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14.What is the most effective specific treatment and the expected clinical response to treatment of FeL due to L. infantum?
The published information on the treatment of FeL is extremely limited because it is available from only 20 case reports and only some of them were followed up (Table 7). Allopurinol is the most frequently used drug followed by meglumine antimoniate, but information is lacking on pharmacokinetic and pharmacodynamic characteristics of these drugs in cats and also about their safety.
Allopurinol is generally well tolerated; however, in one cat, elevation of hepatic enzymes was reported at 10 mg/kg BID and the dose was reduced to 5 mg/kg BID. Clinical improvement was observed in most cases treated with allopurinol – even in FIV positive cats –within a few weeks after treatment was initated [37, 50, 64] or slowly after 6 months. A long term follow-up was available in some cats treated with allopurinol. A clinical cure was obtained in these cats but relapse occurred after discontinuation of treatment, suggesting that they were still infected [14, 37, 55]. Clinical worsening leading to euthanasia occurred in a few cases after a few weeks of therapy [54, 57].
Clinical cure was generally obtained in the few cats that were treated with meglumine antimoniate, but long term follow up are not available from these cases.
Some other oral drugs (fluconazole, itraconazole, metronidazole and spiramycin) administered to one cat at different times were considered as not effective.
Surgical removal of cutaneous nodules (performed in two cats) was followed by relapsing of cutaneous lesions [36, 51].
In conclusion, currently, no scientific evidence concerning the best treatment for FeL is available, but more extensive clinical experience is available for treatment with allopurinol (10 mg/kg BID or 20 mg/kg SID). The drug of choice to be used in FeL should nevertheless be based on the best compliance and safety for the cat with the alternatives of long term oral drug treatment (allopurinol) or a parenteral therapy (meglumine antimoniate). As there are no studies on the safety of these drugs in cats, it is recommended to strictly monitor the health status of animals under treatment by means of regular check-ups including urinalysis, and advising the owner to promptly report any abnormality.
The duration of allopurinol treatment should be evaluated case by case based on clinical response and on parasitological and serological monitoring.
A further strategy is based on the use of epitopes which can be delivered using viral or DNA vectors. Such an epitope-based strategy for coronavirus vaccination has already been reported and the major advantages is the prevention of a possible vaccine reversion to virulence. A further benefit of this technique is the possibility to eliminate any regions of the viral genomic sequence which be associated with a potential autoimmune effects. The limitation of this approach is mainly based on potential variations. In this respect, epitopes which frequently undergo mutations will not protect against the SARS-CoV infections if used in epitope-based vaccines. If the SARS-CoV evolves as a highly variable virus, it will be crucial to identify highly conserved epitopes of the virus.
In summary, the important development of SARS vaccines can be approached using several techniques which should ideally encompass the induction of both humoral and cell-mediated mechanisms. As coronavirus vaccines in animals have partly been reported to cause an enhancement of viral infections, a cautious approach has to be followed. A first study has investigated the ability of adenoviral delivery of a codon-optimised SARS-CoV spike protein S1 fragment, membrane protein, and nucleocapsid protein to induce immunity in rhesus macaques. The immunization with a combination of these three Ad5-SARS-CoV vectors and a booster vaccination on day 28 demonstrated antibody responses against the spike protein S1 fragment. Also T-cell responses against the nucleocapsid protein were found and all vaccinated animals displayed strong neutralising antibody responses in vitro. These results indicated that an adenoviral-based vaccine can induce SARS-CoV-specific immune responses in monkeys.
As most patients develop an immunity against the SARS-CoV and survive the infection, the possibility of creating an effective and safe vaccine seems to exist. There are several options to develop vaccines against the SARS-CoV.
