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This study is a component of the 4S network syndromic surveillance. The principles of the 4S network were approved by the Ministry of Health in its guidelines for influenza surveillance policy, finalized with the support of Pasteur Institute in Dakar and the Strengthening Influenza Sentinel Surveillance in Africa (SISA) project funded by the WHO. The protocol and oral consent were determined as routine surveillance activity, and therefore non-research by the Senegalese National Ethics committee and the steering committee for 4S network, an entity representing MoH, IPD, WHO and Clinicians in compliance with all applicable National regulations governing the protection of human subjects. Data were collected in an objective of surveillance and are anonymous. The information provided to participants was an informal description of the study. Respiratory specimens were collected, only after informed consent was granted, verbally, to local health care workers by the patients or parents in the case of minors. Oral consent was documented in the patient form with two questions about received information and about oral consent. Patients could refuse to participate, no specimen will be taken. For the surveillance activities, written consent is judged not necessary by the Senegalese national ethics committee, which has also previously approved the work of the National Influenza Center. Collections of non-sensitive data or an observation from normal care in which participants remain anonymous do not require ethics committee review. The patients included in this study were of all ages and consulted the sentinel sites due to influenza-like symptoms; the patients, or parents in the case of minors, accept the tests for respiratory viruses largely because they are free and safe.
Our literature review suggests that community-acquired adenoviral pneumonia in immunocompetent adult civilians presents as a non-specific febrile respiratory illness that progresses rapidly to respiratory failure and often requires mechanical ventilation. The laboratory and radiological findings are typical of viral infection but are also non-specific. Novel respiratory virus real-time RT-PCR testing enabled us to rapidly detect adenovirus as the cause of severe community-acquired pneumonia in our patient.
Molecular biology procedures were accomplished in collaboration with the Division of Virology and the Division of Hygiene and Medical Microbiology, at the Medical University of Innsbruck, Austria.
Acute viral respiratory tract infections continue to be a worldwide health concern. The presentation of clinical symptoms does not often enable clinicians to discriminate between viral or bacterial pathogens. The recommended early and easy use of antibiotics is not effective in viral ARTIs, and can only prevent the occurrence of bacterial super-infection. In this context, viral diagnosis can prevent the use of unnecessary costly antibiotics. For this reason, rapid and reliable diagnostic tools are required for intervention with the appropriate infection treatment measures. This study aimed to profile a large number of viruses and the most common bacteria in the respiratory tract of infected children in Sousse area in Tunisia using the multiplex qRT-PCR for virus’s genome detection and the qPCR assays for bacteria identification. The results of the study should provide a better understanding of the viral/bacterial etiology in this region.
Regarding HAdV infection comparisons between age groups were performed using the Fisher’s exact test. P value < 0.05 was considered statistically significant and the 0–5 year age group was used as reference group. HAdV mono-infections were also compared to HAdV co-infections. The R.3.0.1 tool was used to perform the analyses.
Modulation of any human host pathway for the treatment of viral infections has potential drawbacks with respect to toxicity and other side-effects. For example, although interferon is widely used to help combat viral pathogens, the treatment is known to cause an array of side-effects related to toxicity including confusion, lethargy, impaired mental status, numbness, tingling, fevers, chills, headaches, anorexia and sepsis,. Another caveat is that some proteins are beneficial if up-regulated during initial viral infection but have detrimental effects if over-activated for prolonged periods. Thus determining the desired mechanism and direction of therapeutic intervention requires careful study. Although targeting host-pathogen interactions is a challenging therapeutic approach, there are considerable upside benefits with respect to overcoming pathogen-mediated drug resistance and the capability of treating multiple, co-infecting pathogens. Our study suggests several potential human-host proteins that could be targets of future therapeutics as well as some possible drug candidates for further investigations of repurposing against respiratory virus infections.
Our case report and review of the literature provides the first comprehensive review of community-acquired adenovirus pneumonia in immunocompetent adult civilians. Hakim and Tleyjeh published a case report and literature review of adenovirus pneumonia in immunocompetent adults in 2008; however, their cohort was a mix of civilians, military recruits and healthcare-associated cases.
