibition of Hsp90 leads to degradation of viral subunits. Besides, Hsp90 inhibitors reduce the levels of the assembled polymerase complex, resulting in decreased viral RNA levels (Momose et al., 2002). A recent study showed that Hsp90 is also required for the replication of beta-herpesviruses (Burch and Weller, 2005). In the human cytomegalovirus infection model, Hsp90 inhibition resulted in degradation of the viral polymerase and reduction of viral gene expression via downregulation of the PI3-kinase pathway (Basha et al., 2005). Similarly, in the flock house virus, Hsp90 influences RNA polymerase stability (Kampmueller and Miller, 2005). Collectively, pharmacological inhibitors of Hsp90 have potential as broad spectrum antiviral agents. In addition to their universal activity against diverse viral infections, Hsp90 inhibitors show the possibility of overcoming viral drug resistance. Most antiviral agents lead to generation of drug-resistant variants, which is one of the major issues in the development of effective antiviral therapy (zur Wiesch et al., 2011). Interestingly, Hsp90 inhibitors are not reported to induce viral drug resistance till date. Therefore, they might be particularly useful for antiviral therapy against viruses prone to develop drug resistance (Geller et al., 2012).
Hsp90 inhibitors also have potent anti-inflammatory and anti-oxidative actions in vascular tissues (Hsu et al., 2007). Hsp90 inhibitors were shown to extend survival, attenuate inflammation, and reduce lung injury in murine sepsis (Chatterjee et al., 2007). Hsp90 was also suggested to participate in viral capsid protein folding and in the assembly of various picornaviruses including poliovirus, rhinovirus, and coxsackievirus, which renders Hsp90 an attractive candidate for the development of antiviral vaccines (Brenner and Wainberg, 1999). Hsp90 is also important for subcellular localization of specific mRNAs in regions neighboring the mitochondria, which could explain the inhibitory effect of Hsp90 inhibitors on RNA polymerase.
Human rhinoviruses cause common cold in humans, and can sometimes accelerate airway diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis (Zaheer et al., 2014). As an important human respiratory virus, HRV is a non-enveloped positive-sense single-strand RNA virus involved in 50–80% of upper respiratory tract infections and has also been associated with lower respiratory tract disease in high-risk populations, for example in patients with asthma or other airway inflammations (Gern and Busse, 1999). Generally, symptoms of rhinovirus in mice are not severe. However, our present data showed that the levels of pro-inflammatory cytokines such as TNF-α and IL-6 in the lung and BALF of mice were increased upon intranasal HRV1B infection, which is reported to contribute to the pathogenesis of asthma during long-term infection (Liebhart et al., 2002; Jartti and Korppi, 2011; Rincon and Irvin, 2012).
Ribavirin is the only antiviral drug approved by the FDA for treatment of RSV infection (Molinos-Quintana et al., 2013), and is also a broad-spectrum antiviral drug for RNA viruses including FLU-A, HRV 14, RSV, and CVB3 (Shi et al., 2007). Although ribavirin is known to have a broad-spectrum antiviral activity against several respiratory viruses, it has limitations due to its controversial efficacy and toxicity (Kneyber et al., 2000). Indeed, ribavirin did not show efficient antiviral activity against HRV1B infection in our experiment, and 50 μg/ml of ribavirin showed only marginal antiviral activity in Hela cells infected with HRV1B (data not shown).
In the present study, we analyzed the antiviral activity of pochonin D against HRV infection. Although pochonin D is a well-known Hsp90 inhibitor (Moulin et al., 2005; Wang et al., 2016; Choe et al., 2017), it is still uncertain that the inhibition of Hsp90 by pochonin D is directly associated with the antiviral activity of it. We found that treatment with pochonin D lowered the level of pro-inflammatory cytokines in the lung and BALF of mice, which were increased by rhinovirus infection. Furthermore, the virus titers of HRV-infected mice treated with pochonin D were significantly decreased to levels similar to those in naïve mice. We also examined the levels of pro-inflammatory chemokines/cytokines (CCL2, CXCL1, TNF-α, IL-6, and IL-1β) in lung lysates and lung RNA. Their concentrations were decreased by pochonin D treatment in HRV1B-infected mice, and were comparable to the chemokines/cytokines levels in naïve mice. These data suggest that pochonin D may reduce inflammatory damage in rhinovirus-infected mice. We also found that neutrophil infiltration into the inflammatory site was reduced by pochonin D treatment in HRV1B-infected mice. This reduction may be due to the mild viral infection and inflammation in pochonin D-treated group. Finally, we observed the histopathology of the lung and airway, and found that pochonin D treatment ameliorated the damage induced by rhinovirus infection in the lung and airway.