There is insufficient evidence to recommend withholding necessary medications for dogs and cats with IMHA. However, all medications, particularly those previously implicated in immune‐mediated diseases, should be used with caution in patients with IMHA. Every patient should ideally have a complete history recorded, which includes all vaccines and drugs administered, the doses, dates, frequency, duration, and route of their administration, and information about the products being used such as manufacturer, indications, specific lot, and any adverse events. Exposure to toxins should also be documented in any dog or cat with IMHA.
Evidence for cefazedone in dogs168 and propylthiouracil in cats173, 174 suggests that >1 class of drugs may be associated with IMHA in small animals. For most commonly prescribed medications, the evidence is negligible. Specific documentation of vaccine histories and long‐term prospective studies may help determine whether vaccines can trigger IMHA. To date, approximately 8% of dogs with a diagnosis of IMHA and vaccination histories had been vaccinated within 30 days of IMHA diagnosis. However, studies comparing this prevalence to adequate controls are limited and inconclusive. Animals with IMHA are at risk for recurrence of anemia, making careful decisions about the risks and benefits of revaccinating important in every case. Animals receiving immunosuppressive treatment are less likely to mount protective immunity after routine vaccination.
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.
Wide range of viruses is known to be associated with respiratory disease in humans. Adenoviruses, coronaviruses, human enteroviruses (HEV), human rhinoviruses (HRV), influenza viruses, parainfluenza viruses (PIV), and respiratory syncytial viruses (RSV) are well-known causes of acute respiratory tract infections (ARTI) in both industrialized and developing countries. Over the last decade, modern molecular techniques have led to the discovery of several previously unknown respiratory tract viruses, including human metapneumovirus (hMPV), two new human coronavirus types [2, 3], human bocavirus (HBoV), and two new human polyomaviruses [5, 6]. The significance of these novel viruses has been reviewed recently [7, 8].
It is widely accepted that common cold is almost always caused by viruses, most frequently by HRV, and viral infections are considered to contribute to the generation of complications of common cold, such as acute otitis media and sinusitis. Moreover, different viruses, including influenza viruses and RSV, are also frequently detected in samples obtained from patients with lower respiratory tract infection (LRTI), either alone or together with pathogenic bacteria. Several recent reports, including some from Africa, suggest viruses as potential etiologic agents in pneumonia in children [10–13], or exacerbations of asthma [14–16].
Several studies underscore the importance of respiratory tract viruses in Nigerian patients, but these studies were carried out before the introduction of modern molecular diagnostic techniques [14, 17–19]. The present study was designed to identify viral agents associated with respiratory infections among young children in Nigeria using modern, validated molecular techniques. We wanted to explore the presence of different virus groups, including some of the newer ones detected by only molecular techniques.
There is no consensus in the literature on the clinical implications of the viral detection and co-detection. Some studies linked multiple viral detections with fever, or increased hospitalization and intensive care admission, while others described a very similar prognosis as in single infection, or even milder presentations. In this study, the virus-negative patients had fever more often, which may be caused by other pathogens such as bacteria. We also found that rhinorrhea was more frequently present in patients with multiple viruses than in those with a single virus, and some viruses were more (or less) likely to exist in certain age groups or were accompanied with certain symptoms. Since we did not follow the cases, the associated clinical course and outcome (such as hospitalization) remain unknown. A better understanding on the clinical courses of single and multiple viral etiologies requires further studies.
The current study has several limitations. The majority of outpatients enrolled in this study were mild and moderate cases. Therefore, we could have missed pathogens responsible for severe ARIs. As healthy or asymptomatic controls were not included, their viral carriage burdens and the actual role of virus infections could not be elucidated. Following up the cases for clinical burdens and serologic testing would be required in future studies. Air quality indicators such as Ozone and PM2.5, which might influence the host’s susceptibility or virus circulation, should be included to investigate meteorological factors.
