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In summary, this study reports a high viral carriage in pediatric ARI cases with high viral co-detection rates mainly due to EV and HRV. There were overlapping seasonal trends of many viruses throughout the year. Meteorological factors, including temperature, humidity, and wind velocity, were associated with the viral detection rates. Statistically significant associations were present among the viruses. Further studies are needed to address polyviral etiology and viral interaction in multiple virus positive cases.
PIV1 and PIV3 have been reported to be negatively associated with wind velocity. In low wind speed environment, viruses can easily colonize in the epithelium of upper respiratory tract. An increased wind velocity is correlated with RSV activity in Germany. In our study, PIV3, PIV4, and RSV were inversely associated with the wind velocity. Although we observed higher rates of EV and FluB but lower rate of HRV in low wind velocity, we could not confirm these associations by logistic regression analysis.
The underlying reasons for the observed associations between virus circulations and meteorological factors are unclear. Climate could have a direct or indirect effect on viral survival, transmission efficiency, host immunity, and social behavior change. Cold and dry conditions might favor the transmission of viruses, and cold or rainy days could decrease outdoor activities of children and increase the probabilities of close contact and transmission of infections. Holidays (supported by our data with less cases in February as Chinese new year and July-August summer holiday, Figure 2 and Additional file 3), could also play a role in an annual epidemic cycle. It is likely that several factors interact in complex ways in the development of observed epidemics under optimal climatic conditions and that the contributions of individual factors vary for different viruses. Further investigations such as time series model over many years are needed to account for their inherent autocorrelations, and thus the observed associations between meteorological parameters and viruses in this exploratory analysis should be interpreted with caution.
This study revealed that all major respiratory virus groups tested for can be detected in Nigerian children with respiratory tract infection. All viruses investigated, including the more recently discovered HMPV and HBoV, were present in at least some of the specimens. This wide variety is somewhat surprising because the specimens have been collected only during a four-month period, mostly in the dry season of the year. It is known from the previous studies that circulation of some viruses is at a low level during the dry season [25–27]. Seventy-seven percent of the children harboured at least one viral pathogen in their samples. This figure is relatively high taking in account that we did not have the possibility to test the specimens for human coronaviruses, and comparable to or higher than in some recent similar studies performed elsewhere [29–31]. Human rhinoviruses and PIV were the two most frequently detected virus groups. Simultaneous infections with two or even three viruses were also found, similar to observations by others in comparable studies [9, 25].
In individual cases, a demonstration of viral nucleic acids in a nasal or throat swab does not necessarily prove an etiological role of this pathogen in the concurrent disease as positive test results have also been obtained from a proportion of healthy individuals. Etiological diagnosis of LRTI is especially difficult without invasive procedures, but when searched for, for example, rhinovirus has been found in alveolar lavage specimens from pneumonia patients establishing the role of HRV as a significant respiratory pathogen beyond being the major causative agent of common cold.
Previous studies on respiratory tract viruses in Nigeria have not tested for the presence of rhinoviruses. In our study, HRV accounted for 39% of the virus findings in the study population reiterating the observations done elsewhere [9, 29, 30, 34]. Our test for the related HEV revealed a positive result in about 10% of the specimens, but two thirds of these specimens also tested positive for HRV. Because of the well-established cross-reactions, these double-reactive specimens were recorded as HRV, and only those 7 HEV-positive specimens that were HRV negative were scored as “true HEV positives.” However, we cannot exclude concomitant infection with both HEV and HRV in these double-reactive cases.
A high incidence of parainfluenza viruses was found in our study population with PIV3 being the most frequent of the three serotypes tested for. Interestingly, PIV3 has also been one major agent in some previous studies done in Nigeria [14, 17] and one in Cambodia. Respiratory syncytial virus was a rare finding in our study, likely due to the seasonal occurrence of this virus. Our specimens were collected in February-May which has been a low season for RSV in Nigeria in previous studies [26, 27].