In vitro, 10 μM of pochonin D did not influence cell viability; however, slight toxicity was observed at pochonin D concentrations greater than 50 μM (data not shown). Adverse effects were also observed in mice treated with pochonin D at 1.75 mg/kg and 600 μg/kg, but not with 200 μg/kg (Data not shown). The dose of 200 μg/kg pochonin D was non-toxic to mice and was also more effective at controlling HRV infection compared to the dose of 600 μg/kg. Therefore, it is necessary to use an appropriate dose of pochonin D ensuring both safety and efficacy in antiviral therapy.
Collectively, blocking Hsp90 with pochonin D induces an antiviral effect against rhinovirus infection, and reduces the inflammatory response. As a result, treatment with pochonin D enables recovery from HRV1B virus infection in mice.
The specimen was shaken vigorously for 5 minutes in phosphate‐buffered saline solution, centrifuged at 9.6 g for 20 minutes, and the supernatant was aspirated. About 50 µL of RNA was extracted from 140 µL supernatant using the QIAamp Viral RNA extraction kit (QIAGEN, Hilden, Germany), according to the manufacture's instruction and was stored at −80°C.
An infectious etiology for cancer was first documented in animals during the early part of the nineteenth century with the diagnosis of pulmonary adenocarcinoma in sheep (later attributable to jaagsiekte sheep retrovirus) (5). Animals are the host species for many oncogenes. Among the most studied are rodent (Abl, Int1/Wnt1, Int2, Notch1, Pim1/2, Runx, Tpl2), fowl (Erb-b, Fos, Myc, Src), feline (Myc), and fish (cyc) (6). For example, reticuloendothesliosis virus readily induces cancer in chickens (avian leucosis/sarcoma). The virus has been found in eggs intended for human consumption and vaccines prepared in eggs (7). A wide variety of viruses, mirroring their human analogs, are ubiquitous among animals in nature and their habitat (e.g., fecal coliform contamination) (8–10). Common types include viruses in the polyoma, adeno, retro, and papilloma family.
Animal viruses potentially express oncoproteins in human cells even though stringent replicate restrictions exist in the latter (11). The “hit and run” hypothesis posits that certain viruses interfere with the hosts immune system to cause cancer, yet do not integrate into the victims DNA (leaving no detectable fingerprints) (12). Newborn hamsters infected with polyoma virus have been shown to develop cancers, even though the cells of this species do not support virus replication (13). Similarly, tumors induced in immunocompetent mammals with Rous sarcoma virus do not present neutralizing antibodies (14). In contrast, some animal viruses [e.g., feline leukemia virus (FeLV)] have been observed to replicate in vitro in human cells (15, 16). Sera collected from 69% of 107 persons among 46 households with at least 1 FeLV gs-a positive cat tested positive for antibodies against FeLV (15). Although it is unclear exactly how antibodies directed toward animal viruses could have oncogenic or mitogenic effects on host cells, these findings support the idea that long-lasting “biological memory” of animal virus exposure can exist within the host in the absence of direct effects on host DNA.
Animal bacteria also have been implicated in cancer. The occurrence of gliomas in the brain of fowl have been noted in several reports (17–19) and these tumors have been described as having the pathognemonic encephalitic features of a pleomorphic parasite infection (e.g., hypertrophy and hyperplasia of blood-vessels; perivascular infiltration by lymphocytes, plasma cells, and monocytes; and the presence of A-D bodies) (20). Chickens spontaneously and experimentally infected with toxoplasma have been observed to develop glioma-like tumors (21, 22). A study of 16 human brain tumors observed bodies indistinguishable from the C and D phases of the fowl parasite (23). Epizootic outbreaks of toxoplasmosis have been reported in various avian species and mammals (22, 24, 25). Furthermore, toxoplasma antibodies have been isolated in the blood of exposed sheep farmers, flock animals, herder dogs, mice, and rats (26). Potential cellular mechanisms by which animal viruses and bacteria lead to tumorgenesis are shown in Figure 1.
