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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.
The prominent gross findings of all necropsied cats revealed severe hemorrhage in various organs, including the brain, lung, liver, intestinal tract and lymph nodes. Their morphological diagnoses were severe acute diffuse pulmonary hemorrhage (Fig. 2a), segmental hemorrhagic enteritis with massive hemorrhagic lymphadenopathy (Fig. 2b,c), petechial hemorrhagic encephalitis (Fig. 2d), and necrotizing hemorrhagic hepatitis. The degree of severity of systemic hemorrhage varied among the cats. With respect to the histopathology, representative sections of the brain, lung, heart, tongue, stomach, small and large intestine, liver, spleen, and lymph nodes from three cats (no.1–3), designated 18R217C, 19R81C and 19R124C, respectively, were examined.
The morphological findings were broadly similar among all three examined moribund cats. Changes in the small intestine were severe and included extensive villous blunting and fusion along with crypt necrosis. Large, amphophilic, intranuclear inclusion bodies were observed in rare crypt epithelial cells (Fig. 2f). In the ileum, spleen, and lymph nodes, the numbers of lymphoid follicles were markedly depleted with variable accumulations of fibrin, karyorrhectic debris, and aggregates of histiocytes within the center of the remaining lymphoid follicles (Fig. 2h). In cat nos. 2 (19R81C) and 3 (19R124C), the lungs were markedly congested with multifocal to diffuse histio-lymphocytic interstitial pneumonia, and the perivascular spaces of many pulmonary vessels were markedly edematous (Fig. 2e). In cat no. 1 (18R217C), the lung was edematous, and had patchy hemorrhages with increased numbers of circulating neutrophils throughout the alveolar capillaries. In cat no. 3, the liver had random foci of hepatic necrosis with multifocal areas of lobular collapse. In addition, biliary hyperplasia was noted. In cat nos. 1 and 2, the microscopic findings of the liver were within normal histologic limits. For the heart, there were small hemorrhagic foci observed in cat no. 1.
Interestingly, cat no. 1 had vascular changes in the cerebrum and cerebellum that were characterized by engorged blood vessels containing variable numbers of PAS- and Alcian blue-negative, eosinophilic, homogenous globular material and an expansion of the perivascular Robin-Virchow space (Fig. 2i) with open spaces containing variable amounts of eosinophilic flocculent material intermixed with rare, small, proteinaceous droplets (Fig. 2i, inset). The affected blood vessels were lined by plump reactive or occasionally pyknotic endothelial cells.
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.
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.
Acute respiratory infections (ARIs) are a major public health problem causing approximately 1.9 million child deaths in 2000. About 20% of child mortality (<5 years) is due to pneumonia, bronchitis or bronchiolitis and 90% of them are attributed to pneumonia. Community acquired pneumonia (CAP) is a major cause of morbidity and hospitalization in developed countries and a major cause of mortality among children living in developing countries where socio-economic issues such as malnutrition are aggravating factors. To date, it is difficult to reliably predict the pathogen based on clinical signs and symptoms. Respiratory pathogens etiologies could help to better understand acute febrile illnesses in malaria endemic area and orient adapted therapies. Viruses were considered as causative agents of acute lower respiratory infections (ALRIs) and have been investigated in several studies. Since 2001, several new respiratory viruses have been described such as metapneumovirus (HMPV), human coronavirus (HCoV), NL63 and HKU1 and human bocavirus (HBoV). Various respiratory viruses that caused epidemics and pandemic, such as swine lineage influenza ALRIs A (H1N1) virus infection, in 2009, have heightened the need to develop sensitive and rapid diagnostic test. The development of molecular methods such as multiplex Real-Time PCR (RT-PCR) greatly facilitates the etiological study of respiratory infections but it does not, especially in developing countries, assist the clinician in the care of patient. Madagascar is a country with low HIV prevalence (estimated at 0.2% in 2010) and malaria is endemic with stable transmission during all year on the East Coast. Recent studies on surveillance of fever among child result in malaria over-diagnosis with consequent under diagnosis of other fever-causing disorders such as pneumonia. Little is known about the pathogens responsible for ARIs especially in rural areas. A recent study shows that respiratory viruses play an important role in children under 5 years old consulting in public and private clinics in Antananarivo with Influenza-Like Illnesses (ILIs) symptoms. In Ampasimanjeva, a small village located in a rural area endemic for malaria, a recent study showed that 68% of acute fever illnesses among children are not explained by malaria (Ratsimbasoa, personal communication). The objective of this study is to determine the prevalence and seasonal distribution of a large panel of respiratory pathogens including viruses and atypical bacteria among a well clinically defined cohort of acute febrile children between 2 to 59 months of age presenting clinical ARIs symptoms in Ampasimanjeva.
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.
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.