Little was known about the epidemiology of influenza viruses in West Africa until very recently. A report from Senegal published, while this manuscript was in preparation, showed a September-October peak of influenza A and RSV. Hence, seasonality might have again been contributing to the paucity of influenza virus A and B findings among our patients. The two influenza A viruses further characterized were of the H3N2 subtype, that is, the same subtype that was predominant during the 2008-2009 influenza season (http://apps.who.int/globalatlas/dataQuery/default.asp). In contrast, influenza virus type C was found surprisingly often. With an incidence of almost 5%, influenza C was as common as adenoviruses in this study.
Double or triple infections were detected in 16% of the virus-positive children, which is comparable to that observed by others [31, 34]. We do not have any explanation for the observed relatively higher frequency in the older children. One could speculate about increasing number of contacts by age in the community, but we did not collect information enabling further analysis of this possibility.
In conclusion, the findings of this study emphasize the presence of all major groups of viruses in association with respiratory illnesses in young children of West Africa. Rhinoviruses and parainfluenza viruses were the most prevalent virus groups while influenza A and B viruses, as well RSV were rarely detected, possibly due to low season of these viruses during the time of sample collection.
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.
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.
In the Figure 2, the seasonality of the mainly respiratory pathogens found in each defined clinical group was represented. The HMPV circulated from February to July with a peak in April/May 2010 corresponding to the end of the hot and rainy season. This season is correlated to the highest number of CAP cases (group I). The end of the cool and dry season was correlated to the peak of the other ALRIs cases (group II), where we observed the HPIV and HCoV co-circulation (data not shown). RSV A/B infection shows a significant seasonal variation with a peak in the month of February 2011, during the hot and rainy season correlating with Flu-like illnesses cases (group III) and Influenza virus infection peaks (data not shown). The beginning of the cool and dry season represents the seasonality where we found the largest number of cases in each defined clinical group, except for the group III. Finally, HRV (data not shown) and HAdV circulated throughout the year without any epidemic peak.
In the present study, of the viruses evaluated, RV was most frequently related to exacerbations of asthma in Korean adult asthmatics. RV was responsible for one-third (32.4%) of the viral infections in exacerbated cases. This frequency is in good agreement with previous reports on Caucasians. RV URTI and LRTI were frequently associated with asthma exacerbations in child and adult Caucasian asthmatics.14 Nicholson et al.8 reported that 33% of 229 acute exacerbation cases were RV-positive by RT-PCR. A longitudinal study of adult patients reported an RV detection rate of 20% in 30 asthmatics.7 In a study of 42 adult asthmatics hospitalized due to respiratory viral infection, RV was detected in 30% of the subjects by RT-PCR.10 Thus, RV constitutes ~30% of the respiratory viruses related to asthma exacerbations in both Korean and Caucasian adult asthmatics. The prevalence of respiratory viruses differs markedly according to age and geography.13 The frequency of RV detection is significantly lower in adult asthmatics than in child asthmatics. Indeed, viruses were detected in ~80% of child asthmatics with acute exacerbations; RV caused 70%–90% of these episodes.6
Interestingly, the frequency of RV detection was higher in non-exacerbated stable LRTI cases than in exacerbated LRTI cases in our study (45.5% vs 32.4%), although the difference was not significant. Thus, because RV was detected in almost half of the stable LRTI cases, the relationship between RV infection and asthma exacerbations seems to be inconsistent: detection of RV in sputum does not always induce an asthma exacerbation. Other studies of adult asthmatics have reported similar results. In an 11 month longitudinal study of 31 atopic asthmatics aged 15–56 years, 30 viruses were detected, 18 (60%) of which were associated with asthma exacerbations.7 In these populations, RV was detected in 14/30 (46.6%) asthmatics and 3/9 (33.3%) non-asthmatics. A longitudinal study of adult asthmatics reported that one-third of symptomatic respiratory tract viral infections were not associated with worsening of asthma symptoms.10 In a recent longitudinal cohort study of couples (1 with asthma and 1 without) that evaluated the presence of RV in nasal secretions at 2-week intervals over a 3-month period, the incidence of RV infection was similar in those with asthma and those without (10.1% vs 8.5%).9 In a study of children, the rate of RV isolation was almost identical in those with wheezy bronchitis (28.6%) and those with upper respiratory illness (29.5%).20 These studies and our data suggest that RV is related to both acute exacerbations of asthma and stable LRTI without asthma exacerbations.