The 21 cases we identified in the literature demonstrate that patients with adenovirus pneumonia usually present with several days' history of a non-specific febrile respiratory illness. These patients frequently have respiratory compromise with hypoxia at the time of presentation, while the classical features of adenoviral infection, such as pharyngitis, conjunctivitis, rash or diarrhea, are usually absent. The clinical condition of most patients deteriorates rapidly during admission and requires intubation and ventilation, a pattern commonly seen with primary influenza pneumonia. Laboratory findings are also typical of viral infection, with a normal total white cell count, relative lymphopenia, thrombocytopenia and elevated transaminases and CPK being frequently observed. The most commonly seen radiological pattern on admission is widespread bilateral interstitial shadowing, which is consistent with the results reported in a case series describing the radiological appearance of adult patients with confirmed adenoviral pneumonia. It is noteworthy that several patients, including our own case, had normal initial chest radiography results. Lobar consolidation, a pattern considered more suggestive of bacterial infection, was observed in around one-fourth of patients with adenoviral pneumonia. All of these radiological patterns (including normal initial chest radiographs) have been described in patients with primary influenza pneumonia. Although the overall mortality rate in this series was 24%, only two patients who were reported on after 1979 have died, possibly representing improvement in supportive care over this time period.
Our present case report of an immunocompetent adult civilian patient with sporadic adenoviral pneumonia is the first case to be reported in the literature in which molecular diagnostic methods were used. Nucleic acid detection has the advantages of increased sensitivity and rapid availability of results compared to the conventional diagnostic techniques of viral culture and antigen detection. In addition, multiplex real-time reverse transcriptase PCR (RT-PCR) assays are increasingly being used by diagnostic laboratories to detect a wide range of respiratory viruses in a single reaction. While it is well-recognized that influenza virus and adenovirus can cause pneumonia, there is increasing evidence that other respiratory viruses, such as RSV, human metapneumovirus, PIV, human rhinovirus and human coronavirus play an important role in the etiology of community-acquired pneumonia in adults. The increasingly widespread use of multiplex real-time RT-PCR for the detection of respiratory viruses in clinical practice will allow us to accurately determine the burden of respiratory viral infection in patients with community-acquired pneumonia and may demonstrate that adenoviral pneumonia in immunocompetent adults is more common than previously thought.
The advantages of rapidly diagnosing respiratory viral infection in patients with community-acquired pneumonia include the institution of appropriate infection control measures, the rational use of antibiotics in the absence of bacterial co-pathogens and, in some instances, the use of specific antiviral therapy. Two patients in our series received the antiviral agent cidofovir, and while there are no randomized, controlled trials demonstrating its efficacy in adenoviral infection, it has been used successfully in immunocompromised patients with severe adenoviral pneumonia.
This study did not involve human experimentation; the only human material used in this study was throat swab specimens collected from cases with respiratory tract infection during the implementation of the surveillance project on viral aetiology of acute respiratory infection. This study was approved by the second session of the Ethics Review Committee of the National Institute for Viral Disease Control and Prevention in China CDC. Written informed consent for the use of the clinical samples has been obtained from all patients involved in this study.
Pharynx and tonsil secretions of the patients were wiped with disinfection long cotton swabs with gently action, and after samples collection, all samples were transported under a cold chain and preserved at −80°C for further identification. A multiplex one-step reverse transcription-polymerase chain reaction (PCR) was performed to screen for 15 different respiratory viruses (respiratory syncytial virus A and B, influenza virus A and B, parainfluenza virus 1–4, human adenovirus, human enterovirus, human rhinovirus, human metapneumovirus, human bocavirus, and human coronavirus NL63-229E and OC43-HKU1) simultaneously by using a commercial kit (Seeplex RV 15 ACE Detection kit; Seegene, Inc., Seoul, Korea). Adenovirus-positive specimens were cultured and further analysed.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and under animal biosafety level 2 (BSL-2) conditions with personnel using BSL-3 practices as approved by the Institutional Biohazards Committee of the Texas Biomedical Research Institute (TBRI). The protocol for marmoset study 1305-CJ, “The pathogenicity of the novel adenovirus TMAdV in a marmoset animal model”, was approved by the Institutional Animal Care and Use Committee and the Institutional Biohazards Committee of the TBRI. TBRI is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International and operates in accordance with the NIH and U.S. Department of Agriculture guidelines and the Animal Welfare Act. Marmosets were kept healthy and well-nourished with strict feeding protocols and close monitoring of their health status prior to the start of the study and during the entire study period. One week before inoculation, animals were transferred to the biosafety level-3/4 facility at the TBRI and housed individually in cages specifically developed for marmoset work. Marmosets were sedated and humanely euthanized by administration of a sodium pentobarbital solution by a licensed veterinarian at the TBRI.