The mean incubation period of SARS was estimated to be 6.4 days (95% confidence interval, 5.2 to 7.7). The mean reported time from the onset of clinical symptoms to the hospital admission varied between three and five days.
Main clinical features of the disease are in the initial period common symptoms such as persistent fever, myalgia, chills, dry cough, dizziness, and headache. Further, although less common symptoms are sore throat, sputum production, coryza, vomiting or nausea, and diarrhea. Special attention has been paid to the symptom of diarrhea: Watery diarrhea has also been reported in a subgroup of patients one week after the initial symptoms.
The clinical course of the disease seems to follow a bi- or triphasic pattern. In the first phase viral replication and an increasing viral load, fever, myalgia, and other systemic symptoms can be found. These symptoms generally improve after a few days. In the second phase representing an immunopathologic imbalance, major clinical findings are oxygen desaturation, a recurrence of fever, and clinical and radiological progression of acute pneumonia. This second phase is concomitant with a fall in the viral load. The majority of patients is known to respond in the second phase to treatment. However, about 20% of patients may progress to the third and critical phase. This phase is characterized by the development of an acute respiratory distress syndrome (ARDS) commonly necessitating mechanical ventilation.
Viral respiratory tract infection, with rhinovirus accounted for two thirds, was found to be associated with greater than 80% of asthma exacerbations in children in studies in the 1990s. However, the prevalence of respiratory viral infection varies greatly across different places; for example, influenza is associated with more hospitalization among children in Hong Kong compared with temperate region. Recent observational studies have shown that influenza infection can be associated with asthma exacerbations. Nevertheless, a meta-analysis failed to support the protective effect of influenza vaccination in asthma exacerbations. Newly discovered respiratory viruses such as human metapneumovirus may also play a role. Triggers other than respiratory tract infection, like air pollutions may become more prevalent over time and supersede respiratory tract infections as a major trigger. Thus, we carried out a prospective study to delineate the current role of different viral respiratory tract infections including newly discovered respiratory viruses in asthma exacerbation in children in our locality.
Children aged 6–14 years who attended regular follow-up at the asthma clinic were invited to participate. Children with physician-diagnosed asthma, symptoms of asthma in the preceding year, no hospital admission for exacerbation, and on regular inhaled steroid equivalent to beclomethasone ≤400 μg daily for at least 3 months prior to enrolment were recruited. Exclusion criteria were those with other known chronic respiratory disease and oral steroid therapy given within 4 weeks of enrolment. The participants were followed up to cover a full calendar year to reduce potential biases associated with temporal and age-related differences in respiratory tract infections.
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.
Prior to the development of therapeutic regimes based on molecular mechanisms of the disease, the causative agent had to be isolated and analysed. Soon after the fast establishment of the international WHO laboratory network, rapid progress was made in the identification process of the causative agent, and it was reported that SARS is most probably caused by a novel strain of the family of coronaviruses. These viruses are commonly known to cause respiratory and gastrointestinal diseases of humans and domestic animals. The group of coronaviruses is classified as a member of the order nidovirales, which represents a group of enveloped positive-sense RNA viruses consisting of coronaviridae and arteriviridae. Viruses of this group are known to synthesize a 3' co-terminal set of subgenomic mRNAs in the infected cells.
More than half of the patients (132/246, 53.6%) were less than one year of age, and almost one half of the remaining children, whose age was recorded (40/83, 48.1%), were under two years of age (Table 1). Respiratory tract viruses were detected in 189 (77%) of the 246 children. The overall rate of virus positivity was similar in children less than 1 year of age (102/132, 77.3%), between 1 and 2 years (32/40, 80.0%), and above 2 years of age (33/43, 76.7%) and slightly lower among the 13% of children whose age was not recorded (22/31, 71.0%). The most frequently detected virus groups, both found in about one third of the children each, were HRV and PIV, with type 3 of PIV being the most prevalent serotype in the latter group (Table 1). Adenoviruses, influenza virus C, hMPV, and HBoV were also found in considerable numbers. Influenza virus types A and B and RSV were detected less frequently. Seven specimens tested positive for HEV and negative for HRV (“true HEV”), whereas 16 other additional specimens yielded a positive result by the HEV and the HRV test. While the overall proportion of virus-positive specimens was similar in children aged under or over two years, as well as in the group with unrecorded age, all adenovirus, influenza virus A, RSV, and all but one HBoV and “true HEV” detections were in the youngest age group (Table 1).
Altogether 224 virus findings were obtained from the 246 children, if only those HEV-positive results were accounted where the same specimen was negative in the HRV test (“true HEV”). The number of findings was 240 if all positive test results for HEV were included. Twenty-nine specimens contained two different viruses and another two specimens three different viruses. No obvious pattern could be seen in the mutual associations of two or three viruses (Table 2). However, there were more multiple infections in children older than 2 years than in the younger than 2 years group (33% versus 9% of children, P = 0.0001 (2-sample t-test for equality of proportions), data not shown.
