In the host, initial infection occurs at epithelia of Harderian gland, trachea, lungs, and air sacs. The virus then moves to the kidney and urogenital tract, to establish systemic infection [33, 35]. In this regard, the severity and clinical features of IB depend on the organ or system involved. Infection of the respiratory system may result in clinical signs such as gasping, sneezing, tracheal rales, listlessness, and nasal discharges. Affected birds appeared listless and dull with ruffled feathers (Figure 1). Other signs may include weight loss and huddling of birds together under a common heat source.

Other clinical outcomes associated with IB infection include frothy conjunctivitis, profuse lacrimation, oedema, and cellulitis of periorbital tissues. Infected birds may also appear lethargic, with evidence of dyspnoea and reluctance to move. Nephropathogenic IBV strains are most described in broiler-type chickens. Clinical signs include depression, wet droppings, and excessive water intake. Infection of reproductive tract is associated with lesions of the oviduct, leading to decreased egg production and quality. Eggs may appear misshapen, rough-shelled, or soft with watery egg yolk (Figure 2). Unless effective measures are instituted, decline in egg production does not return to normal laying, thus contributing to high economic loss [1, 37].

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.

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.

Pathological changes observed grossly at necropsy include congestion and oedema of tracheal mucosa and extrapulmonary bronchi (Figure 3) [38, 39].

Histopathological changes include loss of cilia, oedema, rounding and sloughing of epithelial cells, and infiltration by lymphocytes (Figure 4). Presence of Russell bodies in Harderian cells has been observed following infection with H120 IBV serotype.

Nephropathogenic IBV strains cause nephritis characterized by swelling and congestion of the kidney (Figure 5), sometimes with pallor of ureters that contain urate deposits. Coinfection with bacterial pathogens such as E. coli may lead to a more complex outcome, usually associated with high morbidity and mortality. Similarly, infection with nephropathogenic IBV strains may result in pale, swollen, and mottled kidneys [39, 41]. Histological findings include interstitial nephritis, tubular degeneration, and infiltration by heterophils. In some cases, necrotic and dilated tubules are filled with urates and casts. Experimental studies have shown that IBV-T-strain causes necrosis of the proximal convoluted tubule and distension of distal convoluted tubule. In addition, necrotic foci, heterophils, and lymphocytes are observed in the interstitial spaces. Oedema of Bowman's capsule and granulocytic infiltration has been reported in the collecting ducts and spheroids [42, 43].

When the reproductive system is affected, there may be nonpatent and hypoglandular oviduct, especially in severely affected chickens [43, 44]. Large accumulation of yolk fluid may be seen in the abdominal cavity (Figure 6), often associated with bacterial infection in laying hens [45, 46]. Cystic oviduct has also been observed in young layers following infection with certain IBV strains (Figure 7).

Acute respiratory infections (ARIs) are the most common infections in humans of all ages. Children and infants are one of the most vulnerable groups of the population, and ARIs are the most common cause of children's hospitalization worldwide. Although bacteria, fungi, and parasites can cause ARIs, respiratory viruses cause the majority of infections. Most respiratory virus infections in early childhood are confined to the upper respiratory tract. About one-third of infants develop lower respiratory tract infection (LRTI). The most common causative viral agents of ARIs in children, respiratory syncytial virus (RSV), human metapneumovirus (HMPV), influenza viruses (Flu), and adenoviruses (AdV), were the subject of intensive research for years; therefore, clinical characteristics and regional epidemiological features of those ARIs in Croatia are well known [3–6]. However, the list of respiratory viruses is growing due to the rapid advance of laboratory diagnostic methods. In the last ten years, newly discovered viruses have been identified including human bocavirus (HBoV), coronaviruses NL63 (HCoV-NL63) and HKU1 (HCoV-HKU1), new enterovirus (HEV), parechovirus (HPeV), and rhinovirus (HRV) strains. Additionally, despite the fact that some of the respiratory viruses have been well known for a long time, particularly parainfluenza type 4 (PIV-4), the technically demanding cultivation methods and unavailability of commercial tests made it difficult to diagnose PIV-4's infection [8, 9]. Infections caused by some of the newly discovered viruses (i.e., HBoV, HCoV-NL63, and HCoV-HKU1) as well as those difficult to cultivate (PIV-4) have not been recorded in the country yet. There are few recent studies from the region providing valuable but still insufficient data regarding regional epidemiology of infections caused by the abovementioned viruses [10, 11]. Furthermore, the issue of multiple respiratory virus detection, which occurred because of high sensitivity of molecular methods, complicates the interpretation of laboratory diagnosis. The aim of this study was to determine the viral etiology for sixteen viruses tested by multiplex PCR method among children with ARI admitted to the hospital in Zagreb region in two respiratory seasons, in order to demonstrate the need for molecular diagnostics introduced in routine practice. Also, we aimed to investigate the characteristics of infections with single and multiple virus detection, especially regarding the type of virus involved and severity of infection.