Virus isolation for the adenovirus-positive specimens was performed by using HEp-2 cell lines (from American Type Culture Collection, ATCC Number CCL-23) following the standard protocol. Cells inoculated with clinical samples were incubated at 37°C for 7 days. If no cytopathic effect was observed, the culture was used to inoculate fresh cells for up to 2 additional passages; the cultures with adenovirus-like cytopathic effects were passaged again to confirm the presence of the virus.
The 4 human cell lines (HEK293, CaCo-2, A549, and HFDL) and 4 monkey cell lines (PMK, B95a, Vero, and BSC-1) used in this study have been previously established and are routinely maintained at the Viral and Rickettsial Disease Laboratory (VRDL) branch of the California Department of Public Health for use in viral diagnostic studies,. Viral supernatants for cell culture passaging or the generation of infectious stocks were subjected to 3 freeze-thaw cycles and clarified by centrifugation for 10 min×4000 g. After cells achieved 80–90% confluency, cell culture media were changed to maintenance media with 2% FBS and were inoculated with 100 µL of passaged viral supernatant. Viral replication was monitored over 14 days by visual inspection under light microscopy for cytopathic effect (CPE).
Early passages of TMAdV were not suitable for generation of infectious viral stocks for marmoset inoculation because of poor growth and insufficient titers in both monkey and human cell lines. Thus, in vivo work with experimental marmoset infection using wild-type or early passages of TMAdV, while desirable, was not practical. Robust growth was only observed after passage 7 in A549 cells, with the appearance of the 3202 C→G in the TMAdV E1B-55k resulting in the 388P→R amino acid change (Fig. 1), reflecting TMAdV adaptation. Viral stocks at titers of ∼107 TCID50/mL were prepared from passage 10 TMAdV initially isolated in BSC-1 and PMK cells and serially passaged in A549 cells (Fig. 1A). Infectious viral stocks were quantitated by an end-point dilution assay.
Adenoviruses, first isolated in the 1950s from explanted adenoid tissue, are double-stranded nonenveloped DNA viruses that naturally infect many vertebrates, including humans and nonhuman primates. The human adenoviruses in the Mastadenovirus genus, comprised of all mammalian adenoviruses, are classified into 7 species A-G, and at least 51 different serotypes (and 5 proposed types, HAdV-52 to HAdV-56) have been described to date,. Adenoviruses are the cause of an estimated 5–10% of febrile illnesses in children worldwide. Some serotypes, such as human adenovirus type 14 (HAdV-14), have been associated with severe and potentially fatal outbreaks of pneumonia in residential facilities and military bases. Adenoviruses have also been associated with other clinical syndromes including conjunctivitis, hepatitis, and diarrhea. In nonhuman primates, most epidemiologic studies of adenoviruses have focused on their identification in fecal samples from asymptomatic animals,,. Overt respiratory disease associated with simian adenoviruses has also been observed. Although adenoviruses are significant pathogens, genetically modified strains are being actively explored as potential vectors for vaccines and gene therapy.
Infection by adenoviruses has generally been thought to be species-specific. Human adenoviruses do not usually replicate in monkey cells in the absence of helper viruses, and do not productively infect rodents (and vice versa). Studies of sera from animal handlers and zoo workers exposed to chimpanzees in captivity fail to detect antibodies to chimpanzee adenoviruses,. However, recent serological surveys have found antibodies to New World and Old World monkey adenoviruses in donor human sera from regions where the monkeys are endemic,. In addition, phylogenetic analyses of adenoviruses from greater apes reveal that they fall precisely into “human” adenoviral species B, C, and E. The high degree of sequence relatedness within members of each species suggests that at least some adenoviral strains may be capable of infecting both nonhuman primates and humans.