Although the prevalence of virus infections is similar in asthmatic children with vs without exacerbations, respiratory symptoms are more severe in the presence of viral infections, in that cold and asthma symptoms are more than 2-fold longer in duration and loss of control is more frequent in virus-positive compared with virus-negative respiratory tract illness in children (47% vs 22%).5 Adults with asthma are not at increased risk of RV infection, but those infected with RV have more severe and longer-lasting LRTI symptoms and greater decreases in peak expiratory flow rate than do healthy individuals.9
In the present study, IFV A was the second most frequently detected virus. Thus, RV and IFV constituted 51.5% of the viral infections in subjects with exacerbated asthma. A similar frequency has been reported in adult asthmatics.21 However, RV and IFV infections are more prevalent among subjects with asthma exacerbations compared with those without exacerbations in Korean children.22
In the present study, respiratory viruses were detected in the sputum of subjects with exacerbated (26.3%) and stable (17.2%) asthma. This detection rate is lower than that initially expected. In a 1979 study of children, the detection rate was 26.4%.20 In that study, virus isolation was performed by cell culture.20 In contrast, respiratory viruses can now be identified by virus culture, serology, immunofluorescence antigen detection, and PCR-based tests.13 Since the development of PCR assays in the 1990s, their sensitivity has improved markedly. Thus, our detection rate is relatively low, likely due to inadequate sputum samples. Virus detection is usually performed in the upper airway because collection of sputum from young children and some adult asthmatics is not feasible. In our study, viruses were detected in sputum samples, not in nasopharyngeal washings or swabs. Use of this method may have resulted in the low frequency of RV detection in our study, because RV usually infects the upper respiratory tract.9 However, in our comparison study of sputum and nasal swabs in asthmatics with LRTIs,23 the concordance rate of virus detection was 95.2%, and the detection rate was higher in sputum than in nasal swabs. Thus, use of sputum did not seem to be a cause of the low frequency of RV detection in our study. The second reason is the delayed sampling of sputum in our study. An exacerbation was defined as aggravation of pre-existing symptoms of dyspnea and wheezing within 14 days before the study and a post-bronchodilator FEV1 <80% of the predicted value or the personal best.18 RV has an incubation period of 2 days and is shed for 7 days after development of symptoms.2425 FEV1 decreases significantly after infection, reaching a minimum at 2 days after experimental RV inoculation.26 Because our study was a cross-sectional design, we did not analyze the lag time between the appearance of LRTI symptoms and sputum virus analysis. Therefore, a lag time of >7 days in some patients may have resulted in the low detection rate.
A subset of asthmatics is particularly susceptible to recurrent exacerbations.2 In our study, the initial sputum samples (exacerbation) were positive for virus, while the second sputum samples (stable state) were negative in 10 of 14 cases (71.4%). This frequency was higher than that in the exacerbated/exacerbated group (17.1%; P=2.50×10-4). These data indicate that a subset of patients is susceptible to asthma exacerbation in the presence of viral LRTI. In addition to viruses, asthma exacerbation can be caused by other agents, including allergens (dust mites, pollen, and animal dander),27 occupational exposure (grains, flours, cleaning agents, metals, irritants, and woods), hormones (menstrual asthma), drugs (acetylsalicylic acid [ASA], nonsteroidal anti-inflammatory drugs [NSAIDs], and beta-blockers), exercise, stress, smoking exposure,28 and air pollutants.2930 The factors that trigger exacerbations differ among individuals. Thus, exacerbation of asthma may be a result of the complex interplay among respiratory viruses, host airway susceptibility factors, and environmental modifiers. A case-control study of 60 adult patients compared those hospitalized with acute asthma with 2 control groups: patients with stable asthma and patients hospitalized for non-asthma conditions.2 Compared with the controls, a significantly higher proportion of acute asthmatics were both sensitized and exposed to allergens, including dust mites, cat, and dog allergens. Intriguingly, the combination of high exposure to 1 or more allergens and virus detection significantly increased the risk of hospitalization for asthma compared with controls with stable asthma.27 These results indicate synergism among allergen sensitization, exposure to a high level of a sensitizing allergen, and viral infection in inducing asthma deterioration.3132 In our study, the frequency of virus detection was not different between atopics and non-atopics (data not shown). Although all of these factors are expected to predispose asthmatics to viral infections,33 determining whether the exacerbation is due to viral infection or other causes is not feasible at present.