This study reports an outbreak of severe lower respiratory illness caused by two subgenotypes (C3 and B) of human coronavirus NL63 (HCoV-NL63). The new subgenotype C3 with enhanced viral entry into host cells accounts for half of patients, which alerted that HCoV-NL63 which consist of multiple subgenotypes, is undergoing continuous mutation, and has the potential to cause large-scale severe infections in humans.

Disease manifestations are often more severe in children under five and adults over 20 years of age. Patients who are immunocompromised may present atypically and may not develop a rash. During a measles outbreak, clinicians should advise patients with viral syndromes who are being discharged from the ED to monitor for appearance of a rash, especially one that first appears on the face. If a rash develops, children or adult patients should avoid public places and seek immediate medical advice.

Acute gastroenteritis (AGE) is a common illness affecting all age groups worldwide. Even in developed countries, most of the population will average one episode of AGE per year. Among young children and the elderly, the rate is higher and the disease more severe,[3]. Globally, approximately 1.5 billion episodes and 1.5 to 2.5 million deaths annually in children under age five are estimated to be associated with AGE, the majority occurring in developing countries. Among the elderly, underlying immunological, physiological and functional deterioration exacerbate severity, resulting in increased morbidity and mortality attributed to diarrheal disease. Mortality may exceed 50% in the above 73 years age group in the US. However, for such an important disease, our understanding of causality remains incomplete. The importance of bacteria and parasites in AGE has been known for many years, although the roles of campylobacter (1972) and cryptosporidium (1976) in human infection were recognized more recently. Viruses such as rotavirus and enteric adenovirus were also first described over 30 years ago and shown to have an association with diarrheal disease. Rotavirus is the most common cause of viral AGE in children under age five. The recent widespread availability of molecular diagnostics has allowed an increased understanding of the role of other viruses in causing AGE. The human calicivirus genus Norovirus (NoV, formerly Norwalk-like virus, NLV) is now recognized as the most common cause of epidemic outbreaks of AGE across all age groups with noroviruses belonging to genogroup 2 (NoV GII) more frequent than those belonging to genogroup 1 (NoV GI). Recent reports suggest prevalence rates of 9 to 20% for NoV in sporadic AGE in children–[11]. Similarly, associations with AGE have also been made for both a second calicivirus genus Sapovirus (formerly Sapporo-like virus, SLV or classic calicivirus) and astrovirus. A limited number of reports suggest sapoviruses are less frequently found in cases of AGE than NoVs, are mainly restricted to children less than age five years and may cause less severe symptoms than infections with rotavirus and NoV–[14]. Human astroviruses are reported to have an association with AGE, especially in children, with prevalence rates up to 8.6% documented–[17]. Higher rates (39%) have been reported in children less than 1 year old and in areas lacking adequate sanitation. Other viruses such as torovirus, picobirnavirus, picotrinavirus, pestivirus and coronavirus have also been implicated–[23]. Lately, there have been reports of human bocavirus (HBoV) sequences in feces of children with respiratory infections or AGE–[26]. However, many of these observations are from uncontrolled trials and in the absence of large prospective controlled studies, their association with AGE is uncertain. Despite this wide range of known and suspected pathogens, in both pediatric and adult populations, the pathogen causing AGE remains unidentified in up to 67% of cases,[14],[27],[28].

In order to further investigate the causes of AGE in children, in late 2000 we commenced a prospective controlled study of patients presenting to a children's hospital in Adelaide, South Australia. Unexpectedly, even with extensive molecular testing, we could not find a cause in 28% of cases (Ratcliff, R.M. unpublished). Proposing that novel viruses may be the cause of the undiagnosed cases, we developed a method for ‘non-selective’ detection of viral DNA in feces. This method uses degenerate oligonucleotide primed (DOP) PCR amplification to non-specifically amplify DNA from diarrheal samples. Previously, DOP amplification had been utilized to replicate DNA from minute samples such as single cell nuclei, and forensic and archaeological sources. An alternative ‘non-specific’ amplification method used nuclease digestion to specifically purify virus nucleic acid by exploiting the protection of the virus nucleic acid from digestion afforded by the virus capsid. Combining nuclease digestion with DOP amplification ought to facilitate the sole replication of viral nucleic acid from nucleic acid ‘soups’ such as feces. The DNA produced from such virus enrichment followed by non-specific amplification can be cloned and sequenced to assess the presence of viral DNA and provide a method to screen for novel enteric viruses. We initially investigated a ‘summer’ cluster of sporadic AGE cases occurring between January and April 2001 and who were part of the case control study into the etiology of pediatric AGE. We detected a previously unknown parvovirus, which we have named Human Bocavirus species 2 (HBoV2). Following the development of specific amplification assays to detect HBoV2, we screened all samples collected from cases and controls in the study during 2001 and assessed their association with pediatric AGE. During this screening, we discovered a second novel parvovirus which we have named Human Bocavirus species 3 (HBoV3).