The study was approved by the Ethical Committee of the Oyo State Ministry of Health. Participation of children in the study was voluntary and required informed consent from the parents. Inclusion criteria were recent onset of symptoms suggestive for respiratory tract infection, such as cough, coryza, repeated sneezing, and/or difficulty in breathing. Patients were recruited between February and May, 2009, and included hospitalized patients, children seen at emergency departments, and outpatient clinics at 3 different children's hospitals in Ibadan, Oyo state. Demographic and clinical information, including age, sex and clinical symptoms, was recorded during the medical visit by means of a structured questionnaire.
A nasal swab sample was obtained from children by inserting a sterile nylon swab (Regular Flocked swab, Cat. No. 520CS01, Copan Diagnostics Inc., Murrieta, Calif USA) into the nostril to a depth of 2–4 cm, and retracting it with a rotating motion, in order to trap epithelial cells in the swab. With a second swab, a throat specimen was collected by rubbing the tonsils and the posterior wall of the pharynx. The 2 swabs were then placed in a vial containing 2 mL of RNAlater solution (RNAlater Tissue Collection, Applied Biosystems, Espoo, Finland). The specimens were transported to the laboratory on the same day in an ice pack and stored at −70°C until further processing.
The study was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster. The study was conducted in accordance with the Declaration of Helsinki. Verbal consent was obtained from the participants and written consent was obtained from their parents or legal guardians.
It is not known whether HCoV-EMC is going to be fully established in humans. Extensive efforts have been made and will continue to be needed to fight against this possible epidemic. If we are “lucky” enough to control this novel disease, more resources should be allocated to different areas of coronavirus studies. Currently, we know some animal coronaviruses in wildlife only at the nucleotide level. In fact, the number of bat species tested for coronaviruses is only a fraction of the total number (>1,200) of bat species. In addition, there is a lack of biological/biochemical characterization of these animal viruses. Ideally, we should develop an effective universal strategy to treat and prevent human infections caused by animal coronaviruses. The phylogenetic relationships of coronaviruses (Fig. 1) suggest that there have been a number of introductions of animal coronaviruses (e.g., SARS-CoV and 229E) into humans in the past. The great diversity of coronavirus in bats will surely increase the odds of yet another zoonotic event occurring in the future.
Although the data on FeL supported by consolidated evidence-based studies are limited, these guidelines constitute a baseline for educating and informing feline practitioners with the most comprehensive and updated data set on this important neglected feline protozoal disease.
Further studies need to elucidate gaps in knowledge on this infection in cats and to provide evidence-based information on the management of this disease.
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.
For the cultivation, purification and titration of recombinant baculovirus (r-virus), Spodoptera frugiperda-9 (Sf-9) cells were used in this study. The Sf-9 cells were cultured in suspension at 27°C at densities ranging from 0.5-2 × 106 cells/ml in HyQ SFX-Insect Media (HyClone, Logan, UT, USA) containing 5% (v/v) fetal bovine serum (FBS) (PAA Laboratories GmbH, Pasching, Austria) and 10 μg/ml gentamicin.
Felis catus whole fetus-4 (Fcwf-4) cells were used for the propagation of the type II FCoV strain NTU156 and maintained in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, USA) supplemented with 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin in 5% CO2 at 37°C.
In late December 2018, all ten cats kept at household A were brought to a veterinary hospital with reported acute depression, bloody diarrhea and bloody respiratory discharge. All 10 cats, aged from 1–3 y, had been up-to-date vaccinated for FPLV, feline calicivirus (FCV), FeLV and rabies virus (RV). Later, in the beginning of January 2019, four core-vaccinated cats, aged from 1–2 y, from household B showed clinical signs of depression, followed by diarrhea, acute hemoptysis and ataxia; while three 1-month-old non-vaccinated kittens in household C were carried to the hospital in late February 2019 due to the acute onset of depression, anorexia, bloody diarrhea, hemoptysis and seizure.