The predominant respiratory virus depends on the season.34 In our study, the number of cases was highest in late fall and early winter. However, the virus detection rate was ~45% in February and March and <20% in May, July, and August. These data indicate that symptoms of LRTI with asthma exacerbation in early spring may be due mainly to viral infections, and that those in late spring to summer may be due mainly to other environmental factors. In addition, virus prevalence varied markedly: RV was predominant during fall and early winter, while IFV A was predominant during winter. IFV A is the predominant respiratory virus in winter in Korea.35 RSV was predominant during fall, winter and early spring, and PIV was predominant during late spring and early fall. The seasonal variation reported in the present study is in agreement with that previously reported.34
In summary, the presence of respiratory viruses was analyzed in 323 sputum samples from asthmatics with manifestations of LRTI to evaluate their contribution to asthma exacerbations. Virus was detected in approximately one-fifth of the subjects with exacerbated and stable disease. In both states, RV was the most frequently detected virus, followed by IFV A, which is comparable with those of Caucasian studies. Forty-nine patients underwent an examination for viruses during 2 episodes of exacerbation and at the time of each exacerbated and stable episode. The virus detection rate at the second examination was significantly higher in cases with 2 exacerbation episodes than in those with sequential exacerbation and stable episodes, suggesting a presence of susceptible asthmatics to exacerbation in case of LTRI. Seasonal variations in detection rates and types of virus show the similar patterns to those of Caucasian asthmatics.
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.
Respiratory infectious phenotypes are associated with LOS and severity of symptoms in AECOPD. Furthermore, respiratory viral infection plays an important role in exacerbations of COPD. CAT may be a predictor of longer LOS with coinfection.
Data of FEV1 is shown in Table 1. We found no statistically significant difference in FEV1 among different infectious phenotypes (P>0.05). Moreover, there was no statistically significant difference in the classification of patients according to the new GOLD staging system among the four groups.
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.
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.
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.
Bocavirus (BoV), a linear single stranded DNA (ssDNA) virus with an approximately 5.5 kb length genome belongs to the genus Bocaparvovirus in the family Parvoviridae. The BoV genome possesses three main open reading frames, ORF1-3, encoding the non-structural protein NS1, capsid protein VP1/2 and nuclear phosphoprotein NP1, respectively1. The BoVs have emerged and caused diseases in various animals and humans, including canine BoVs (CBoVs), porcine BoVs (PBoV), bovine parvovirus, gorilla BoV, California sea lion BoV, rodent BoV, feline BoVs (FBoVs) and human BoVs (HBoVs), and so suggests a potentially wide host range of BoVs1–3. Many researches have indicated the association of BoVs with various clinical symptoms. Respiratory and intestinal diseases have been observed frequently in BoV-infected hosts4, but other uncommon clinical presentations have also been reported. These include encephalomyelitis in a PBoV-infected pig, necrotizing encephalitis in HBoV-infected humans and hepatitis in CBoV-infected dogs5–7. Likewise, previous studies have reported the detection of the BoV genome in liver, lymph node, feces and blood of infected hosts, suggesting that BoVs could cause systemic infections2.