Influenza A virus is an enveloped virus belonging to the Orthomyxoviridae family. It can cause annual epidemics and infrequent pandemics. The Spanish flu pandemic of 1918 as well as the Asian flu of 1957 and the Hongkong flu in 1968 pandemics caused the death of millions of people. In 2009 the pandemic swine origin influenza A H1N1 virus as well as the outbreak of H7N9 in China in 2013 has reminded the world of the threat of pandemic influenza [3–6].
The genome of influenza virus consists of eight segmented negative RNA strands. The envelope bilayer harbors the two spike glycoproteins hemagglutinin (HA) and neuraminidase (NA), and the M2 proton channel. The homotrimeric HA is the most abundant protein on the viral surface. It mediates attachment to the host cell surface via binding to sialic acid (SA) residues of cellular receptors, and upon endocytic virus uptake it triggers fusion of the envelope with the endosomal membrane releasing the viral genome into the cytoplasm. NA cleaves glycosidic bonds with terminal SA facilitating the release of budding virions from the cell.
In diagnostics, antibodies against spike proteins are the preferred tool for identification and serotyping of viruses. Development of therapeutic antibodies against influenza is a challenge, as the high viral mutation rate (antigenic drift) and genetic reassortment of the virus genome (antigenic shift) continuously lead to new strains escaping from neutralization by antibodies [7, 8]. This goes along with adaptation to small molecule inhibitors (e.g. oseltamivir).
Vaccines can only temporarily control the recurring epidemics of influenza, because antigenic changes are typical for HA and NA. 16 avian and 2 bat serotypes of influenza A virus HA (H1—H18) are known, but only three (H1, H2, and H3) have been adapted to humans. Antibodies binding to regions of hemagglutinin conserved among serotypes have been developed which demonstrated broad specificity and neutralization potency [10–15]. However, development, production and quality control of antibodies is expensive and time consuming.
As an alternative, short peptides binding specifically to the spike proteins can be produced in automated high-throughput synthesis at low costs. HA-binding peptides have been recently obtained by phage display, lead structure optimization of natural products and specific toxins, bioinformatics tools and discovery from side effects of known anti-inflammatory peptides [16–23]. Some of them showed antiviral activity [17, 19–23]. A more epitope-oriented accession to binding peptides is the search for paratope-derived peptides from variable regions of specific antibodies. Antibodies against HA have been described, and at least 6 antigenic sites (A-F) on the HA-trimer have been identified, localized either at the receptor binding site, the interface of the three HA-monomers, or at other sites like the stalk [8, 11, 25]. Several structures of HA–antibody complexes have been published deposited in the protein data bank (PDB) [11–14]. Indeed, an antibody was described, whose HA binding is mediated mainly by one CDR, namely HCDR3.
Inspired by this finding, we chose linear peptides corresponding to the CDRs of VH of monoclonal antibody HC19, having the majority of contacts with the HA1 domain of the strain A/Aichi/2/1968 [26, 27]. The antibody and the derived peptides bind to HA at the SA binding site, in particular to the 130-loop and the 190-helix, which belong to the antigenic sites A and B, respectively. This binding site is conserved among several HA serotypes providing a basis for a peptide with broader specificity.
We used complementary experimental and theoretical approaches to select HA binding VH-CDR peptides and to improve their potential to inhibit binding, and finally, infection of cells by influenza A virus. The inhibitory potential of the most efficient CDR-peptide was improved by microarray-based site-directed substitutions of amino acids. We could demonstrate a broader specificity of the selected peptides as they bound to HA of human and avian pathogenic influenza strains.