Beginning in May of 2009, a deadly outbreak of fulminant pneumonia and hepatitis occurred in a closed colony of New World titi monkeys of the Callicebus genus at the California National Primate Research Center (CNPRC). Routine microbiological testing for an infectious etiology was negative. We previously developed the Virochip (University of California, San Francisco) as a broad-spectrum surveillance assay for identifying viral causes of unknown acute and chronic illnesses,,,,,,. The Virochip, a pan-viral microarray containing ∼19,000 probes derived from all viral species in GenBank (n∼2500),, has been previously successful in detection of novel outbreak viruses such as the SARS coronavirus, and the 2009 pandemic H1N1 influenza virus. Here we apply the Virochip to identify a novel and highly divergent adenovirus as the cause of the titi monkey outbreak. In addition, we present clinical and serological evidence that this virus may have infected a researcher at the CNPRC and a family member, thus demonstrating for the first time the potential for cross-species infection by adenoviruses.
Nested PCR targeting the HAdV hexon gene’s hypervariable region was employed for genotyping. The outer primers used were forward 5′-GCCACCTTCTTCCCCATGGC.
− 3′ and reverse 5′-GTAGCGTTGCCGGCCGAGAA-3′, and the internal primers were forward 5′-TTCCCCATGGCCCACAACAC-3′ and reverse 5′-GCCTCGATGACGCC.
GCGGTG-3′. Nested PCR was conducted in 25 μL volume comprising 2.5 μL of 10 × EXTaq buffer, 1.0 μL (10 pM) of each primer, 2.0 μL of dNTP Mix, 0.5 μL of EXTaq DNA polymerase, 2.0 μL of viral nucleic acid extract or first nested-PCR product, and 16 μL of double-distilled water. The mixtures were amplified with an initial denaturation at 94 °C for 10 min, followed by 36 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min, and a 7 min extension at 72 °C. PCR products were analyzed on 1.50% agarose gels, purified, and then confirmed as authentic by sequencing. Samples that failed to amplify are defined herein as untyped.
In hindsight, only one individual at the CNPRC reported becoming ill during the titi monkey outbreak, the researcher in closest, daily contact with the animals. Symptoms began near the onset of the outbreak, although whether they began prior to or after identification of the index case is unclear. The researcher, with a past medical history of multiple sclerosis, initially developed symptoms of a viral upper respiratory infection (URI), including fever, chills, headache, and sore throat, followed by a dry cough and “burning sensation in the lungs” that was exacerbated by a deep breath or coughing. The researcher endorsed a history of recurrent upper respiratory infections, and did not regard the illness as related to the titi monkey outbreak. Although symptoms persisted for 4 weeks, at no time did the researcher seek medical care, and no antibiotics were taken during the illness.
We carried out contact tracing to identify family members and other individuals in close contact with the researcher. Interestingly, two family members in the household also developed flu-like symptoms about 1–2 weeks after the researcher initially became sick. Their symptoms – fever, cough and muscle aches – appeared milder than those of the researcher and completely resolved within 2 weeks. Neither individual sought medical care for these symptoms, and notably, neither had ever visited the CNPRC.
Sixty-two of the patients (86.11%, 62/72) from the 72 HAdV-positive cases were diagnosed with pneumonia, including 46 with bronchopneumonia, 4 with asthmatic bronchitis, 3 with refractory bronchopneumonia, 8 with pneumonia (3 with mycoplasma pneumonia, 3 with bacterial pneumonia and 2 with viral pneumonia) and 1 with refractory pneumonia. Viral pneumonia and bacterial pneumonia accounts for 3.23% (2/62) and 4.84% (3/62) of the pneumonia, respectively. In addition, the remaining 10 cases were diagnosed with acute tonsillitis (5), acute upper respiratory infection (2), acute bronchitis (2), as well as 1 with Epstein-Barr virus-associated infectious mononucleosis. The main clinical signs in the HAdV-positive patients were fever (97.22%, 70/72) and cough (75%, 54/72), while the other clinical presentation were vomiting (18.06%, 13/72), gasping (15.28%, 11/72), diarrhea (4.17%, 3/72) and angina (5.56%, 4/72). Furthemore, vomiting didn’t associate with specific HAdV types by Fisher’s exact probability (P = 0.069). In this study, of the 72 HAdV-positive children with RTIs, 4 (5.56%, 4/72) had asthma, the other 68 had no pre-existing chronic conditions.
TNF has a wide range of biological functions including modulation of immune response to pathogen assault. Mouse TNF knock-out phenotypes include abnormal immune system physiology, increased susceptibility to viral infection, and both increased and decreased susceptibility to bacterial infection–. In our study, TNF is mostly up regulated in infections by CMV, CORON, COX, and FLU but directionally ambiguous for MPNEU and not expressed under RSV. While total disruption of TNF function would be deleterious to the host, there are instances where partial TNF inhibition provides a clinical benefit in patients with viral complications,.