Essential diagnostic tests showed severe anemia and marked leukopenia (ranging from 1,200–3,500 cells/µL) without significant changes in the blood chemistry panels in all cats. Neither protozoa nor parasitic eggs were found by microscopic fecal examination. The FeLV and FCoV antigen and FIV antibody tests all revealed negative results, while the FPLV antigen rapid test kits were positive. No evidence of detectable warfarin and organophosphate derivatives were observed in both the urine and feces samples. Bacterial cultures from nasal and fecal swabs in randomized cats from households A and B showed isolated Klebsiella sp. and Escherichia coli growth, respectively. However, no relevant findings in the aerobic bacterial cultures of the oral swab samples of cats from household C were found.
All 10 cats from household A did not respond to supportive treatment with antibiotics and fluid therapy, and decompensated over the course of 48 h. Including the cats and kittens in households B and C, 13 of the 17 affected cats died. Based on the availability of the owner’s consent, three of these moribund cats (one from each household, where cat no.1 to 3 was from household A to C, respectively) were submitted for necropsy and pathological examination. The FBoV-1 infected cat status, household of origin and sampling procedure are summarized in Fig. 1.
In late 2002, severe acute respiratory syndrome coronavirus (SARS-CoV) crossed the species barrier from animals to humans. SARS first struck in Guangdong Province, China, and was officially recognized by the World Health Organization (WHO) in February 2003. After its introduction into human populations in Hong Kong in February 2003, the virus spread across the globe within weeks. A number of superspreading events occurred in several health care settings during the epidemic. When SARS was declared to have been contained (5 July 2003), there were 8,098 confirmed SARS cases, and 774 of these patients died from the disease. After this catastrophic event, one of the most frequently asked questions was whether SARS would come back.
Ten years after the introduction of SARS, yet another novel coronavirus (NCoV, or HCoV-EMC hereafter) jumped from animals to humans (1, 2). At the time of writing, there are 17 confirmed human cases, including 11 deaths. Recent findings related to this novel virus are alarming. The virus can readily infect cell lines from multiple hosts, including humans, swine, monkeys, and bats, suggesting that it might have a relatively weak species barrier. In addition, many of these human cases/clusters are not epidemiologically linked. The first detected case dates back to April 2012 in the Middle East. It is not known whether there is a low level of circulation of this novel coronavirus in asymptomatic human carriers or if there is an animal reservoir that allows for multiple introductions of HCoV-EMC into humans. While the transmission route (or routes) from animals to humans is not yet identified, a recent report of three human cases from a family cluster in the United Kingdom indicates that this virus is transmissible between humans.
Previous studies showed virus identification rate ranged from 32% to 85% of asthma exacerbation in children [14, 19]. The conservative estimation of virus detection rate of 50% would give the largest sample size estimate of 96 exacerbations with level of confidence at 95% and precision of detection rate of 10%. As the number of urgent visits due to asthma was 1.2 per person-year in children on regular inhaled steroids in another study, the number of subjects required would be around 80. We performed simple descriptive analyses of demographic data. The frequencies of presenting symptoms and physician diagnoses of unscheduled visits, virus detection rate and the distribution of different types of viruses were described. Student T test (+− Mann–Whitney U test) was used to compare continuous variables; for example, age and Pearson’s chi-square test (with Yates’correction/Fisher’s exact test) was used to compare categorical variables; for example, sex (female or male), atopic status (yes or no) between children with and without unscheduled visits. A p value less than 0.05 was considered to be statistically significant. All statistical analyses were carried out by the SPSS 11.0 software (SPSS Inc., Chicago, IL).
Several samples were collected from the cats enrolled in this study, including whole blood, plasma, swab samples (rectal, nasal, oral and conjunctival swabs), body effusions and internal organ samples, and were screened for FCoV by reverse transcription-nested polymerase chain reaction (RT-nPCR). FCoV-positive samples were subsequently subjected to genotyping of the virus according to the procedures reported by Addie et al..