This study was approved by the National Ethics Committee of the Malagasy Ministry of Health (CE/MINSAN n° 019). A briefing note explaining the purpose of the project and the informed consent form was given to each of the parents involved in the study who signed the consent forms to provide written informed consent.
6 × 105 TCID50 of purified PRV3M was resolved by SDS-PAGE on a 12% gel. The proteins were transferred onto a PVDF membrane, and blocked for 1 h in 5% BSA in Tris-buffered saline containing 0.05% Tween-20 (TBS-T). Membranes were incubated with monkey sera diluted to 1:500 in 5% BSA in TBS-T for 1 h at room temperature. Membranes were washed 3 times in TBS-T, then HRP-conjugated anti-monkey antibody (Santa-Cruz, USA) at a 1:10,000 dilution was added for 1 h. The immuno-signal was developed using Amersham ECL western blotting detection reagent (GE healthcare, UK) and images were captured using ChemiDoc MP imaging system (Bio-Rad, USA).
For infection and cytotoxicity assays Madin-Darby Canine Kidney Epithelial Cells (MDCK II, NBL-2, CCL-34) were used (ATCC). Cells were cultivated under standard cell culture conditions with DMEM (supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine) in humid atmosphere at 37°C and 5% CO2. Hemagglutination inhibition assays (HAI) were performed using either human erythrocytes (α-2,6’-sialosugars) for Aichi H3N2, or turkey erythrocytes (α-2,3’-sialosugars) for Rostock H7N1.
In addition to molecular detection of viruses, this study also presented a look at perceptions of biosecurity practices. Considering the 12 PPE questions, there were clear differences between perception and use for safety glasses, flu vaccinations, showering out of work, and disposable boots. While wearing safety glasses, flu vaccinations, and disposable boots were seen as efficacious but not used, showering out was often done by those who did not see it as an effective means of preventing cross-species infection. These differences could be due to training both for employers and workers, as the majority of farm biosecurity and disease prevention literature does not discuss worker protection against disease risks [34, 38, 39]. While not all 12 PPE items studied have proven effect in preventing cross-species infection or improving farm biosecurity, some listed interventions, such as rubber gloves, have shown marked differences in preventing the spread of zoonotic influenza virus.
Serum neutralization tests were performed by incubating 200 TCID50 of PRV3M with 2-fold serial diluted macaque serum for 30 min at 37°C. The virus-antibody mixture was then added to a monolayer of Vero cells and incubated for 1 h at 37°C. After incubation, the inoculum was removed and replaced with DMEM supplemented with 2% FBS. Virus-induced CPE was observed at days 4 post-infection and the titre was recorded at the highest dilution where CPE was absent.
The pre-publication history for this paper can be accessed here:
This study describes for the first time the etiology of a clinically defined cohort of children with fever and acute respiratory infections living in a malaria endemic rural region of Madagascar. The rural community of Ampasimanjeva consists of seven villages located in south-eastern Madagascar (coordinates 21° 44 ′0 “South/48° 2′ 0” East) dominated by an equatorial climate with high humidity. Recently, 1,549 children under 5 years of age were referenced and among them, 937 presented a fever and were examined at the hospital during the study period. Of these children, 295 presented ARIs clinical signs and a negative test for malaria. To better understand the role of the viral and atypical bacteria agents in nasopharyngeal swabs, we stratified the respiratory clinical symptoms into 4 major groups to obtain defined ARIs cohorts. Seventy five percent (n = 220) of the acute febrile children had a nasopharyngeal sample containing at least one virus or atypical bacterial agent. The low rate of negative specimens shows that the technique used in this study is highly sensitive and the spectrum of pathogens is large, covering most of those implicated in the ARIs. In this study, the frequently encountered pathogens were the HRV, HMPV A/B, HCoV, RSV A/B and HPIV. The most representative pathogens into the defined clinical groups were HMPV, HPIV, HRSV and HAdV for pneumonia, other ALRIs, Flu-like illnesses and URTI, respectively. The human rhinovirus, HMPV A/B and RSV A/B were single detected while HCoV, HPIV and HBoV were most often co-detected. Human rhinovirus was found largely in all clinical manifestations.