Pranlukast is a cysteinyl leukotriene receptor-1 antagonist that reduces bronchospasm caused by an allergic reaction, usually with asthmatic individuals. This drug inhibits TNF-alpha by blocking macrophage cysteinyl leukotriene 1 (cysLTC4, D4) receptors or suppression of NF-kappa B activation. Pranlukast has been recently shown to be beneficial not only in cases of respiratory syncytial virus postbronchiolitis, but also in a wide variety of other diseases with strong inflammatory complications such as cystic fibrosis, cancer, atherosclerosis, eosinophils cystitis, otitis media, capsular contracture, and eosinophilic gastrointestinal disorders.
Amrinone is a type 3 pyridine phosphodiesterase inhibitor used in the treatment of congestive heart failure and is an inhibitor of TNF. Phosphodiesterase inhibitors have been shown to alter immune response– and, in one case, specifically through TNF. Amrinone is known to modulate pro- and anti-inflammatory factors in endotoxin-stimulated cells. Type 4 phosphodiesterase inhibitors have been used to treat RSV-induced airway hyper-responsiveness and lung eosinophilia. Therefore, indirect evidence suggests that Armirone may be beneficial in respiratory viral infection situations by inhibiting TNF via type 4 phosphodiesterase, although this has yet to be seen in clinical studies.
Diarrhea is the second most common cause of death worldwide and accounts for about 8 to 9% of the 5.9 million yearly deaths in children under the age of 5 (1, 2). Most of these deaths occur in Southeast Asia and sub-Saharan Africa (3, 4). The chances of infection with enteric viruses are higher in developing countries than developed countries, probably due to suboptimal sanitation and hygienic conditions and low quality of drinking water, especially in rural areas (5). In Cameroon, a limited number of studies have investigated the prevalence of enteric pathogens as the cause of gastroenteritis in humans. These studies mainly focused on the epidemiology of a limited number of pathogens such as rotavirus, norovirus, and enteroviruses, revealing significant differences in the prevalence of these viruses in different settings and time periods (4, 6, 7). In parts of Cameroon, a high prevalence of several enteric viruses such as enterovirus, norovirus, rotavirus, and adenovirus was found in children and adults (8). Generally in Africa, many episodes of gastroenteritis remain unexplained as no etiological agent is determined (9, 10). A proportion of the unexplained gastroenteritis cases are likely due to other known viruses, for which no tests were performed. However, a part of these gastroenteritis cases could also be caused by novel viral agents.
Transmission of these enteric viruses is predominantly fecal-oral, and humans are constantly exposed to these viruses through various routes (11). One of these routes is zoonosis from reservoirs in wild or domestic animals, either by insect vectors or by exposure to animal droppings or tissues. One rich but, until recently, underappreciated reservoir of emergent viruses is bats. Of the ∼5,500 known terrestrial species of mammals, about 20% are bats (12). Several viruses pathogenic to humans are believed to have originated in bats over the last several years, including severe acute respiratory syndrome (SARS)- and Middle East respiratory syndrome (MERS)-related coronaviruses, as well as filoviruses, such as Ebola and Marburg viruses, or henipaviruses, such as Nipah and Hendra viruses (13–18).
In the Southwest region of Cameroon, bats are hunted and eaten. Such close interactions provide ample opportunity for zoonotic events to occur (19).
Previously, we identified a plethora of known and novel eukaryotic viruses in Cameroonian fruit bats using a viral metagenomics approach, including viruses known to cause gastroenteritis in humans (sapovirus, sapelovirus, and rotaviruses A and H) and those not yet associated with gastroenteritis (bastrovirus and picobirna-like viruses) (20–23). In the current study, we metagenomically screened 221 human fecal samples collected in the same region (where bats are hunted and eaten), to assess (i) if any viruses of animal origin could be identified and (ii) which known human gastrointestinal viruses were present. These fecal samples were collected from children less than a year old to adults of more than 60 years who had gastroenteritis and/or were in contact with bats. Additionally, since the gut virome typically contains both eukaryotic and prokaryotic viruses (phages), of which the latter usually represents the largest fraction of the gut virome, we also analyzed the phageome of these samples.