The FBoVs, comprised of FBoV-1 to −3 and belong to Carnivore bocaparvovirus 3 to 5, respectively, were recently discovered in domestic cats. Firstly, the novel FBoV-1 was detected in various samples, such as feces, blood, kidney and nasal swabs, collected from asymptomatic cats in Hong Kong1. Thereafter, FBoV-2 and −3 were discovered during high throughput metagenomic study of fecal viromes in healthy cats8,9. However, neither the pathological roles of these FBoVs associated with intestinal disease nor other systemic diseases have been established. To date, the emergence of FBoVs has been reported in Belgium, China, Japan, Portugal and USA8–13, where the FBoV genomes were detected in the feces of cats with and without clinical signs. Later, the FBoV-1 genome was detected in cats with severe enteritis12, but the relationship between FBoV-1 detection and clinical presentations with its pathogenesis in infected cats is still limited. Furthermore, a recent study revealed that the FBoV-1 genome was the most co-infected virus with other viral pathogens. For example, the FBoV-1 genome was detected in the brain of feline panleukopenia virus (FPLV) infected cat showing neurological signs10. So far, the reports have addressed the potential role of FBoV-1 infections, yet the role of FBoV-1 when co-infected with FPLV is still unknown.
It is known that mutation accumulation and genetic recombination can both contribute to genetic diversity and virus evolution. Recombination allows viruses to quickly change their properties and results in novel genetic variants. For BoVs, genetic recombination has been focused on as a potential mechanism for virus evolution. For example, the evidence suggests that HBoV-3 emerged as a result of genetic recombination between HBoV-1 and HBoV-214. Likewise, the HBoV-4 genome carried the admixture genome between HBoV-2 and HBoV-315–17. Furthermore, homologous genetic recombination among CBoV-2 strains was also evident2. These findings indicated that genetic recombination is likely to play an important role in the diversity of BoVs.
In this study, novel FBoV-1 strains were identified in 17 FPLV-infected cats from three different households with an acute onset of depression, systemic hemorrhage, and respiratory dysfunction as well as intestinal problems. In situ hybridization (ISH) on three of these cats that died (one from each household) revealed a systemic FBoV-1 viral DNA with the signals localized in various cells of intestinal tissues and endothelial cells at intestinal mucosa and serosa, as well as in various lymph nodes. Genetic analysis of the full-length coding genome of the obtained Thai FBoV-1 strains indicated three separate strains (one per cat) and evidence of natural genetic recombination. The clinical presentation, through the pathological findings of FBoV-1 infected cats, is described and the potential roles of co-infection in the affected cats are addressed.
Feline coronavirus (FCoV), a common pathogen in cats, is an enveloped, positive-sense, single-stranded RNA virus. Together with canine coronavirus (CCoV), transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus and human coronavirus 229E (HCoV 229E), FCoV is classified into the genus Alphacoronavirus. Two pathotypes of FCoV have been well demonstrated, i.e., feline enteric coronavirus (FECV) and feline infectious peritonitis (FIP) virus (FIPV). The former causes mild enteric infections, and the latter causes a fatal immune-mediated disease known as FIP.
Infection with coronavirus is determined by the interaction between the receptor binding domain of its spike (S) protein and corresponding receptors on target cells. S protein is the main determinant of cell tropism and of the induction of neutralizing antibodies. FCoVs can be divided into two serotypes, types I and II, which differ in the neutralizing antibody reaction and have distinct S protein sequences. These two types of FCoV differ in growth characteristics in vitro. Type II FCoV is antigenically related to CCoV and TGEV. The receptor usage of the two types of FCoV is different. Feline aminopeptidase N was identified as a receptor for type II FCoV, but not for type I FCoV. The main receptor for type I FCoV infection remains unclear. A feline dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin serves as a coreceptor for both type I and II FCoV. Both types of FCoV can infect domestic and wild Felidae and cause FIP. Seroprevalence studies for the detection of the two types of FCoV infection have been performed using various methods, and type I FCoV was found to be predominant in the field, with a seropositive rate of 83-98%, whereas the type II virus accounted for only less than 10% of infections-.