Acute respiratory infection (ARI) is an important cause of morbidity and mortality with a worldwide disease burden estimated at 112 900 000 disability adjusted life years (DALYs) and 3·5 million deaths.[1], A strict definition of ARI would include all infections of the respiratory tract. However, in practice, acute lower respiratory infection accounts for most of the serious disease burden. ARI causes about 20% of all deaths in pre-school children worldwide, with 90% of these deaths being due to pneumonia. Risk factors for severe ARI include malnutrition, low birth weight, passive smoking, non-breastfeeding, low socio-economic status, overcrowding, immunodeficiency and HIV infection, and consequently, most of the morbidity associated with ARI is found in the developing world.[3],,

ARI causative organisms are predominantly bacterial (most commonly Streptococcus pneumoniae and Haemophilus influenzae) or viral, although it is not possible to differentiate between them based on clinical signs or radiology. New pathogens are frequently being reported in the literature, including Coronaviruses (NL63 and HKU1), human Metapneumovirus (hMPV) and human Bocavirus (hBoV).[6],,, This expansion in the number of pathogens, combined with an increased ability to simultaneously test for multiple organisms, has highlighted the potential role of co-infection, with the detection of more than one pathogen from a single sample, although their significance with regard to disease severity remains debatable.[10],

The causes of ARI in children under-5 years have been studied in different environments and populations. Respiratory syncytial virus (RSV), hMPV, Rhinoviruses (hRV) and Para-influenza viruses (PIV) have consistently been shown to predominate, with some displaying strong seasonal peaks and with co-infection with more than one viral pathogen occurring in 4–33% of children.[11],,, Few studies however have attempted to determine whether particular viruses are associated with differing severities of disease. Those that have, have concentrated on the more severe end of the disease severity spectrum i.e. those children with RSV disease hospitalized or ventilated on intensive care.[15] Similarly, few studies have examined in detail the viral causes of specific clinical conditions in the under-5 age group.[16]

The aim of this study was to determine the prevalence and seasonal distribution of viral and atypical bacterial pathogens in a cohort of well-characterised children under the age of 5 years presenting to a paediatric A&E department in North-Eastern Brazil with various severities and clinical manifestations of ARI.

Measles classically presents with a high fever (often >104°F [40°C]), generally of 4–7 days in duration. This initial sign occurs after an incubation period of 1–2 weeks following exposure (average 10–12 days). During this prodromal phase, a classic triad of cough, coryza and conjunctivitis (the “3 Cs”) is often present.5 Patients may have photophobia. The eyes have a characteristic appearance, typically showing erythema of the palpebral conjunctiva with nonpurulent discharge (Figure 1) and sometimes periorbital edema. Patients may also report malaise, myalgias, anorexia, and diarrhea. Adults often develop transient hepatitis.6

Koplik spots, when seen, are pathognomonic of measles (Figure 2). If present, they manifest 1–2 days prior to the rash and last for 3–5 days. They appear as bluish-gray enanthema (“small grains of sand”) on a red base and are typically seen on the buccal mucosa opposite the second molars. Therefore, it is essential to have proper lighting to visualize them. During a measles outbreak, after donning appropriate respiratory protection, emergency physicians (EP) should carefully assess the oropharynx in patients presenting with non-specific viral syndromes and assess for the presence of Koplik spots.

The rash of measles generally erupts about 14 days after exposure, which is usually 2–4 days after onset of symptoms. Unlike rashes of some infectious diseases that start on the lower extremities or trunk, the rash of measles begins on the face and progresses cephalocaudally to the torso and extremities. Thus, assessing the pattern of rash evolution is essential to identify measles patients. Erythematous macules and papules coalesce into patches and plaques within about 48 hours (Figure 3). Petechia and ecchymosis can also be seen. By the time a rash develops, within 1–2 days, patients will be ill appearing. After 5–7 days, the exanthem begins to fade, forming coppery-brown hyperpigmented patches that may desquamate. The rash initially disappears at the location where it first appeared. The rash can be more difficult to detect on dark-skinned patients (Figure 4).

Respiratory syncytial virus (RSV), of the family Paramyxoviridae, is a nonsegmented, negative-strand RNA virus that expresses 11 proteins. RSV is amongst the most important pathogenic infections of childhood and is associated with significant morbidity and mortality, especially in developing countries. The clinical manifestations of RSV infection range from mild upper-respiratory-tract illnesses (URTIs) or otitis media to severe and potentially life-threatening lower-respiratory-tract illnesses (LRTIs). The most common form of LRTI in RSV-infected infants is bronchiolitis, but pneumonia and croup are also seen [2–6]. There are two major antigenic subgroups of RSV, A and B, based on antigenic differences in their glycoproteins G and F [7–10]. The epidemiological features of RSV-A and RSV-B have differed in previous studies, and although strains of both subtypes often cocirculate, one subtypes generally predominates, depending on the region and climate [5, 6, 11, 12]. Therefore, the in-depth epidemiological characterization of RSV in specific areas is required worldwide, and especially in developing countries.