Acute respiratory infections (ARIs) are one of the illnesses of highest morbidity and mortality in children worldwide. The pathogens causing ARIs vary geographically and by season, but globally viruses play a major role. Respiratory syncytial virus (RSV) is by far the most common pathogen associated with severe respiratory diseases as bronchiolitis, exacerbation of asthma, or pneumonia in early life, and is a leading cause of hospitalization in children under two. Influenza viruses have the greatest potential to cause severe respiratory diseases in the very young, the elderly and those with underlying chronic conditions. Enteroviruses including human rhinoviruses (HRV) and human enteroviruses (EV), previously identified in childhood upper respiratory tract infections, are commonly associated with milder ARIs and have been suspected as major etiological agents of lower respiratory tract infections leading to bronchiolitis and pneumonia in infants. It has also been reported that human metapneumovirus (hMPV) causes approximately 5-10% of all ARIs in children and adults and adenoviruses (ADV) account for 5-15% of respiratory infections in children. Respiratory illnesses can be attributable to other viruses such as parainfluenza viruses (PIV) and human coronaviruses hCoV-229E, OC43. With rapid progress in molecular diagnostics, newly discovered viruses including human bocavirus (hBoV), human coronaviruses (hCoV-NL63, hCoV-HKU1), human parechoviruses (hPeV), and polyomaviruses WU (WUPyV) and KI (KIPyV) have also been detected in children with respiratory infections, with varying levels of proof of causation.
Hospital-based studies in children published over the last decade worldwide have identified viruses in up to 95% of ARI episodes, with a single virus found in 40-60% and multiple viruses in 1-40% of infected patients. Co-infection is reportedly related to the time of year when circulations of multiple viruses occur. Some studies have shown that the prevalence of co-infections is not related to the absolute prevalence of individual viruses. Factors such as young age, male gender, and history of immunosuppression are associated with an increased chance of viral co-infections. There could be likely interactions between climatic, environmental, and behavioral factors, and complex interplay between circulating viruses and population-level immunity regarding viral co-infections. Understanding these factors may help us prevent transmission of these infections.
Recent etiologic studies on pediatric respiratory infections mostly report the prevalence in hospitalized children and the seasonality of viruses without elaborating viral co-infection. Therefore, the significance of the detection of multiple viral pathogens in ARIs is unclear. Here, we investigated fourteen common respiratory viruses among pediatric outpatients in southern China during 2010–2011 and their associations with meteorological factors.
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.
Cell monolayers were treated with 1% formaldehyde solution for 10 min at room temperature. Cells were then harvested and subjected to sonication. The lysed samples were centrifuged for 10 min at 13,000 rpm at 4°C and the supernatant was diluted 10-fold with RIPA buffer containing protease inhibitor. Immunoprecipitation was then performed with Dynabeads Protein A (Invitrogen, Cat#: 100.01D) with antibodies against CREB (Abcam#ab32096) and NFкB (Abcam#ab7970). To analyze immunoprecipitated DNA, PCR amplification was performed with primers against Egr-1 promoter: 5'-TGG GGG GCT TCA CGT CAC TC-3' and 5'-AAG TTC TGC GGC TGG ATC TCT C-3''. The products were analyzed by 2% agarose gel electrophoresis.
All FFPE tissues from the three necropsied cats were subjected to IHC analysis using the 1.B.450 mouse monoclonal anti-canine parvovirus antibody (Abcam AB59832, Cambridge, UK) as the primary antibody. Briefly, after deparaffinization and rehydration, tissue sections were pretreated by 0.1% (w/v) trypsin at 37 °C for 25 min, followed by blocking endogenous peroxidase activity with 5% (w/v) skim milk at 37 °C for 40 min. After washing three times with PBS, the sections were incubated with primary antibody (1:200 dilution) at 4 °C overnight followed by detection using the Dako REAL EnVision Detection System (Dako, Glostrup, Denmark) at RT for 45 min. After triplicate washings with PBS, a positive antigen-antibody reaction was observed by labeling with DAB and counterstained with Mayer’s hematoxylin. The FPLV-positive intestinal tissue from a PCR-positive FPLV-infected cat served as a positive control. Samples treated with distilled water (DW) instead of the primary antibody and non FPLV-infected samples served as negative controls.