In this study, a type-specific partial S protein-based immunofluorescence assay (IFA) was established to distinguish between the two serotypes of FCoV. The seroprevalence of FCoV in Taiwan was determined, and the correlation between the genotypes and serotypes of FCoV infection was assessed.
Although human coronaviruses are characteristically causing self-limiting short diseases, the question of potential chronic SARS infections is of major importance for a future disease control. If the SARS-CoV is able to cause a chronic persistent infection, chronic carriers may serve as sources for new SARS outbreaks. However, the detection of SARS-CoV in stool of patients for longer periods than 6 weeks after hospital discharge has not been reported so far. Therefore, the danger of chronic carriers may not be relevant. In contrast to common human coronavirus infections with short durations, most animal coronaviruses cause persistent infections. As an example, the feline coronavirus FIPV infects animals which then continue to shed virus for periods reaching up to seven months after infection without carrying disease symptoms. Also, TGEV and MHV tend to cause chronic infections as these viruses may be found in the airways and small intestine (TGEV) or the nervous system (MHV) several months after infection. Although the SARS-CoV has jumped to humans it may still have this property of inducing chronic infections. Thus, SARS-CoV RNA was found in patients' stool specimen more than 30 days after the infections.
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.
There were totally 122 participants recruited from the end of September to the end of December 2003 and were followed up until the end of December 2004. Eight of these participants withdrew early as their parents found it inconvenient to attend unscheduled visit. One hundred and fourteen children aged 6 to 13 years completed the study. They were followed up for 12 to 15 months. Their baseline characteristics were tabulated in Table 1. Among these 114 children, 16 children (14.0 %) did not report any exacerbations or respiratory illnesses. Children with respiratory illnesses were younger than children without respiratory illnesses (p < 0.05) and there was greater proportion with normal pulmonary lung function test at the time of recruitment (p = 0.02). Fifteen children had reported 20 episodes of mild respiratory illness with symptoms with scores ≤3 that did not warrant unscheduled visits. The remaining 83 children had experienced ≥1 episode of respiratory illnesses with symptoms score >3 and the maximum number of episodes per children was seven in two children. There were a total of 211 episodes with a symptom score >3. Nasal swab specimens were obtained in 166 and the interval between onset of respiratory symptoms and nasal swab collection ranged from 0.5 to 6 days. Nasal swab specimens were not available in the remaining 45 episodes as the children attended general practitioner (GP) instead. There were 74 episodes of mild respiratory illnesses with symptom score ≤3 reported in these 83 children that were also managed by GP. The distribution of these episodes of respiratory illnesses among the children was illustrated in Fig. 1. Thus, there were a total of 305 episodes of respiratory illnesses including asthma and non-asthma related episodes in our study cohort over the 14-month study period. The mean number of asthma exacerbations, other respiratory illnesses, and all episodes as diagnosed at unscheduled visits were 0.69, 1.6, and 2.29 per person-year, respectively.
The presenting symptoms of 166 episodes of unscheduled visits with nasal swab specimens obtained are tabulated in Table 2. Ninety-two episodes were diagnosed as asthma exacerbations and 74 non-asthma related. Among 92 episodes of asthma exacerbations, physician also made a diagnosis of concomitant respiratory tract infection in 69 (59 with upper respiratory tract infection, 5 with lower respiratory tract infection, and 5 with sinusitis) of these episodes based on history and physical findings.
Respiratory viruses were detected in 61 of these 166 episodes (36.7 %) (Table 3). There was no significant difference in virus detection rate between asthma (32 out of 97 episodes, 34.8 %) and non-asthma related episodes (29 out of 74 episodes, 39.2 %). Rhinovirus was detected in 41 episodes, influenza in 7, coronavirus in 6, parainfluenza virus in 2, RSV in 1, and mixed viruses in the remaining 4. The patterns of distribution of respiratory viruses were quite similar in asthma exacerbations and non-asthma related episodes. (Table 4)
A seasonal pattern was noted in the rates of detection of respiratory virus. RV was prevalent in September to December, IFV in January to April, PIV in May to September, and RSV A/B in September to April (Fig. 2).