HRV has long been considered to be a benign virus causing mild upper respiratory tract infections, but there is evidence that HRV is also involved in ALRIs more specifically in bronchiolitis, but its role is not yet well defined in pneumonia,. HRV is frequently found as asymptomatic carriage,,, a comprehensive study on genotypes from HRV-positive samples would be interesting. As, it seems that the new and potentially more pathogenic HRV-C which has been recently discovered, is correlated with the severity of ARIs,.
Our molecular results showed a large number of respiratory pathogens associated with the different clinical manifestations including a large part in pneumonia. Viral pneumonia is also increasingly described in the literature but it is nevertheless still underestimated,. In children, RSV, HRV and HMPV have become important pathogens most frequently encountered in children with pneumonia in developed countries–. In Ampasimanjeva, although detected in all ARIs clinical manifestations, the human metapneumovirus was better associated with community-acquired pneumonia with a higher prevalence than those described in the literature,,. HMPV is, however, considered to have an important role in pneumonia increasing child morbidity worldwide,. In a study done in Antananarivo (2008/2009), RSV A/B was mainly represented among children with Influenza-Like Illnesses and similarly, our results showed that this virus was primarily associated with group II which we defined here as Influenza-like syndrome.
Atypical bacteria pathogens were also investigated. We detected a low rate of M. pneumoniae (1.7%) and C. pneumoniae (0.7%) distributed in all four clinical groups. Our results correlate to those described in several studies, which show that atypical bacteria prevalence in acute respiratory infections may vary depending on the age of children,–.
Our study has several limitations. Firstly, we did not investigate the viral and atypical respiratory pathogens in control population as asymptomatic carriers. This could limit our interpretation to ascribe the etiological agents to the defined clinical groups. However, some of the potential etiological agents described here are rarely identified or less common in asymptomatic patients, except for rhinoviruses,,. A longitudinal case/control study would be interesting to validate the relevance of these agents in acute respiratory infections despite the difficulty to obtain approval requirements from ethical committee. Our results highlight a significant proportion of viruses among children with ARIs. However, the viral etiology of an infection does not exclude the coexistence of a bacterial infection nor superinfection, often observed, for example, with pneumococci following influenza virus infection. The bacterial etiology could not be evaluated in this study but remains important enough to be investigated further. Using information gathered from clinical records, it appears that 87.5% of children received antibiotics as a result of auscultation. If only viruses are actually mostly the cause of ALRIs in Ampasimanjeva, simple recommendations such as those proposed by WHO and UNICEF could reduce the morbidity and mortality. Secondly, we did not evaluate the proportion of respiratory pathogens among children with malaria (n = 21). In the absence of appropriate diagnostic tools in low income countries, integrated management of childhood illnesses (IMCI) at health facilities is presumptive and symptom-based: fever for malaria, and fever/cough/difficult breathing for pneumonia. These overlapping symptoms, compatible both with malaria and pneumonia, necessitating dual treatment, need to be evaluated in further studies. Unexplained acute febrile illness is the only common factor in children living in endemic malaria region. In this study, we showed that the fever cannot be considered alone to guide clinical diagnosis. Moreover, respiratory pathogens do not seem to be related to fever prognosis except for Influenza viruses. Finally, the interpretation of the fever may be biased by the use of antipyretic treatment, and 24.1% of our patients had evidence of antipyretic use before study enrollment. Finally, this study was conducted over one year, covering both hot/rainy and cold/dry seasons, to get a preliminary description of respiratory pathogens and respiratory pathogens spread during this period. This short time period should be prolonged to deepen our knowledge about these pathogens and perhaps anticipate epidemics in light of improving the health care of children.
In conclusion, the use of molecular assays has allowed us to refine our understanding of the viral etiologies of ARIs among children living in a rural area of Madagascar. Further studies are needed to properly distinguish between infection and colonization. They will lead to comparing findings in different respiratory samples and reference standards. Viruses seem to be commonly involved in pneumonia. While some viruses such as HRV are mainly represented in all clinical presentations, some such as hMPV and RSV are most often associated with pneumonia or influenza-like illnesses. A better understanding of the biodiversity of each pathogen correlated to well-defined clinical ARIs manifestations could be explored.