Acute respiratory viruses cause substantial morbidity and mortality worldwide. Most respiratory viral infections induce self-limiting disease. However, the disease range can vary from common cold, croup, and bronchiolitis to pneumonia, with an array of possible etiological agents, such as parainfluenza, influenza, RSV, adenovirus, rhinovirus, bocavirus, human metapneumovirus and coronavirus. Coronaviruses (CoV) are responsible for a broad spectrum of diseases, including respiratory and enteric illnesses, in humans and animals. Human coronaviruses (HCoV) were identified as the cause of acute respiratory tract disease in the early 1960’s, but their correlation with mild respiratory tract infection outweighed the importance of severe forms of the infection. The emergence of SARS-CoV in humans in 2003 increased scientific interest in CoVs and emphasized the ability of highly pathogenic CoVs, most importantly those of animal origin, to infect humans. Consequently the importance of monitoring circulating coronavirus strains in humans has been reemphasized with the emergence of SARS and Middle East Respiratory Syndrome (MERS) CoV in humans.
The family Coronaviridae was recently subdivided into four genera according to their antigenic and genetic characteristics: Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus (http://ictvonline.org/virusTaxonomy.asp?version=2012). Alphacoronavirus (HCoV-229E and HCoV-NL63) and Betacoronavirus (HCoV-OC43, SARS-CoV, HCoV-HKU1 and HCoV-MERS) infect a wide range of mammals [4,7–11], whereas members of the genus Gammacoronavirus and Deltacoronavirus usually infect birds, although a Gammacoronavirus was isolated from a Beluga whale. Feline CoV, an Alphacoronavirus, infects wild and domestic cats causing mild enteritis. However, a lethal systemic disease known as feline infectious peritonitis (FIP) is also associated with FCoV. Feline CoV is closely related to CCoV, TGEV and human coronavirus HCV-229E, especially the Feline aminopeptidase N, which can be used as a functional receptor by these viruses.
The CoVs have a positive-sense, single-stranded RNA genome of 27–32 Kb. Nine to fourteen open reading frames (ORF) have been identified in the CoV genome. ORF1a and ORF1b encode the highly conserved replicase complex. Most RT-PCR assays described in the literature to screen for CoV target the ORF1b region. CoVs show a high frequency of nucleotide mutation and RNA recombination through copy-choice mechanism which, associated with broad receptor and co-receptor usage allow the virus to increase pathogenicity and possibly shift its host range.
Before the SARS-CoV outbreak, only two HCoV respiratory strains, HCoV-229E and HCoV-OC43, had been described. Due to the increased interest highlighted by the SARS outbreak, three new strains were described afterwards; HCoV-NL63, HCoV-HKU1 and HCoV-MERS. This study aimed to investigate the circulation of human respiratory CoV and to determine the genetic variability of HCoV in Arkansas.
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.
The overnight culture of S. pyogenes, grown on MH agar at 37°C, was added to a saline solution, containing 0.5% Tween-80 (Sigma-Aldrich). Different concentrations of the A. vestita essential oil (5, 25 and 50 μg/ml) were prepared and added to the suspension, which was incubated at room temperature. After 12 h, the bacterial cells were centrifuged at 15,000 × g for 20 min. The bacterial cells were washed with 0.5 mol/l Tris-acetate buffer (pH 7.1; Sigma-Aldrich), fixed in Tris-acetate buffer containing 1.2% glutaraldehyde (Sigma-Aldrich), and were then freeze-dried. The bacterial culture of S. pyogenes was then visualized using SEM (Hitachi S-3000H; Hitachi, Ltd., Tokyo, Japan) at magnification, ×10,000. The bacterial cell suspension in saline with no essential oil treatment served as a negative control.