RSV is detected with direct or indirect immunofluorescence, enzyme-linked immunosorbent assay, viral culture, or increasingly, with reverse transcription–polymerase chain reaction (RT–PCR), especially real-time RT–PCR [6, 13]. Real-time PCR has many advantages in the detection of viruses, including its high specificity, high sensitivity, quantifiability, and simplicity of operation. It has been used for early diagnosis and the evaluation of drug efficacy and therapeutic effects, although the quantitative analysis of viruses in respiratory specimens is still impracticable. The results are usually simply positive or negative, although quantification is possible from Ct values or with plaque assays [14, 15]. The results from two independent respiratory specimens, such as throat swabs, nasopharyngeal swabs, or bronchial alveolar lavage fluid, can differ, even when both specimens are taken from the same individual at a same time, because they are dependent the sampling skill and features of the samples themselves. Thus, inconsistent results can be produced. However, comparative quantification PCR using a housekeeping gene, such as β-actin (ACTB) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH), might be suitable for the analysis of these types of respiratory specimens.

In this study, we investigated the epidemiological features of RSV (subgroups A and B) in pediatric patients (≤ 14 years old) hospitalized with respiratory illness in Guangzhou, China, between January 2013 and December 2015, using a newly established multiplex real-time PCR, including the housekeeping gene, human ACTB.

Viruses cause most respiratory tract infections, yet the specific infectious agent often remains unknown,. Comparison of the viral causes of infection provides a useful starting point for an understanding of illness following respiratory infection. It also provides data relevant to the development of prevention strategies. The following viruses (in no particular order) have been detected during acute respiratory infections (ARIs),: influenza virus (Flu), parainfluenza virus (PIV), adenovirus (ADV), picornavirus (PIC, including rhinovirus and enterovirus), respiratory syncytial virus (RSV), human metapneumovirus (hMPV), and human coronavirus (HCoV). Respiratory virus infections are diagnosed in four principal ways: virus culture, serology, immunofluorescence/antigen detection, and nucleic acid/PCR-based tests. Nucleic acid tests are significantly more sensitive than the other methods described, which may have an impact on the viruses detected,. Nucleic acid tests are now being multiplexed, allowing rapid simultaneous detection of many viruses,.

In China, several groups have reported the prevalence and clinical presentation of viral infections–[5], particularly those of HCoV infections by reverse transcriptase PCR (RT-PCR) assays performed on clinical specimens taken from adults with ARTIs from 2005 to 2009 in Beijing. However, more precise data regarding their epidemiology and clinical characteristics are lacking in mainland China after the 2009 H1N1 pandemic. Moreover, to the best of our knowledge, there is no published report that describes the potential impact of viral agents on adults with ARTIs admitted to an ED in China. To directly address this situation, we screened for the presence of 13 respiratory virus in adults with ARTI admitted to Peking Union Medical College Hospital from May, 2010, to April, 2011, in an effort to gain a better understanding of the seasonality, epidemiology, and clinical profile of these viruses in a city with a population of more than 22 million.

Lower respiratory tract infections (LRTIs) are an important cause of mortality among children worldwide.1 Viruses are the causative agent in a sizable proportion of pediatric LRTI cases, especially among children less than 1 year of age.2 In affluent societies, the burden of such infections may be even higher, but deaths from respiratory syncytial virus (RSV) and other associated LRTIs are several times more frequent in developing than in developed countries.3 The frequency of LRTI from different respiratory viruses is clearly influenced by a seasonal pattern that may vary according to geographic location and climate conditions.4,5 However, the relative importance of RSV as the causative agent in LRTI varies widely in different geographic locations, and there is limited information on RSV seasonal patterns in tropical areas.4,6

In Brazil, RSV is the most common viral pathogen found in episodes of LRTI; other frequent viruses include influenza, rhinovirus, parainfluenza, adenovirus, metapneumovirus, and bocavirus.7–16 Some studies conducted in Brazil have suggested a clear seasonal pattern for RSV infections, with predominance from March to July, and a smaller number of cases in the summer months, especially November to February.14–19 Other viruses have been investigated to a lesser extent, but a seasonal pattern was found for different respiratory viruses in Southern and Southeastern Brazil, where the winter is cold and dry.10,13,20 Only a few studies were conducted to investigate the prevalence and seasonal patterns of different viral infections among children with an episode of LRTI seen at hospitals from Northeastern Brazil, where the winter is warm and wet.14,16,21–24 Such studies were typically conducted in only 1 major city in the region and have not allowed for concurrent assessments within different areas of the Northeastern region of Brazil. Moreover, they have used different eligibility criteria and methods for viral identification. In the current study, we adopted standardized criteria and methods to assess the frequency of infection with RSV and 7 other viruses in children aged less than 2 years old hospitalized due to an LRTI in 4 capitals (Aracajú, Salvador, Recife, and Maceió) from Northeastern Brazil over a period of 1 year. We also seek to evaluate the association between the frequency of LRTI in general, of RSV, and of the other viruses and meteorological data (temperature, precipitation, humidity, and solar radiation).

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.

Human parainfluenza viruses (HPIVs) are RNA viruses in the genus Paramyxoviridae. Four HPIV types have been identified. HPIVs are important causes of upper respiratory tract illness (URTI) and lower respiratory tract illness (LRTI), especially in children. An estimated five million LRTI occur each year in the United States in children under 5 years old, and HPIVs have been isolated in up to one third of these infections. The HPIV-1, HPIV-2 and HPIV-3 are second only to respiratory syncytial virus (RSV) as a cause of hospitalizations (2%–17%) for acute respiratory infection among children aged younger than 5 years in the United States.