In contrast to several widely used antibiotics, antiviral drugs typically target a single virus. The rising number of diverse emerging and endemic viral diseases that pose a significant health risk to human populations underscores the need to develop broad-spectrum antivirals. However, the few broad-spectrum antivirals that are licensed have limited potency and are poorly tolerated. Theoretically, a broad-spectrum antiviral compound could target a conserved host pathway or protein that supports infection by multiple viruses or a common viral structural feature. As an example, host proteases are potential targets for broad-spectrum antivirals since many viruses such as Ebola, reovirus and coronavirus all require host proteases for entry [2–4]. In addition, the host enzyme IMP dehydrogenase, the target of ribavirin, is required for the replication of several RNA and DNA viruses. Currently in phase II clinical trials for treatment of influenza, T-705 (favipiravir), also inhibits several RNA viruses - likely through selective inhibition of the viral RNA-dependent RNA polymerase. In a recent PNAS study, Wolf et al. describe the discovery and characterization of a promising broad-spectrum antiviral compound, LJ001, which shows activity against an impressive number of enveloped viruses.
LJ001 is an aryl methyldiene rhodanine derivative that was found during a high-throughput cell-based screen for inhibitors of Nipah virus (NiV) entry using a vesicular stomatitis virus (VSV) luciferase reporter pseudotype system. LJ001 caused minimal cellular toxicity at in vitro inhibitory concentrations. The authors employed several independent approaches to show that LJ001 acts at the level of virus entry. For example, inhibition was only achieved if LJ001 was added before or during virus absorption onto cells - if it was added after virus adsorption, infection occurred normally. A beta-lactamase content mixing assay further demonstrated a role during entry, though a binding assay showed that viral attachment occurred normally, thus implicating virus-membrane fusion as a potential target.
To this point, the activity of LJ001 resembled that of other inhibitors that act at very early stages of the viral lifecycle. However, it became evident that LJ001 showed antiviral activity not just against Nipah virus, but also against an impressive array of viruses including representatives of the filoviridae, orthomyxoviridae, arenaviridae, bunyaviridae, paramyxoviridae, flaviviridae, retroviridae, poxviridae and rhbdoviridae, but not against members of the adenoviridae, picornoviridae and reoviridae. As a result, its antiviral activity was independent of the viral glycoprotein responsible for mediating virus entry. The only unifying, structural feature evident amongst the diverse viruses that were inhibited by LJ001 is the presence of a viral membrane: LJ001 inhibited all enveloped viruses tested, but failed to inhibit nonenveloped viruses. Aided by its intrinsic fluorescence, the authors showed that LJ001 associates with liposomes, and that liposomes can compete with NiV-pseudotyped VSV for LJ001 unless virions were pretreated with LJ001 before addition of liposomes. The authors suggest that LJ001 intercalates into viral membranes in an essentially irreversible manner, preventing membrane fusion in a manner that is independent of the viral membrane fusion protein. To further support a proposed viral glycoprotein-independent mechanism of action, the authors attempted to generate resistant HIV mutants through serial passaging, yet resistant virus failed to develop after four weeks. Longer passaging studies as well as the use of additional strains of the viruses tested thus far may provide clues for a mechanism of action and address the important issue of whether resistance can be generated. Another unanswered question is whether LJ001 intercalates preferentially into viral membranes, or whether it associates equally well with any lipid bilayer regardless of specific protein or lipid content. Without some degree of membrane specificity, it is difficult to envisage how LJ001 will achieve high enough levels in vivo to prove effective. If systemic administration becomes impractical, a topical formulation may be feasible, as with the anti-herpesvirus drug Docosanol (Abreva). Importantly, the association of LJ001 with viral membranes was potent enough to protect mice infected with lethal doses of Ebola or Rift Valley Fever virus, but only when virions were pre-treated with LJ001 prior to inoculation of the animals.
Though LJ001 associates with both viral and host membranes, it is only the viral membrane whose function appears to be impaired: LJ001 inhibits virus-cell fusion, but it fails to inhibit cell-cell fusion reactions mediated by the same viral glycoprotein. The authors hypothesize that this is due to the fact that host membranes are continually remodeled and can repair themselves by metabolizing or extracting membrane-active agents, thus escaping gross membrane perturbation. Consistent with this, synergistic disruption of plasma membrane integrity was observed when cells were exposed to an inhibitor of fatty acid synthesis (TOFA) and LJ001. This again suggests that simply associating with lipid membranes is not sufficient for antiviral activity, though the precise mechanism has not yet been determined. One mechanism by which LJ001 could potentially prevent fusion is by altering membrane curvature, as a variety of non-bilayer lipids that impact membrane curvature have been shown to inhibit fusion.