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.
Rapid progress has been made in understanding the clinical presentation of SARS in adults and children. In comparison to adults, SARS seems to be less aggressive in younger children, with no children in one case series requiring supplementary oxygen while in adults, systemic infection as well as respiratory infection may be the rule. SARS is much milder with non-specific cold-like symptoms in children younger than 12 years than it is in adolescents and adults. The reason for the milder clinical presentation of SARS in children is most likely due to differences in developmental stage of the immune system.
The course of the disease in teenagers more likely resembles adults in concerning clinical presentation and disease progression. SARS may also develop severe illness requiring intensive care and assisted ventilation in these adolescent patients. The common presenting features are fever, malaise, coryza, cough, chills or rigor, headache, myalgia, leucopaenia, thrombocytopaenia, lymphopaenia, elevated lactate dehydrogenase levels and mildly prolonged activated partial thromboplastin times. The radiographic findings are non-specific: However, high-resolution computed chest tomography in clinically suspected cases may prove to be an early diagnostic aid when initial chest radiographs appeared normal. While rapid diagnosis with the first-generation RT-PCR assay was not satisfactory, improved RT-PCR assays may help to diagnose SARS in early stages. In this respect, a sensitivity approaching 80% in the first 3 days of illness when performed on nasopharyngeal aspirates may be achieved. The best treatment strategy for SARS among children still has to be determined while no case fatality has been reported in children. In comparison to the prognosis in adults, there is a relatively good short- to medium-term outcome. However, it is crucial to emphasize that continued monitoring for long-term complications due to the disease or its treatment is of major importance.
Currently, no strong evidence exists for a causal link between cancer and IMHA in cats; further studies are needed to determine if such an association exists. Nevertheless, retrospective evidence suggests a relatively high prevalence of concurrent cancer in cats with IMHA.
The evidence for pancreatitis causing IMHA is negligible in dogs and negligible to low in cats. Additional studies would be required to establish a causal relationship.
The evidence for necrosis as a cause of IMHA is negligible in dogs and is not reported in cats.
Anecdotal reports suggest that generalized inflammatory processes induce IMHA in dogs and cats, but direct evidence is lacking. Well‐designed studies to determine whether non‐infectious inflammatory processes cause IMHA are warranted.
Seventeen studies described dogs with IMHA that had been exposed to drugs or toxins,1, 10, 12, 27, 34, 80, 116, 127, 135, 142, 143, 160, 168, 169, 170, 171, 172 but only 11 reported cases with sufficient primary data for the calculation of an IME value.10, 12, 27, 34, 80, 142, 143, 168, 169, 170, 172 The majority of cases (35/36) were dogs exposed to antimicrobial drugs.10, 80, 142, 143, 168, 170 For these cases, IME values ranged from 1.70 to 7.09, with a median of 1.87 (Figure 9). The highest level of evidence, with an IME value of 7.09, came from 1 unblinded, randomized, prospective clinical trial in which 6 of 14 dogs given escalating doses of cefazedone acquired anti‐erythrocyte antibodies.168 The remaining reported cases were associated with low or negligible evidence to support other drugs or toxins as a cause for IMHA in dogs (Figure 9).
All respiratory samples positive for coronavirus by real-time RT-PCR were screened for other respiratory viruses. Among the real-time RT-PCR CoV positive samples, 13% were also positive for Respiratory Syncytial Virus, Parainfluenza 2 and 3, and Rhinovirus (Data not shown). Among the patients harboring respiratory CoVs, detected by qRT-PCR and confirmed by RT-PCR, six (17%) were co-infected with a second respiratory virus.