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.
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.
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 natural reservoir of influenza A virus (infAv) is considered to be aquatic birds because all known subtypes (H1-H16 and N1-N9) of infAv have been isolated from waterfowl. InfAv can, however, infect many other mammalian species, including humans, swine, horses, ferrets, and sea mammals. There are several specific swine influenza A virus (SIV) subtypes (H1N1, H1N2 and H3N2) circulating in the pig populations in Europe, including Denmark. The Danish SIV subtype H1N2 differs from the European SIV H1N2 subtypes, in that it is a re-assortment between two circulating Danish SIV strains of the subtypes H1N1 and H3N2. The first known Danish H1N2 isolate occurred in 2003 and is therefore a relatively new strain in Denmark.
It has been described that pigs have receptors for both human and avian strains of influenza A viruses in the upper respiratory tract and therefore are susceptible to infections by both. Based on this finding, it has been proposed that pigs can act as a mixing vessel when infected by both a human and avian influenza A virus (AIV) strain to make a new reassorted virus with zoonotic and even pandemic potential. In recent years, however, there have been examples of infAv crossing the species barrier without the involvement of pigs. Infections with infAv are initiated by interactions between virus haemagglutinin and sialic acid (SA) molecules on target cells. AIV strains prefer SA-α-2,3-terminal saccharides whereas human and swine influenza virus strains prefer SA-α-2,6-terminal saccharides as receptors. A few studies have shown that the epithelial cells of the upper respiratory tract of pigs express both receptors. However, recent studies have shown a more variable distribution of the specific receptors in the deeper lung areas whereas in the trachea the SA-α-2,6-terminal saccharides are abundant. It has been described that after infections with AIV in pigs and humans the virus has shifted receptor specificity from SA-α-2,3 to SA-α-2,6 as a part of the adaptation to the new host by the virus. This shift in receptor specificity has been linked to specific amino acid substitutions in the HA molecule, but the exact determinants of the host specificity of infAv have not been fully elucidated.
Specific lectins have been the chosen method for detecting SA receptors. The Sambucus Nigra (SNA) lectin is specific for SA-α-2,6 bindings and the Maackia Amurensis (MAA) lectin is specific for SA-α-2,3 bindings of the SA molecules. In order to detect SA-α-2,3-terminal saccharides it is necessary to use two isoforms of MAA lectin: MAAI and II because the two isoforms are different in the way they recognise the inner sugar structures of SA-α-2,3. A more thorough investigation of the receptor distribution in the respiratory tract of pigs would give a more nuanced picture of the infection dynamic of different infAv in pigs. This, together with investigation of the predilection site of different infAv in the respiratory tract tissue, would enable us to improve our understanding of the mechanisms of infection regarding pathogenesis and host range determination.
The aim of the study was to investigate the tissue and cell predilection sites of avian and swine influenza A viruses, respectively, and SA-α-2,3/2,6-terminal saccharide receptor distribution in the respiratory tract of pigs by the use of immunohistochemical methods and lectin staining.
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.
6.What are the most common clinical findings of FeL due to L. infantum?
Detailed case reports of FeL have been available in recent years mainly from European countries where pet cats typically have a higher standard of health care. In the New World, other Leishmania spp. are endemic and may co-infect cats and complicate the clinical picture. Therefore, we have only reviewed case reports or case series originally from European countries. A total of 46 clinical cases have been published between 1989 and 2014, where the diagnosis of FeL was confirmed by serological and/or parasitological methods [11–14, 21, 26, 36, 37, 50–67].
The most common clinical signs reported in FeL include skin or mucocutaneous lesions and lymph node enlargement, and they have been described in more than half of the cases (Table 4). Some cats showed only dermatological lesions alone [13, 52, 56, 58], while others with skin lesions showed a combination with systemic signs [12, 14, 21, 26, 36, 51, 60, 62–64, 68]. Conversely, other cats did not have any skin detectable lesions on clinical presentation [11, 36, 50, 54, 55, 57, 66, 69, 70].
The cutaneous and mucocutaneous lesions are described in Question 7. Lymphadenomegaly may be solitary or multicentric. Ocular lesions have been reported in approximately one third of the affected cats. Uveitis, either unilateral or bilateral (Fig. 1), is the most common ocular lesion described, with occasionally a pseudotumoral granulomatous pattern and eventually progress to panophthalmitis [50, 53, 55, 64, 69]. Blepharitis and conjunctivitis have also been described in a number of clinical cases [66, 68, 70]. Amastigotes have been found by cytology in conjunctival nodules, corneal infiltrates and aqueous humor, and by histopathology after enucleation of the eye or post mortem even in uveal tissue [50, 53, 55, 64, 69]. Chronic gingivostomatitis is also a common clinical finding and has been found in about one fourth of the cats so far studied with leishmaniosis (Fig. 2) [11, 26, 53, 55, 63, 66, 70]. Nodular lesions are unfrequently seen on the gingival mucosa or the tongue [60, 66, 69, 71], where infected macrophages may be visualized in lesion biopses [60, 69].