Rapid identification of newly emerging viruses through the use of genomics tools is one of the major challenges for the near future. In addition, the identification of critical mutations that enable viruses to spread efficiently, interact with different receptors, and cause disease in diverse hosts through, for instance, enhanced viral replication or circumvention of the innate and adaptive immune responses, needs to be further expanded. Although microarray-assisted transcriptional profiling can provide us with a wealth of information regarding host genes and gene-interacting networks in virus–host interactions, future research should focus on combining data obtained in different experimental settings. Therefore, the careful design of complementary sets of experiments using different formats of virus–host interactions is absolutely needed for successful genomics studies. Special attention should be addressed to the comparative analysis of the host response in diverse animal species. Thus far a limited number of laboratory animal species has been studied, but the recent elucidation of the genome of several other animal species will provide tools to decipher the virus–host interactions in the more relevant natural host. Recent developments in the sequencing of the RNA transcriptome may aid this development. Ultimately, microarray technology may also extend to genotyping of the human host by SNP analysis, to identify markers of host susceptibility and severity of disease, that can be used in tailor-made clinical management of disease caused by emerging infections. Comparative analysis of host responses to emerging viruses may also point toward a similar dysregulated host response to a range of emerging virus infections, enabling the rational design of multipotent biological response modifiers to combat a variety of emerging viral infections. By focusing on broad-acting intervention strategies rather than on the discovery of a newly emerging pathogen that is not characterized yet, we may be able to protect ourselves from several unexpectedly emerging infections with the same clinical manifestations. This approach may readily reduce the burden of disease and time will be gained to design preventive pathogen specific intervention strategies such as antiviral therapy or vaccination. Clearly, for all stages of combating emerging infections, from the early identification of the pathogen to the development and design of vaccines, application of sophisticated genomics tools is fundamental to success.
Influenza viruses pose major threats to public health, as they are responsible for epidemics and pandemics resulting in high morbidity and mortality worldwide. Several pandemics, such as the Spanish flu (1918), Asian flu (1957), and Hong Kong flu (1968), caused millions of deaths in the last century (1). Currently, only two therapies, targeting the viral proteins neuraminidase and M2, are approved to treat influenza. Therefore, novel viral or host targets for antiviral strategies to block viral replication or inhibit cellular proteins necessary for the virus life cycle are urgently needed (2). In this context, host proteases are a group of very promising antiviral targets, because proteolytic cleavage of the precursor hemagglutinin (HA0) into HA1 and HA2 subunits by host proteases is essential for fusion of HA with the endosomal membrane and thus represents an essential step for infectivity of the virus (3, 4). Due to the potential risk of side effects after application of broadband protease inhibitors, the specific inhibition of a single enzyme would convey a huge therapeutic benefit.
The majority of influenza viruses, including low pathogenic avian and human influenza viruses, carry a single arginine (R) residue at the cleavage site. These HAs are cleaved by host trypsin-like proteases (5–8). In vitro studies with cultured human respiratory epithelial cells demonstrated the involvement of several membrane-associated proteases (9). Cell culture studies further identified, among others, the transmembrane serine proteases TMPRSS2, TMPRSS4, and TMPRSS11D as enzymes able to cleave the HAs of influenza virus subtypes H1 and H3 (10–12). We previously showed that deletion of Tmprss2 in knockout mice strongly limits viral spread and lung pathology after H1N1 influenza A virus infection (13). An essential role for TMPRSS2 in cleavage activation and viral spread was also reported for H7N9 influenza A virus (14, 15). We also demonstrated that deletion of Tmprss2 slightly reduced body weight loss and mortality in mice after H3N2 virus infection compared to those for wild-type mice but did not protect mice from lethal infections (13, 15). Therefore, it is likely that in addition to TMPRSS2, other trypsin-like proteases of the respiratory tract are able to cleave the hemagglutinin of H3 influenza viruses.
In this study, we investigated the role of Tmprss4 in the context of influenza A virus replication and pathogenesis in experimentally infected mice. We showed that knockout of Tmprss4 alone did not protect mice from lethal H3N2 influenza A virus infections. In contrast, Tmprss2−/−
Tmprss4−/− double-knockout mice showed massively reduced viral spread and lung pathology and also had reduced body weight loss and mortality.