Compared with studies of HPIV infection in children, less is known about infections in adults. Most HPIV infections in adults cause mild upper respiratory tract symptoms, but the elderly or those with compromised immune systems are at increased risk for severe HPIV infection. Compared with types 1−3, only a small number of reports have studied HPIV-4, and the lack of epidemiologic data on HPIV-4 prevents a clear understanding of the full clinical pattern of HPIVs. In addition, any differences in the clinical presentation of the four HPIV types are still largely unknown.

The aims of this study were to explore the epidemiologic features and clinical manifestations of HPIVs and other common respiratory pathogens in children and adults with acute respiratory tract illness (ARTI) in Guangzhou, southern China, and to uncover clues that might help to establish clinical distinctions between different HPIV types.

Recent scientific and clinical evidence has indicated that the virus is found during upper and lower respiratory tract infections, causing symptoms and signs that do not differ greatly from the symptoms described for the 'old' viruses HCoV-229E and HCoV-OC43. Other systems involvement is still controversial.

Table 1 shows that patients diagnosed with the virus have presented with mild symptoms, indicating upper respiratory tract infection such as fever, cough and rhinorrhoea. On the other hand, the disease is also known to cause significant more alarming lower respiratory tract infection. One of the most alarming symptoms is bronchiolitis, an inflammation of the membranes lining the bronchioles. This symptom was reported by several research groups [25, 50, 64], and although a population-based study in China did not report an association of HCoV-NL63 with bronchiolitis, it is still believed to be one of the presenting symptoms. Several research groups have linked HCoV-NL63 to croup [26, 53]. Croup children present with pharangitis, sore throat and hoarseness of voice, and are considered for hospitalization. Of the rare findings, a group has reported the association between HCoV-NL63 and Kawasaki disease, a form of childhood vasculitis that is presented as fever, polymorphic exanthema, oropharyngeal erythema and bilateral conjuctivitis. However, others fail to report on this association [66, 67].

It is noteworthy to say that the report of symptoms in young children, who represent the majority of patients, is based mainly on parental observations, where other possible subjective signs and symptoms fail to be recognized by the parents. Moreover, most of the studies were conducted on patients reporting to hospitals suffering from acute respiratory tract infection. To date, there are only few population-based studies and the question arises whether larger numbers of such studies might reveal the involvement of other body systems.

Samples in this study were taken as part of standard care. The First Affiliated Hospital of Guangzhou Medical University Ethics Committee approved the experimental design and patient involvement in this study. Written informed consent was obtained from the patient for publication of this report and any accompanying images.

Throat swab samples were collected from patients with ARTI (presenting with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, coryza, sneeze, dyspnoea) at three hospitals in Guangzhou, southern China between July 2009 and August 2011. The samples were refrigerated at 2 to 8°C and transported on ice to State Key Laboratory of Respiratory Diseases and analysed every working day or stored at −80°C before testing. Over 97 samples were collected and tested during each month in our study.

Clinical presentations were collected and categorized retrospectively into the following six groups from the patients’ medical records using designed presentation cards: URTI, LRTI, systemic influenza-like symptoms, gastrointestinal illness, neurologic symptom and others. Patients with nasal obstruction, coryza, sneeze, cough, pharyngeal discomfort, or hoarseness were categorized as having URTI. Patients with pneumonia, bronchopneumonia, increasing lung markings, dyspnoea, or abnormal pulmonary breath sound were categorized as having LRTI. Patients with high fever (≥38°C), chills, dizziness, headache, myalgia or debilitation were categorized as having systemic influenza-like symptoms. Patients with vomiting, poor appetite, or diarrhoea were categorized as having gastrointestinal illness. Patients with convulsion were categorized as having an neurologic symptom. Patients with other symptoms, including but not limited to rash, were classified as “others”. Some patients were assigned to multiple clinical presentation groups. Pneumonia and bronchopneumonia were diagnosed by chest radiography. Pneumonia was defined as an acute illness with radiographic pulmonary shadowing which was at least segmental or present in one lobe (excluding the bronchi); bronchopneumonia was defined as inflammation of the walls of the smaller bronchial tubes, with varying amounts of pulmonary consolidation due to spread of the inflammation into peribronchiolar alveoli and the alveolar ducts. Other clinical symptoms were identified by common medical examinations and clinical descriptions.

Six coronaviruses are known to infect humans and cause respiratory disease, including human coronavirus (HCoV) 229E, OC43, severe acute respiratory syndrome CoV (SARS-CoV), NL63, HKU1 and Middle East respiratory syndrome CoV (MERS-CoV). SARS-CoV and MERS-CoV are highly pathogenic coronaviruses that caused severe and fatal respiratory infections in humans. The SARS-CoV pandemic infected over 8000 people worldwide. As of 9 September 2019, 2458 MERS cases with 848 deaths (34.5% mortality) were reported to World Health Organization (WHO). HCoV-229E, OC43, NL63 and HKU1 are endemic in humans and mainly cause mild respiratory infections worldwide.