Non specific signs such as weight loss, reduced appetite, dehydration, and lethargy also have been reported. A list of other sporadic clinical manifestations described includes: pale mucous membranes, hepatomegaly, jaundice, cachexia, fever, vomiting, diarrhea, chronic nasal discharge, splenomegaly, polyuria/polydipsia, dyspnea, wheezing, abortion and hypothermia.
The implication of Leishmania as a cause of some of these clinical signs has been associated with the presence of the parasite in cytological or histopathological examinations of liver, spleen, lymph nodes, stomach, large bowel, kidney, oral mucosa, nasal exudate and eye tissues [13, 14, 36, 50, 57, 63, 66, 68, 72]. However, clinical disease is commonly associated with an impaired immunocompetence due to several causes including retroviral infections (FIV and FeLV), immunosuppressive treatment and concomitant debilitating diseases such as malignant neoplasia or diabetes mellitus.
As also found in dogs, FeL does not exclude the possibility of concurrent diseases or co-infections. This fact may influence the clinical presentation and prognosis. The cause-effect relationship between various etiological and pathogenic factors is not always easy to establish.7.What are the most common dermatological findings of FeL due to L. infantum and to other Leishmania species?
Cutaneous lesions predominate in the clinical picture of FeL due to L. infantum. Dermal abnormalities include nodules, ulcerations or more rarely exfoliative dermatitis. They are generalized or localized, symmetrical or asymmetric and may, though less frequently, appear all over the body in a focal, multifocal, regional or diffuse pattern [12–14, 26, 36, 37, 51, 52, 56, 58, 60, 62, 64, 68, 70]. Some cats may harbour different types of skin lesions at the same time or develop them subsequently; they may coexist with mucocutaneous lesions (Fig. 3). Cutaneous and mucocutaneous nodules, of variable size, are more often localized on the head, including eyelids, nose and lips, or on the distal parts of the limbs. Nodules have also been reported in the anal mucosa and they are usually small (less than 1 cm), non painful or pruritic and have a normal, ulcerated or alopecic surface [26, 50, 51, 56, 60, 62–64, 66, 68, 70].
Ulcerations which may be diffuse and superficial or focal and deep (Fig. 4) are localized on the same body sites as nodules, and may be complicated by bacterial infections that explain why they are covered by hemorrhagic crusts and/or purulent material [13, 14, 52, 53, 56, 58, 60–62, 64, 65, 68, 70]. However, ulcerative dermatitis is sometimes diffuse and can be observed on the body trunk or on bony prominences [14, 36, 58, 62, 63].
In contrast to CanL, exfoliative dermatitis (Fig. 5) is rare in the feline disease [36, 52, 68]. Other uncommon dermatologic presentations include hemorrhagic papules and nodules where Leishmania amastigotes can be found [37, 52]. Alopecia (Fig. 6), which is also uncommon in FeL [12, 36, 52, 62, 64], may be associated with other skin diseases concurring in L. infantum infected cats such as demodicosis. Mild to severe pruritus is rare in FeL [58, 64, 65] and in some cases with a pruritic syndrome other compatible causes co-existed such as flea allergy, pemphigus foliaceus (PF) or neoplasia (squamous cell carcinoma).
Clinical disease caused by natural infection with species other than L. infantum is typically reported as nodular or ulcerative dermatitis with no systemic clinical signs. Skin lesions are often single but they can metastatize (Table 5) [73–76].8.What are the most common dermatopathological features of FeL?
Skin histopathology of lesions associated with L. infantum has shown that the most commonly observed alteration is a granulomatous dermatitis [26, 51, 56, 59, 60, 68]. It often has a diffuse pattern and the epidermis may present hyperkeratosis, acanthosis and ulceration [56, 68]. A nodular to diffuse arrangement of the granulomatous dermatitis is also reported [26, 60]. However, in a retrospective case series from Spain, two cats presented different histological findings. The first one had granulomatous perifolliculitis with a high number of lymphocytes and plasma cells surrounding the cutaneous adnexa. It was associated with a marked hyperplasia of epidermis and sebaceous glands. The other cat was diagnosed with a lichenoid interface dermatitis typically represented by infiltration of lymphocytes, plasma cells and a few neutrophils and macrophages at the dermoepidermal junction. In this case, epidermal necrosis and epidermal microabscesses were also observed. A perivascular infiltration of superficial skin layers by macrophages, mast cells, neutrophils and eosinophils was also observed in another case.