(Part of this work was performed as Ph.D. thesis work by Nora Kühn at the University of Veterinary Medicine, Hannover, Germany.)
To assess and verify the in vivo antiviral activity of pochonin D against HRV1B, we first determined the pathological phenotype of mice after intranasal HRV1B infection. BALB/c mice were intranasally infected with 1×108 pfu/30 μl of HRV1B. Mice were killed at 8 h, 1 day, 3 days, and 5 days after virus inoculation, and the levels of pro-inflammatory cytokines including CCL2, CXCL1, IL-1β, TNF-α, and IL-6, and virus titers in the lungs were assessed. Mice infected with HRV1B produced significantly higher levels of CCL2, CXCL1, IL-1β, TNF-α, and IL-6 (Supplementary Fig. 2A–2E) with elevated virus titers (Supplementary Fig. 2F) at 8 h and 1 day after infection than the uninfected control mice. The levels of pro-inflammatory cytokines and virus titers peaked at 8 h after infection, and were reduced by day 5 to those observed in uninfected mice as reported previously (Bartlett et al., 2008). We therefore decided to monitor the lung cytokine levels and virus titers at 8 h after HRV1B infection for evaluating the antiviral activity of pochonin D in mice.
To assess the antiviral activity of pochonin D against HRV1B, mice were intraperitoneally administered 200 μg/kg of pochonin D, at 1 h prior and 4 h after intranasal HRV1B infection. We performed placebo infection in control mice, and administered vehicle intraperitoneally in HRV1B-infected mice as a negative control. After 8 h of infection, we sacrificed the mice and obtained lung samples. Real-time PCR analysis of viral mRNA in lung tissues revealed that the virus titer was significantly reduced in pochonin D-treated mice compared to that of vehicle-treated mice after HRV1B infection (Fig. 2A). We thus confirmed that pochonin D has an anti-HRV activity in vitro as well as in vivo when administered systemically via the intraperitoneal route.
Most of the well-known human viruses persist in the population for a relatively long time, and coevolution of the virus and its human host has resulted in an equilibrium characterized by coexistence, often in the absence of a measurable disease burden.
When pathogens cross a species barrier, however, the infection can be devastating, causing a high disease burden and mortality. In recent years, several outbreaks of infectious diseases in humans linked to such an initial zoonotic transmission (from animal to human host) have highlighted this problem. Factors related to our increasingly globalized society have contributed to the apparently increased transmission of pathogens from animals to humans over the past decades; these include changes in human factors such as increased mobility, demographic changes, and exploitation of the environment (for a review see Osterhaus and Kuiken et al.). Environmental factors also play a direct role, and many examples exist. The recently increased distribution of the arthropod (mosquito) vector Aedes aegypti, for example, has led to massive outbreaks of dengue fever in South America and Southeast Asia. Intense pig farming in areas where frugivorous bats are common is probably the direct cause of the introduction of Nipah virus into pig populations in Malaysia, with subsequent transmission to humans. Bats are an important reservoir for a plethora of zoonotic pathogens: two closely related paramyxoviruses—Hendra virus and Nipah virus—cause persistent infections in frugivorous bats and have spread to horses and pigs, respectively.
The similarity between human and nonhuman primates permits many viruses to cross the species barrier between different primate species. The introduction into humans of HIV-1 and HIV-2 (the lentiviruses that cause AIDS), as well as other primate viruses, such as monkeypox virus and Herpesvirus simiae, provide dramatic examples of this type of transmission. Other viruses, such as influenza A viruses and severe acute respiratory syndrome coronavirus (SARS-CoV), may need multiple genetic changes to adapt successfully to humans as a new host species; these changes might include differential receptor usage, enhanced replication, evasion of innate and adaptive host immune defenses, and/or increased efficiency of transmission. Understanding the complex interactions between the invading pathogen on the one hand and the new host on the other as they progress toward a new host–pathogen equilibrium is a major challenge that differs substantially for each successful interspecies transmission and subsequent spread of the virus.