HCoV-NL63 has been prevalent worldwide for many years. The majority of HCoV-NL63 infections in human are mild, although occasionally NL63 causes pneumonia or central nervous system diseases in susceptible individuals including young children, elderly and immunosuppressed patients. HCoV-NL63 primarily infects upper respiratory tract and most of HCoV-NL63 infections are acquired during childhood. Neutralizing activity directed against HCoV-NL63 is common in sera from adults and rarely in infant’s serum. During 2009 and 2016, HCoV-NL63 accounted for about 0.5% (60/11399) of all acute respiratory tract infections in hospitalized pediatric patients in Guangzhou, China, most of these cases associated with HCoV-NL63 were considered to be evidence of endemic infection and no outbreaks were reported.

Here, we identified a cluster of 23 hospitalized pediatric patients with severe lower respiratory tract infection caused by two subgenotypes (C3 and B) of HCoV-NL63, and half of the patients were caused by a new subgenotype C3 which was first reported here. A unique mutation (I507 L) in receptor-binding domain (RBD) was detected in the new subgenotype of HCoV-NL63 associated with increased viral entry into host cells indicating that HCoV-NL63 was undergoing continuous mutation which potentially could enhance HCoV-NL63 virulence and promote transmission. This study showed that HCoV-NL63 had the potential to cause epidemics in humans and it may be a more important human pathogen than is commonly believed. Efforts should be paid to monitor genetic changes in HCoV-NL63 genome and also its pathogenicity and prevalence in the human population.

The clinical characteristics of patients with FluA, ADV, PIC, and HCoV infection are summarized in Table 3. All patients infected with these viruses presented with respiratory infection symptoms including fever, cough, headache, sore throat, runny nose, and so on; a few also presented with diarrhoea. The three most common symptoms of infection with FluA were cough (82.09%), sore throat (79.10%), and headache (65.67%). The most common symptoms of infection with ADV, PIC, and HCoV were headache, sore throat, and cough, respectively. Most infected patients were aged 20–49 years old, and slightly more were female.

A total of 134 children admitted to Children's Hospital Zagreb during two winter seasons (January to March) in 2010 and 2015 with symptoms of ARI and suspected for viral etiology (normal or slightly elevated inflammatory markers, i.e., white cell count) were included in the study. Patients were categorized into three groups according to age (<1, 1–3, and ≥4 years of age) and two groups according to the localization of infection in those with upper respiratory tract infection (URTI) and lower respiratory tract infection (LRTI). URTI was defined by symptoms of the common cold, coryza, cough, and hoarseness often accompanied with fever. Clinical syndromes of respiratory catarrh, rhinitis, and/or pharyngitis are included in URTI category. LRTI was defined according to the clinical symptoms of tachypnea, wheeze, severe cough, breathlessness, and respiratory distress accompanied by LRTI signs such as nasal flaring, jugular, intercostal, and thoracic indrawings, rarely cyanosis, and, on auscultation of the chest, wheeze, crackles, crepitations, and inspiratory rhonchi or generally reduced breath sounds. Clinical syndromes of bronchitis, bronchiolitis, and pneumonia were included in LRTI category. To avoid unnecessary X-ray exposure, chest radiographs were taken only for some of the patients to exclude or confirm bacterial pneumonia. Severe disease and acute respiratory distress syndrome (ARDS) were defined with need for oxygen supplementation and/or mechanical ventilation. The patients' underlying conditions data were collected retrospectively from medical charts. The most common underlying diseases were asthma, anamnestic recurrent wheezing episodes, neurological disorders, prematurity, and anemia. Written consent was obtained from the children's parents or caretakers. The study was approved by the Ethic Committee of the Teaching Institute of Public Health “Dr. Andrija Stampar.”

Nasopharyngeal and pharyngeal flocked swabs from each patient were collected, combined, and placed in viral transport medium (UTM™, Copan, Italy). Specimens accompanied with demographic data and clinical diagnosis were immediately transported to the Molecular Microbiology Laboratory at the Public Health Institute where they were stored at −80°C until tested. Nasopharyngeal and pharyngeal swabs, blood cultures, and serum for serology were searched to exclude bacterial infection. Patients with samples positive on bacteriology testing were subsequently excluded from the study.

Rhinoviruses (RVs) are small, non-enveloped viruses that belong to the family Picornaviridae, genus Enterovirus. To date, there are 171 rhinovirus (RV) genotypes recognized and classified into the three species as RV-A (83 types), RV-B (32 types), and RV-C (56 types) (Royston and Tapparel, 2016; Pan et al., 2018). RV-A and RV-B were discovered by isolation on monkey kidney cells in 1950s (Price, 1956) while RV-C genotypes, which are not cultivable using ordinary culture methods, have been identified decades after following the rise of molecular techniques (Lamson et al., 2006; Lau et al., 2007). There are also differences between RV species in utilization of cell entry receptor: a majority of RV-A and RV-B attach to the intercellular adhesion molecule (ICAM)-1 (classified as the major receptor group) and the others alternatively bind low density lipoprotein receptor (LDL-R) (minor receptor group), whereas RV-C utilizes human cadherin-related family member 3 (CDHR3) (Bochkov et al., 2015; Royston and Tapparel, 2016).