Leishmania amastigotes have always been identified in the affected skin. A semiquantitative estimation of amastigotes was also performed with the aid of immunohistochemistry (IHC), in which the parasitic load of the skin ranged from high (>50 immunolabelled amastigotes/field at x400) to moderate (10–50 immunolabelled amastigotes/field) in cases of diffuse granulomatous dermatitis. Conversely, it was low (1–9 immunolabelled amastigotes/field) in cases of granulomatous perifolliculitis or lichenoid interface dermatitis .
In biopsy samples taken from cases with ulcerative dermatitis, eosinophilic granulomatous dermatitis with a severe dermo-epidermal necrosis were found without the presence of amastigotes, but with a positive quantitative Leishmania PCR.
In some FeL cases, other dermatological diseases such as eosinophilic granuloma and PF were also diagnosed [52, 56, 68].
Interestingly, amastigotes were also found associated with neoplastic tissue in the lesion of two cats with squamous cell carcinoma (SCC). In one other case, SCC was diagnosed in a cat presenting concurrent Leishmania skin lesions [14, 59].
In two cases of skin disease caused by L. braziliensis, a mononuclear and neutrophilic inflammatory infiltrate of the dermal tissue was seen in histological sections.9.What are the most common differential diagnoses in L. infantum endemic areas for dermatological features?
The commonly seen cutaneous nodular form in FeL cases should be distinguished from nodules caused in cats with cryptococcosis, sporotrichosis, histoplasmosis, sterile or eosinophilic granuloma, mycobacterioses, and a wide variety of cutaneous neoplasms (e.g. feline sarcoids, mast cell tumor, fibrosarcoma, basal cell carcinoma, bowenoid in situ carcinoma and lymphoma). The main differentials of the ulcerative lesions include squamous cell carcinoma with which however it may co-exist [13, 14, 59], idiopathic ulcerative dermatitis, indolent ulcer, mosquito-bite dermatitis, atypical mycobacteriosis and feline leprosy, cutaneous vasculitis, erythema multiforme and cold-agglutinin disease. Finally, skin diseases such as dermatophytosis, systemic or cutaneous lupus erythematosus, exfoliative dermatitis due to thymoma or due to immune-mediated pathomecanisms, PF, sebaceous adenitis/mural folliculitis complex and paraneoplastic alopecia could be included in the differential list of those leishmanial cats that are admitted with the rare exfoliative/crusting dermatitis which may also be alopecic and erythematous. It has been postulated that PF and FeL may share a common pathomechanism (molecular mimicry) when they co-exist in the same cat.10.What clinicopathological findings may alert the clinician to the possibility of FeL due to L. infantum?
Limited information is available about clinicopathological abnormalities in cats and it is only based on case reports. Mild to severe normocytic normochromic non-regenerative anemia is the most frequent haematological abnormality reported in clinical cases. Moderate to severe pancytopenia may be observed [37, 50, 57] in association with aplastic bone marrow, but some of the cats reported with pancytopenia were FIV positive [37, 50, 57]. Curiously, in one of these cases, amastigotes were found in 4 % of neutrophils in buffy coat smears.
Hyperproteinemia with hypergammaglobulinemia is a common finding in FeL as also found in dogs, and hypoalbuminemia is occasionally reported [37, 50].
Renal proteinuria and increased serum creatinine are also reported at diagnosis or during follow-up in some cases [37, 68].
Relative lymphocytosis and an increase in serum ALT activity were significantly associated with seroreactivity to L. infantum.
The type of inflammatory infiltrate found in tissue cytology (aspirates, impression smears) or histopathology in organs such as skin, eye, oral mucosa, liver, spleen and kidney is commonly pyogranulomatous to granulomatous [66, 68, 72]. There was also lymphoid reactive hyperplasia in lymphoid organs such as lymph nodes and spleen, with variable numbers of Leishmania amastigotes observed (Fig. 7).11.What are the most common differential diagnoses in endemic areas for systemic illness caused by L. infantum in cats?
As lymph node enlargement is the most common sign, apart from skin and mucocutaneous lesions, FeL should be included in the differential list when this finding is noted on physical examination as solitary or generalized lymphadenomegaly. This list mainly includes infections with other infectious agents (FIV, FeLV, FCoV, Bartonella, Mycobacteria, T. gondii, Cryptococcus or other systemic mycoses), lymphoma or metastatic involvement from other neoplasia.
FeL should also be considered in cats with ophthalmologic disease, mainly in cats with acute, recurring or chronic uveitis and differentiated from similar clinical conditions caused by FIV, FeLV, FCoV, Bartonella, T. gondii, fungal infections, neoplasia or paraneoplastic syndrome. Some feline uveitis cases are considered idiopatic and treated with corticosteroids. A diagnosis of idiopatic uveitis was initially made in some cases of ocular FeL and corticosteroids worsened the disease [50, 55, 69]. This fact warrants a careful investigation to exclude FeL before treating ocular disease with corticosteroids.