Upper respiratory tract infections (URTIs) are disorders caused by an acute infection, commonly involving the nose, sinuses, pharynx or larynx. The various URTIs include the common cold, sinusitis, laryngitis and pharyngitis (1). URTIs are usually caused by viruses, including rhinovirus, corona-virus, Para influenza virus and adenovirus (2). Certain URTIs also result from bacteria, including Streptococcus pyogenes, Streptococcus pneumoneae, Heamophilus influenzae, Corynebacterium diptheriae, Bordetela pertursis and Bacillus anthracis (3). Bronchitis, which is inflammation of the mucosal membranes of the bronchi, is also caused by certain types of bacteria, with ~10% of cases caused by bacteria, including Mycoplasma pneumoniae, Chlamydophila pneumoniae, Bordetella perturis and S. pneumoniae (4,5). S. pyogenes is a spherical, Gram-positive bacterium, and causes > 700,000,000 infections worldwide each year and >650,000 cases of severe, invasive infections with mortality rates of 25%. S. pyogenes the cause of several important human diseases, ranging from mild skin infections to potentially fatal systemic disorders. Infections typically begin in the throat or skin, with pharyngitis being common in S. pyogenes infection. Infections caused by certain strains of S. pyogenes are associated with the release of bacterial toxins, which in certain throat infections results in scarlet fever (6). Certain strains of S. pyogenes have now developed resistance to macrolides, tetracyclines and clindamycin, indicating the requirement for novel antibacterial agents (7–10).
Essential oils, which are composed of an odoriferous mixture of monoterpenes, sesquiterpenes and aromatic compounds and are used in naturopathic therapy, are well known for their antimicrobial properties. Essential oils were among the first topical and gastrointestinal antimicrobial agents used by humans. Due to the extensive use of conventional antibiotics and synthetic antimicrobial drugs, there has been an increase in the widespread development of drug resistant microorganisms, including methicillin-resistant Staphylococcus aureus (MRSA) and multidrug resistant strains of Klebsiella pneumonia and P. aeruginosa (11,12). Essential oils and their components target bacterial cell walls and cytoplasmic membranes, resulting in permeabilization, which is followed by the loss of ions, reduction of the membrane potential, collapse of the proton pump and depletion of the adenosine triphosphate pool (13–15). Due to their multifunctionality, essential oils have wide applications in medicine and aromatherapy. Essential oils exhibit potent antimicrobial actions against a wide range of Gram-positive and Gram-negative bacteria (16). Essential oils have also traditionally been used for treating respiratory tract infections, and as an ethnic treatment for colds (17–19). Inhalation therapy using essential oils has been used to treat acute sinusitis and acute and chronic bronchitis and it has been reported that inhalation therapy using volatile essential oil vapors enhances the respiratory tract fluid output (20 maintains ventilation and drainage of the sinuses, reduces asthma, and reduces inflammation of the trachea (21–23).
Artemisia vestita Wall, a perennial member of the Artemisia genus in the family Asteraceae is distributed on wasteland and river banks in China and has been widely used in traditional Tibetan and Chinese traditional medicine to treat various inflammatory diseases (24). Several studies have investigated the chemical constituents of A. vestita, and a number of flavones, monoterpenes and sesquiterpenoids have been isolated. The chemical composition of the essential oil of A. vestita has also been investigated (24–26). At present, to the best of our knowledge, the in vivo and in vitro antibacterial activity of A. vestita against respiratory infection-causing bacteria has not been reported previously, and the detailed mechanism of action of A. vestita essential oil on bacteria remains to be fully elucidated. Therefore, the present study is the first on the A. vestita essential oil, in which in vitro and in vivo antibacterial activities against the respiratory infection-causing bacteria, S. pyogenes (ATCC-12344), MRSA, S. pneumoniae (ATCC-2730), K. pneumoniae (ATCC-27853) and H. influenzae (ATCC-33391) are investigated. The effects of the oil on biofilm formation and its architecture, and its effect on leakage of potassium ions (K+) from S. pyogenes was also investigated. Furthermore, the effect of the essential oil and its major component, grandisol, on hepatotoxic- and nephrotoxic-associated biochemical parameters in a S. pyogenes-infected mice model were evaluated.