Rhinovirus genome is a 7.2-kb single-stranded, positive-sense RNA with a single open reading frame (ORF) joined to a 5′ untranslated region (5′UTR) and a short viral priming protein (VPg) (Jacobs et al., 2013). ORF encodes a poly-protein which is cleaved by virally encoded proteases in 11 proteins. Four proteins – VP1, VP2, VP3, and VP4 – make up the viral capsid and account for the virus’ antigenic diversity, while the remaining non-structural proteins are involved in viral genome replication and assembly. A rather conserved 5′ UTR region that harbors internal ribosomal entry site (IRES) is usually utilized for RV detection from clinical samples, while more precise genotyping is based on VP4/VP2 or VP1 sequence analysis.

Rhinovirus have been neglected for decades, primarily because they were considered less virulent and only capable of causing mild common cold, and were not recognized, until recently, as important causative agents of lower respiratory tract infections (LRTIs) and severe respiratory disease (To et al., 2017). Introduction of sensitive and technically simple molecular detection assays, especially multiplex PCR, enabled affordable RV detection in line with other common respiratory viruses in clinical samples. Together with coronaviruses, RVs are indeed responsible for majority of upper respiratory tract infection (URTI), but also for substantial rate of LRTIs in all age groups (Lauinger et al., 2013; Zhao et al., 2018, Čivljak et al., 2019). Many studies showed that RV is one of the leading causes of pneumonia, bronchiolitis and other form of severe respiratory disease (Zhao et al., 2018), standing side by side with respiratory syncytial virus (RSV) in children and influenza in elderly (Esposito et al., 2013; Ning et al., 2017; Čivljak et al., 2019). There are several studies that report increased rates of asthma exacerbations and LRTIs among children with RV-C when compared to those infected with RV-A and RV-B (Bizzintino et al., 2011; Lauinger et al., 2013); however, recent studies did not find any relationship between a specific RV species and the severity of clinical presentation (Jacobs et al., 2015; van der Linden et al., 2016; Zhao et al., 2018).

Data on RV prevalence in Croatia are scarce, and molecular epidemiology was thus far not described. This study aims to determine the RV prevalence, compare it with prevalence patterns of other common respiratory viruses, as well as to explore clinical and molecular epidemiological features of RV infections among hospitalized children with acute respiratory infection.

Viral upper respiratory tract infections remain a major cause of morbidity and mortality worldwide, with influenza infections being an important cause. Influenza viruses A and B cause annual epidemics and produce 3–5 million cases of severe disease and 290,000 to 650,000 respiratory deaths annually. Because influenza epidemics lead to increased social concern each season and the appearance of a novel influenza A subtype virus can cause a pandemic, and disease surveillance is crucial from a public health perspective.

The case definition is a key factor in any standardized system of public health surveillance and, ideally, should be based on a combination of signs and symptoms that characterize the condition of interest.

The clinical characteristics of influenza, known as influenza-like illness (ILI), are similar to those caused by other viruses causing acute respiratory infection (ARI), and only laboratory confirmation permits a specific disease diagnosis. Because influenza is very common and, during seasonal epidemics, affects 10%–20% of the unvaccinated population, it is not feasible to confirm all suspected cases. Due to the lack of specificity of influenza symptoms and because it is necessary to assess the disease burden, quantitative indicators of ILI or ARI are commonly used.

The World Health Organization (WHO) defines ILI as an acute respiratory illness with a measured temperature of ≥38 °C and a cough, with onset within the last 10 days.

The European Center for Disease Prevention and Control (ECDC) defines ILI as the sudden onset of symptoms and at least one of following four systemic symptoms: fever or feverishness, malaise, headache, and myalgia, and at least one of the following three respiratory symptoms: cough, sore throat, and shortness of breath. ARI is defined as sudden symptom onset with at least one of the following four respiratory symptoms: cough, sore throat, shortness of breath, coryza, and the clinician’s judgement that the illness is due to an infection.

Any influenza surveillance system aims to reliably assess the influenza epidemic activity each season. The system should provide robust, continuous data in order to monitor the trends of clinically diagnosed ILI or ARI in the population studied and in specific groups at increased risk of complications and death.

Like any public health surveillance system, the influenza surveillance system must evaluate its own performance in relation to its main purposes. The accuracy of the clinical case definition has traditionally been assessed in terms of sensitivity, specificity, and positive predictive value. More recent studies also include the positive predictive likelihood/negative predictive likelihood ratio, named the diagnostic odds ratio (DOR).

The objective of this study was to investigate the performance of different case definitions and clinical manifestations of ILI and ARI in a primary healthcare influenza sentinel surveillance system.