Proliferative and ulcerative chronic inflammation of the oral mucosa associated with FeL can be included in the list of possible causes of the feline chronic gingivostomatitis syndrome (FCGS). This painful and common immune-mediated disease is considered multifactorial in cats and treated by full mouth teeth extraction for eliminating oral plaque antigenic stimulation. Corticosteroids are frequently used to improve the clinical signs; however, when this was tried in some cats with oral disease associated with L. infantum infection it induced worsening of FeL [11, 66].
Hyperglobulinemia with increased gammaglobulin level reported in FeL is usually found in chronic infections caused by viruses, bacteria or systemic fungi, or inflammation associated with FCGS or inflammatory bowel disease, or in neoplasia such as lymphoma, or multiple myeloma.
Asthma is a highly prevalent, chronic respiratory condition characterized by reversible airflow obstruction, airway hyper-responsiveness and airway inflammation producing frequent exacerbations. There are 300 million people worldwide affected by asthma.1 The public health burden of asthma has increased over the past 2 decades, and acute exacerbation of asthma is a particularly important and costly problem, because morbidity and mortality due to asthma are closely related to the frequency and severity of the exacerbations.2 Identification of causal factors is vital for prevention and management of exacerbations. In Western countries, viral infections are responsible for up to 80%-85% of exacerbations in childhood asthma.3456 In contrast, viral infections are involved in <50% of asthma exacerbations among adult asthmatics.789101112
Among various respiratory tract viruses, including rhinovirus (RV), influenza virus (IFV), adenovirus (ADV), human metapneumovirus (hMPV), parainfluenza virus (PIV), coronavirus, and respiratory syncytial virus (RSV),13 RV, and IFV trigger exacerbations in children with asthma most frequently.14 In Korea, the prevalence of respiratory viruses is reported to be similar to that in Western countries. Of the respiratory viruses that cause asthma exacerbations, up to 60%–70% are RV, while IFV and RSV are responsible for a substantial proportion of exacerbations in children with asthma.1516 However, there have been few reports on the prevalence of viruses related to asthma exacerbations in Korean adult asthmatics.
This prompted us to evaluate the prevalence of respiratory viruses in the sputum of asthmatics with lower respiratory tract illnesses (LRTIs) and to compare the frequencies and types of viruses detected in patients with exacerbations (exacerbated LRTIs) with those in subjects without exacerbations (stable LRTIs) to evaluate the contribution of respiratory viruses to asthma exacerbation.
Viral bronchiolitis is frequent and has an important impact on the children’s health care due to the high rates of hospitalization and mortality, especially of young infants [1, 2]. Human respiratory syncytial virus (HRSV) is the predominant etiological agent, but infections by other respiratory viruses, such as human rhinovirus (HRV), metapneumovirus, parainfluenza, influenza, adenovirus, and coronavirus, also occur. These infections with different respiratory virus present similar clinical characteristics, so etiological diagnosis can be carried out in clinical practice only by virus identification, either by molecular tests, immunofluorescence or culture methods [1–5]. Although the current guidelines do not indicate routine tests to identify the etiologic agent in infants with bronchiolitis, the etiological diagnosis may contribute to the prevention of nosocomial acquisition, since the transmission mechanisms diverge among respiratory viruses. Knowledge on molecular epidemiology also contributes to programming and organizing prophylactic strategies, such as the use of monoclonal antibodies to HRSV and influenza vaccination [3, 6]. Etiological diagnosis may also contribute for developing specific therapeutic approaches for each agent.
Clinical and epidemiological evidences indicated that pathogenic pathways are different in HRSV and HRV infections. HRSV is the main agent of bronchiolitis, responsible for high rates of hospitalization and it is a major cause of mortality, especially in premature infants and those with risk factors. Despite this fact, the therapeutic approach consists mainly in supportive measures. HRV is the most common agent of cold and triggering asthma attacks in atopic individuals, however around 35% of asymptomatic subjects have positive results for HRV tests. Serious infections by both agents in early life are associated with recurrent wheezing in the following years, but this association is stronger with HRV. While HRSV infection leads to structural and functional changes in the airways, HRV infections do not cause as many changes and are more related to atopy and asthma. Clinical studies suggest that the use of corticosteroids during the acute phase of infection with high levels of HRV may reduce the risk of recurrent wheezing in the subsequent year [10, 11].
Differential patient responses to respiratory viruses lead to different clinical outcomes and, interestingly, it has been found that infections with different respiratory viruses (HRSV, HRV, Influenza A), as well as with different genotypes of the same virus (HRSV), present distinctive PBMC transcriptome signatures. Furthermore, PBMC transcriptome profiles can be used to assess disease severity in infants with HRSV and to predict individualized responses to HRV. Thus, besides contributing to clarify the etiology, genomic methods can bring important information on the pathogenic role of the different respiratory virus as single agents, or in codetection, and in symptomatic and asymptomatic patients, which is especially important in HRV infections.