Other membrane targeting broad-spectrum antivirals have been developed with varying degrees of success. Cosalane is thought to associate with viral membranes and showed activity against HIV and some herpes viruses in vitro; however, due to its hydrophobic properties, efforts are focused on maximizing bioavailability and tissue clearance. Early studies with Docosanol (Abreva), a saturated alcohol, showed activity against several enveloped viruses and was found to bind to viral membranes. It is currently licensed as a topical cream for treatment of herpes cold sores. Arbidol is an indole derivative that is currently licensed in Russia to treat acute respiratory infections. Like LJ001, arbidol has a strong affinity for membranes and shows a wide spectrum of activity against not only several enveloped but also some non-enveloped viruses. While the mechanism of action of arbidol is not entirely clear, a recent study with HCV shows some similarities with LJ001.
The discovery of LJ001 serves as an important proof of principle for a more global and unbiased approach towards the screening of antiviral compounds. While traditional in vitro enzymatic screening approaches have led to the development of potent and highly specific antiviral agents, cell-based screens such as undertaken by Wolf et al. may prove even more successful in drug discovery in part because biological activity and toxicity are upfront criteria in choosing lead compounds – factors which often halt the progression of otherwise promising compounds – while sidestepping the expensive undertaking of biological target validation that must precede in vitro-based screens. Live-cell screening approaches also afford the opportunity to identify antiviral agents that target any aspect of the virus lifecycle, including compounds that target conserved host cell components rather than viral proteins themselves. The unprecedented activity of LJ001 against numerous enveloped viruses raises the possibility that effective, broad-spectrum antivirals can be identified.
This research was approved by the Review Board of the Center for Disease Control and Prevention of Henan Province, the Review Board of the Center for Disease Control and Prevention of Xinyang city, the Review Board of Beijing Institute of Genomics, the Review Board of Beijing Genomics Institute in Shenzhen, and the Review Board of the Institute of Microbiology and Epidemiology. All participants gave written informed consent for use of their samples in research.
AdV, adenovirus; ARI, acute respiratory infection; CT, cycle threshold; DNA, deoxyribonucleic acid; EC, extraction control; ERV3, human endogenous retrovirus 3; Flu, influenzae virus; HBoV, human bocavirus; HCoV, human coronavirus; hMPV, human metapneumovirus; PCR, polymerase chain reaction; PIV, paraInfluenza virus; RhV/EV, rhino/entero virus; RNA, ribonucleic acid; ROC, receiver operating characteristic; RSV, human respiratory syncytial virus; SARS, severe acute respiratory syndrome; UTM, universal transport medium
To obtain sufficient viral nucleic acid, SISPA was employed to amplify the dscDNA with the Accuprime Taq DNA Polymerase System (Invitrogen) according to the manufacturer’s protocol. Briefly, a 50 µl reaction system containing 10 µl of the above dscDNA mixture, barcode DNA as primer (20 µM), 10×Accuprime buffer I, Accuprime Taq DNA Polymerase (1 U) and ddH2O was denatured at 94°C for 2 min, followed by 40 cycles of 94°C denaturing for 30 s, 55°C annealing for 30 s, 68°C extending for 1 min with final 68°C extension for 8 min. The PCR products were then purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and dissolved in 50 µl TE buffer (100 mM Tris-HCl, 10 mM EDTA, pH8.0).
The study was observational and non-invasive. Protocols were approved by Institutional Animal Care and Use Committees (IACUC) of Harvard University (protocol 96-03) and University of New Mexico (protocol 14-101186-MCC) and were exempt by Boston University’s and the University of Michigan’s IACUC. Protocols followed the Weatherall Report, NIH Guide for the Care and Use of Laboratory Animals, USDA Animal Welfare Act, Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals, US Public Health Service and National Academies of Sciences National Research Council, and US Centers for Disease Control and Prevention.