Acute respiratory tract infections (RTIs) are the most commonly encountered diseases for hospitalization, particularly in infants and young children. These are the major causes of morbidity and mortality with a worldwide disease burden estimated at 112 900 000 and 3.5 million deaths, respectively. RTIs are divided into two categories, as acute lower respiratory tract infections (LRTIs) and upper respiratory tract infections (URTIs) according to localization of the infection. LRTIs are more harmful than URTIs. URTIs refer to infections of the nasopharynx, larynx, tonsillae, sinuses, ears, including rhinosinusitis, tonsillitis, pharyngitis, laryngitis/laryngotracheitis and otitis media. LRTI refers to infections of the trachea, bronchus, and alveolus, including tracheitis, bronchitis, bronchiolitis, and bronchopneumonia. The etiological agents include many bacteria, viruses and fungi. However, viral agents have a greater role in causing RTI as they are more commonly observed. In recent years, many viruses causing RTI have been discovered, including coronaviruses, human metapneumovirus and human bocavirus. Detection of these viruses relies on the polymerase chain reaction (PCR) in virology laboratories. As the agents of RTIs, viruses have constantly been shown to predominate; this majority of the viral agents may be related to the increased use of the PCR in the detection of viruses. Coinfections with more than one viral etiological agent exist in 4-33% of children. The clinical severity of RTIs which are caused by viruses are viewed differently among viral agents. Coinfection may or may not be a risk factor for disease severity. Evidence has also shown that severe viral infections in infancy and early childhood are related to recurrent wheezing and asthma in later life.

The purpose of current study was to determine the incidence and seasonal patterns of viral etiological agents and to compare their clinical manifestations and disease severity, including single and coinfections.

Throat swab samples from pediatric patients (≤ 14 years old) hospitalized with acute respiratory-tract illness (ARTI) were collected at two hospitals between January 2013 and December 2015 for routine screening for respiratory viruses, Mycoplasma pneumoniae (MP), and Chlamydophila pneumoniae (CP). The samples were collected with established clinical protocols. The samples were refrigerated at 2–8°C in viral transport medium, transported on ice to the State Key Laboratory of Respiratory Diseases, and analyzed immediately or stored at −80°C before analysis as described in detail previously.

The patients’ clinical presentations were collected from their medical records using designed presentation cards, and were categorized retrospectively into the following six groups: URTI, LRTI, systemic influenza-like symptoms, gastrointestinal illness, neurological symptoms, and others. Patients with nasal obstruction, coryza, sneezing, coughing, pharyngeal discomfort, or hoarseness were categorized as having URTI. Patients with pneumonia, bronchiolitis, increasing lung markings, dyspnea, or an abnormal pulmonary breath sound were categorized as having LRTI. Patients with high fever (≥ 38°C), chills, dizziness, headache, myalgia, or debility were categorized as having systemic influenza-like symptoms. Patients with vomiting, poor appetite, or diarrhea were categorized as having a gastrointestinal illness. Patients with convulsions were categorized as having neurological symptoms. Patients with other symptoms, including but not limited to rash, were classified as “others”. Some patients were assigned to several clinical presentation groups. Increasing lung markings, pneumonia, and bronchiolitis were diagnosed with chest radiography. Abnormal pulmonary breath sounds included phlegmatic rales, wheezy rales, bubbling rales, and moist rales. Other clinical symptoms were identified with a normal medical examination and clinical descriptions.

Non-influenza related upper respiratory infections (URI) are universally experienced illnesses that, despite their typically self-limited nature, lead to billions of dollars of lost income, and predispose to serious illnesses including pneumonia.[1] When influenza is responsible, pandemics can result and cause millions of deaths. In 2009, a novel H1N1 influenza virus (2009 H1N1) emerged and rapidly spread worldwide, causing excess mortality in children and young adults. Although the global estimate of deaths has been lower than seen in several previous pandemics, the number of life years lost is estimated to be five times higher than those lost to seasonal H1N1 viruses and comparable to the number lost during the 1968 pandemic.[2], Military trainees, along with other groups of crowded, stressed individuals, are disproportionately affected by respiratory illnesses due to a variety of pathogens. With the exception of the prior adenovirus vaccine era from 1980–1996, adenoviruses have historically been the most common causes of febrile URI in this population, and have also led to serious illness and fatalities.[4]–[7] In one large study of transmission dynamics of adenovirus in a military training setting, approximately one-third of incoming trainees were already immune, one-third developed a febrile URI due to adenovirus, and the remainder seroconverted with subclinical or asymptomatic infection.[8] Large influenza outbreaks are less common, given the universal immunization of basic trainees and routine use of ring antiviral chemoprophylaxis in training units with known influenza cases, if cases occur within the first two weeks after immunization.[9], However, in 2009, type-specific influenza vaccine was not widely available until well into the full wave of illness.[11] With large numbers of concurrently circulating respiratory pathogens occurring year round in this diverse group of individuals, coming from a variety of geographic locations and backgrounds, and living in close contact for months, coinfection with multiple organisms would be expected to be a regular occurrence. However, whether coinfection contributes to differing clinical presentations or outcomes in this young, healthy adult population is unknown. While coinfections with viral pathogens including 2009 H1N1 have been described in patients with respiratory infections, few prospective studies have related these to clinical presentation and outcomes in adults since molecular diagnostics became available, and none in the setting of high background rates of adenovirus.[12]–[17]

We sought to describe the epidemiology of 2009 H1N1 and adenovirus in a basic training population, and to correlate differences in clinical presentations and outcomes with each respective pathogen and in coinfections.