In this study, we conducted a comparative global gene expression analysis of PBMC obtained from patients with acute viral bronchiolitis infected by HRSV (HRSV group) or by HRV (HRV group). We employed a weighted gene co-expression network analysis (WGCNA) which allows the identification of transcriptional modules and their correlation with HRSV or HRV groups. This approach permitted the identification of distinct transcription modules for the HRSV and HRV groups. Moreover, differentially expressed genes in the PBMC expression profiles presented significant high fold-changes between HRSV and HRV groups and could be potential etiological markers.
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.
Since the first discovery of HCoV-EMC as the etiological agent of this novel severe respiratory disease (1, 2), researchers have used various strategies to understand virus entry and its replication capability in mammalian cell lines (3, 4). In the absence of autopsy reports of patients who died from HCoV-EMC, our understanding of virus tropism and pathogenesis in humans is inadequate. Kindler et al. (5) describe the efficient infection and replication of HCoV-EMC in differentiated human primary airway epithelial (HAE) cell cultures in vitro. This model was used to culture other human coronaviruses such as HCoV-OC43 and HCoV-NL63 (6). This well-characterized primary culture system exhibits high physiological relevance, reflecting the anatomy of the human conducting airway (7), and allows better understanding of the cellular tropism of this newly emerged
Kindler et al. report that HCoV-EMC has faster replication kinetics than SARS-CoV in HAE cells (5). SARS-CoV has a mean incubation period of about 5 days in humans. This relatively “long” incubation period allowed the identification and isolation of SARS patients before they became highly infectious during the SARS epidemic. If HCoV-EMC has a very efficient replication rate in the upper human respiratory tract, the potential use of quarantine and isolation policy to contain a possible epidemic would be extremely limited.
In terms of cellular tropism, Kindler et al. are the first to show the cellular preference of HCoV-EMC for human non-ciliated bronchial epithelial cells. So far, susceptibilities of the human pseudostratified bronchial epithelial culture to coronaviruses causing the common cold (e.g., HCoV-229E, HCoV-OC43, HCoV-NL63) (6) and to SARS-CoV are comparable to that observed after HCoV-EMC infection. Interestingly, these human coronaviruses seem to have different cell specificities; non-ciliated cells are infected by HCoV-229E and HCoV-EMC, while ciliated cells are more prone to infections by HCoV-OC43, HCoV-NL63, and SARS-CoV. Nevertheless, this finding alone is not sufficient to explain their differential pathogenicities in humans. As acute pneumonia is one of the key presentations in HCoV-EMC infection in humans, the characterization of HCoV-EMC in tissues from lower respiratory tracts such as alveolar epithelial cells or alveolar macrophages might provide a more comprehensive picture of this novel disease.
The unusual severity of HCoV-EMC in humans, combined with its high fatality rate, is reminiscent of the clinical presentation of the SARS outbreak in 2003. SARS-CoV has strategies to limit host antiviral mechanisms by evading interferon (IFN) responses (8). On the other hand, evidence from clinical and experimental studies suggests that SARS-CoV can induce cytokine dysregulation. Kindler et al. (5) delineate the immune response triggered by HCoV-EMC in HAE cultures by studying the proinflammatory gene expression profile upon HCoV-229E, HCoV-EMC, and SARS-CoV infection. The authors observed that both HCoV-229E and highly pathogenic coronaviruses can only marginally induce IFN and interferon-stimulating gene responses. In particular, HCoV-229E is found to be more capable of inducing proinflammatory gene expression, including tumor necrosis factor alpha (TNF-α) and CXCL10, than the more pathogenic human coronaviruses. In contrast to the findings of Kindler et al., another previous study using well-differentiated normal human bronchial epithelial cells indicated that SARS-CoV induced higher cytokine and chemokine levels than HCoV-229E (9). This discrepancy may be attributed to different sampling times for cytokine and chemokine detection. In addition, one should note that SARS-CoV is known to modulate cytokine production by other key cells responsible for innate immunity in the lung (e.g., macrophage and dendritic cells) (10). It is therefore essential to study HCoV-EMC-infected macrophages and dendritic cells. Furthermore, due to the nature of purified cell culture (e.g., HAE) models, cross talk between epithelial cells, macrophages, and dendritic cells cannot be evaluated. Additional experimental systems, such as ex vivo respiratory organ cultures and animal models, may provide further understanding of diseases caused by HCoV-EMC. In addition, clinical parameters determined from HCoV-EMC-infected patients would be extremely valuable for understanding the disease process in humans and could be used as a reference for data generated from in vitro and animal experiments.