The inhibitors and chemicals used in this study were diluted in DMSO to a stock concentration of 10 mM and were subsequently diluted in media to make working solutions. Monolayers of RAW264.7 and CRFK cells grown in 6- or 12-well plates were treated with chemicals or inhibitors as described previously: mock-treatment, pre-treatment, post-treatment, and pre-post-treatment. Briefly, confluent cells pretreated with various concentrations of the inhibitors for 24 h were infected with MNV (MOI, 1 TCID50/ml) or FCV (MOI, 1 FFU/ml) strains for the pre-treatment groups. For the post-treatment groups, cells were adsorbed with MNV (MOI, 1 TCID50/ml) or FCV (MOI, 1 FFU/ml) strains and then treated with different concentrations of inhibitors. For the pre-post-treatment groups, cells were pretreated with different concentrations of inhibitors for 24 h, adsorbed with MNV (MOI, 1 TCID50/ml) or FCV (MOI, 1 FFU/ml) strains, and treated again with the same concentration of inhibitors described above.
The World Health Organization (WHO) Global Polio Eradication Initiative (GPEI) was established in 1988 and successfully prevented wild-type poliovirus (WPV) transmission in the Americas, the Western Pacific (WPR), and Europe (EUR) [1–3]. The Southeast Asia Region (SEAR), home to a quarter of the world's population, was also certified polio-free in March 2014. WHO certified Taiwan, along with the entire WPR, as polio-free in 2000 and Taiwan changed its immunization strategy from oral (OPV) to inactivated polio vaccine (IPV) in 2010. To date, WPV remains endemic in Afghanistan, Nigeria, and Pakistan. Numerous outbreaks in heretofore polio-free regions have been reported recently in China (2011), Somalia (2013), Ethiopia (2013), and Kenya (2013) caused by importation [5–7]. Besides WPV, cases of circulating vaccine-derived poliovirus (cVDPV) causing acute flaccid paralysis (AFP) have risen since 2000, and have been identified in eight countries in 2013 and in two countries in May 2014.
Normally, acute flaccid paralysis (AFP) surveillance is the gold standard for poliovirus surveillance in eradication initiatives; under certain circumstances, environment surveillance is also employed to monitor the circulation of poliovirus in populations in order to better understand its evolution and transmission [9–13]. For instance, although certified as polio-free in 2002, Israel isolated WPV in routine environmental sewage samples in early February 2013, and immediate steps were taken to implement national supplementary immunization with OPV to prevent its spread. Recently, the WHO included environmental poliovirus surveillance in a new strategic plan as part of its global eradication initiative to supplement AFP surveillance. In Taiwan, AFP surveillance has long been established for poliovirus surveillance of the population, but environmental surveillance is not routinely performed.
Besides poliovirus in populations, enteroviruses, adenoviruses, reoviruses, and noroviruses are often found in environmental raw sewage [16–19]. These groups of viruses can cause a broad range of asymptomatic to severe gasterointestinal or respiratory infections, or even more acute conditions such as meningitis and paralysis, thus constituting a considerable public health problem in the community. Among these fecal-oral viral pathogens, reovirus is usually the most abundant virus detected in environmental water [22, 23]. Mammalian orthoreovirus (MRV), which belongs to the family Reoviridae and the genus Orthoreovirus, are a common class of enteric viruses capable of infecting a broad range of mammalian species, including humans. Previous studies have indicated that reoviruses have a high endemic infection rate in humans and seroconversion was found in more than 70% of 4-year-old children. Although reovirus infection in humans usually induces mild respiratory or gastrointestinal symptoms, there are reports of human reovirus-associated neurological disease [26, 27]. Several studies also described the isolation of reovirus strains directly from cerebrospinal fluid (CSF) or neural tissues obtained from patients with meningitis or encephalitis [28–31]. In addition, immunocompromised, pediatric, and elderly populations may become susceptible to severe bacterial respiratory disease due to an initial reovirus infection [32, 33].
In response to the international threat of WPV importation and the changes to the national vaccination policy, we adopted the WHO guidelines for environmental surveillance of circulation in Taiwan. Two-phase Dextran 40/Polyethylene glycol (PEG) separation and cell culture were performed to monitor environmental viral circulation. We successfully isolated enteroviruses, adenoviruses, and mammalian orthoreoviruses, but no poliovirus was detected in sewage collected islandwide. Our results showed a high incidence of MRV, which may cause human disease, and thus further research is warranted.
This study was reviewed and approved by the Centers for Disease Control, Department of Health, Taipei, Taiwan. Permission was obtained from the wastewater treatment plants before conducting the study.