Based on the lack of IFN response found upon HCoV-EMC infection in HAE cultures, Kindler et al. conducted a key experiment and demonstrated the effective suppression of HCoV-EMC replication by administration of type I or type III IFN in the cultures. Zielecki et al. have recently confirmed that HCoV-EMC is sensitive to the antiviral action of type I IFN by using primary non-differentiated tracheobronchial epithelial cells and other epithelial cell lines (11). Again, the confirmation of IFN effectiveness in other models, particularly those relevant to lower respiratory tract infections, will strengthen the use of IFN as a possible therapeutic strategy to control HCoV-EMC infection. Nonetheless, Kindler et al. have pointed out a promising therapy for treating HCoV-EMC infection.
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.
Coronaviruses are enveloped, plus-stranded RNA viruses that cause widespread disease in humans and animals. They tend to infect just one or a few closely related species and in their natural host exhibit marked tissue tropism. Often, the respiratory or gut epithelia are specifically targeted, causing localized infection, but disseminated infections leading to systemic disease are also seen. Together with toroviruses, arteriviruses and roniviruses, coronaviruses are members of the order Nidovirales (Siddell et al., 2005).
Feline coronavirus (FCoV) infection is extremely common in cats. Natural infections with FCoV are usually transient, although a significant percentage of infections may become persistent (Addie & Jarrett, 2001). Most infections are asymptomatic or result in mild, self-limiting gastrointestinal disease, and in these cases the causative agent is known as feline enteric coronavirus (FECV). In a small percentage (<5 %) of animals, however, a fatal multi-systemic, immune-mediated disease occurs and this is known as feline infectious peritonitis (FIP) (Pedersen, 1995). Virus associated with FIP is referred to as feline infectious peritonitis virus (FIPV).
There are two types of FCoV that can be distinguished by serology and sequence analysis. Type I viruses are most prevalent in the field and account for approximately 80 % of all infections (Addie et al., 2003; Hohdatsu et al., 1992). Type II viruses are less prevalent and are characterized by recombination events that result in the replacement of the FCoV spike (S) glycoprotein gene with the equivalent gene of canine enteric coronavirus (CCoV) (Herrewegh et al., 1998). There is no evidence that either type is more commonly associated with FIP in natural infections (Benetka et al., 2004). The majority of research on FCoV to date has concentrated on the investigation of type II strains, most notably FIPV 79-1146, because they replicate well in cell culture.
The species specificity of coronaviruses is, to a large extent, determined by the recognition of a functional receptor on the surface of the host cell (Kuo et al., 2000). Coronaviruses use a variety of cellular receptors (Masters, 2006) and, even within a single host species, different receptors are used by different coronaviruses (Li et al., 2003; Yeager et al., 1992). It is accepted that type II FCoV strains use feline aminopeptidase N (fAPN) as a receptor for host attachment and entry (Tresnan et al., 1996). Consequently, they can be readily propagated in cell lines such as Crandell feline kidney (CrFK) cells, which express fAPN on their surface (Miguel et al., 2002). In contrast, there is conflicting evidence regarding the receptor for the attachment and entry of type I FCoV. Tresnan et al. (1996) have reported that the UCD-1 strain of FIPV, which is a type I virus, also uses fAPN as a receptor, albeit inefficiently. Their conclusion was based upon the ability of the UCD-1 strain to infect and express viral antigens in both hamster and mouse cells (which, normally, cannot be infected) that had been stably transfected with fAPN cDNA. In contrast, Hohdatsu et al. (1998) have concluded that fAPN is not a receptor for type I FCoV. Their conclusion is based upon the ability of an fAPN-specific monoclonal antibody (mAb), R-G-4, to block the infection of Felis catus whole foetus (Fcwf-4) cells with type II viruses, whereas the same antibody was not able to block infection with type I viruses.
The aim of the studies reported here was to provide evidence for or against the involvement of fAPN as a receptor for type I FCoV. Our approach was to produce retroviral pseudotypes that bear type I or type II FCoV S glycoprotein and produce a green fluorescent protein (GFP) reporter gene signal in transduced cells. We chose this approach because human coronavirus S glycoproteins have been successfully pseudotyped onto similar retroviral vectors and then used to analyse the recognition of cellular receptors (Hofmann et al., 2006; Simmons et al., 2004; Temperton et al., 2005). In our case, the pseudotypes were used to screen a range of feline cell lines for the expression of a functional receptor for attachment and entry. Our results clearly show that type I FCoV S glycoprotein fails to recognize fAPN as a functional receptor on three continuous feline cell lines. This suggests fAPN is not the receptor for type I FCoV. Our results also demonstrate that these retroviral pseudotypes can be used to screen for cells that are permissive for attachment and entry with FCoV, and we conclude that they can be used to identify and characterize the cellular receptor for type I FCoV. This would allow for the development of cell lines that efficiently replicate and propagate type I FCoV, which, in turn, would facilitate the investigation of these more clinically relevant viruses and aid in the development of a type I FCoV reverse genetics system.