Of the 72 HAdV-infected cases, 27 (37.50%) comprised co-infections with other respiratory viruses including 21 (77.78%, 21/27) with one other virus, 3 (11.11%, 3/27) with two other viruses, 2 (7.41%, 2/27) with three other viruses and 1 (3.70%, 1/27) with four other viruses. The most frequently identified mixed infection was PIV (12.50%, 9/72) and HRV (9.70%, 7/72), as shown in Table 2. Among the HAdV-positive specimens, the log number of HAdV genome copies ranged from 3.30 to 9.14 copies per mL of NPA, as determined by the qPCR assay measurements (Fig. 2). The log number for the HAdV genome copies was 6.14 ± 1.52 copies/mL of NPA in the children infected with HAdV only, which is slightly higher than the 5.70 ± 1.39 copies/mL of NPA from those co-infected with HAdV and other respiratory viruses; there was, however, no significant difference in the viral loads between the HAdV mono- and co-infections (P = 0.220).

The present study was carried out between April 2017 and March 2018 among hospitalized children with RTIs in Beijing, China. Herein, we describe (i) the prevalence of HAdVs in hospitalized children with RTIs presenting at Beijing Friendship Hospital during a one-year period; (ii) the clinical spectrum of the HAdV-positive RTI patients; (iii) the viral co-pathogens present in the HAdV infections; and (iv) the genetic diversity of the HAdVs.

The clinical characteristics of the RTIs caused by HAdV are very similar to those of influenza, PIV and other respiratory tract pathogens, making it difficult to clinically diagnose this type of infection. Therefore, effective diagnostic methods are needed for rapid identification and genotyping of HAdV. The qPCR assay used herein was established to detect and quantify HAdV. A total of 1276 NPA specimens were screened for the presence of HAdV and 72 specimens (5.64%, 72/1276) were confirmed to be positive for HAdV, which is consistent with prior reports (1.70–13.90%) [16–18]. The detection rate for HAdV varies from region to region in China, and the rate for hospitalized children with acute lower RTIs in Zhejiang province from 2006 to 2012 was 0.63, and 2.24% in Shenzhen city from 2012 to 2015 [19, 20]. However, it is worth mentioning that such discrepant HAdV detection rates can be caused by methodological differences, the number of patients tested, the periods during which the samples are collected, and even a study’s duration.

Previous studies have shown that HAdV detection rates are positively correlated with the monthly mean temperature and sunshine duration, and negatively correlated with wind speed; in fact, the higher the air temperature, the higher the detection rate. Our study also revealed that HAdV infections occurred throughout the year with the highest prevalence in the summer (9.52%, Jun to Aug), peaking in August (10.91%), which is similar to what has been found in Tianjin, a northern Chinese city, where HAdV infections are concentrated during the summer. However, this finding is discordant with other studies that have reported seasonal peaks for HAdV infections in spring in Northern China and Mexico [23, 24].

We found that the HAdV infections occurred predominantly in children under 6 years of age (86.11%), demonstrating that HAdV is an important pediatric pathogen. Previous studies drew identical conclusions that most children become infected by HAdV at an early age [18, 25, 26]. HAdVs can be easily transmitted through fomites contaminated with infectious body fluids. In our study, the HAdV detection rate (3.18%) was lowest among children under 1 year of age, and the reasons need to be further explored.

Many studies have reported that the most common HAdV species causing RTIs in children are B (B3, B7, B21), C (C1, C2, C5, C6) and E (E4) worldwide [27, 28]. HAdV 2, 3 and 7 are the most prevalent species and are associated with severe pneumonia in China [29, 30]. In the present study, a total of 66 samples were identified and phylogenetically analyzed based on the hexon gene sequence, which revealed six HAdV genotypes and showed that HAdV species B and C were the most prevalent, accounting for 62.12 and 37.88% of isolates, respectively. Similarly to what has been found in Asia by other authors, HAdV-B3 was the most common type (56.06%, 37/66) followed by HAdV-C2 (19.70%, 13/66) and C1 (10.61%, 7/66). HAdV-B3 has been identified as the causative pathogen for severe acute respiratory illness outbreaks in Korea, Brazil and Taiwan, and it was the main type of respiratory HAdV infections from 1981 to 2002. Moreover, HAdV-B3 was the pathogen causing epidemic respiratory disease outbreaks in Europe, America, and Oceania. Last, HAdV-B3 is known to cause a characteristic syndrome in older children and adults, as manifested by acute pharyngo-conjunctival fever, especially after contact with summer camps and swimming pools. HAdV- C1 and HAdV-C2 have been frequently reported to cause endemic, sporadic and epidemics cases [35, 36].

In the present study, the most common diagnosis (86.11%) in the HAdV-positive cases was pneumonia, with the common signs and symptoms of fever and cough, which is consistent with previous reports [16–18]. HAdV infection is often accompanied by other virus infections. Our study showed that PIV (12.50%, 9/72) was the major co-infecting pathogen identified followed by HRV (9.70%, 7/72). Although the viral load from children mono-infected with HAdV was slightly higher than those co-infected with HAdV and other respiratory viruses, no significant difference was seen between the two groups. The severity of HAdV infection varies according to age, socioeconomic status, environmental status and, above all, the immunological characteristics of the patient. Therefore, the etiological significance of co-infections with HAdV and other respiratory viruses and its association with disease severity require further study. It was reported HAdV can cause more severe illness in immunocompromised patients, so it would be very valuable to know about pre-existing conditions in HAdV-positive children. Except for 4 cases of asthma, there were no other comorbidities in our study. Taken together, our results provide a foundation for further clarification of the role played by HAdVs in RTIs and for defining the clinical and public health significance of HAdV infections.

Of 6381 specimens tested, 1967 (30.8%) were positive for HAdV (Table 2). Detection rates over the study period are almost similar in the first 3 years (2012, 2013 and 2014) while in 2015 there is a marked decrease in adenoviral infections. The mean age of infected patients was 8 years 7 months and median age was 3 years.

From the 1967 adenovirus positive cases, 1561 (79.4%) were found in co-infection with at least one respiratory virus. The most common were influenza viruses (53.1%; 1045/1967), rhinoviruses (30%; 591/1967), enteroviruses (18.5%; 364/1967) and RSV (13.5%; 266/1967).

Regarding the viral detection per age group, most of HAdV infected cases (62.2%; 1224/1967) were under 5 years patients, a statistically significant finding (p <0.05). However, the detection rates in the other groups including the elderly (above 50 years old) remain high. No significantly gender distribution of adenoviral infection was observed.

The comparison of symptoms prevalence between ILI patients with adenoviral infection and patients without adenoviral infection showed that cases of myalgia (P = 0.0014), cough (P = 0.0028), diarrhea (P < 0.001), rhinitis (P < 0.001) and headache (P = 0.01) are significantly higher in patients infected by adenoviruses (Table 3).

The Fig 1 shows the temporal distribution of HAdV positivity rate per month in Senegal from 2012 to 2015. We noted that HAdV was detected throughout the year at a high level with detection peaks of different amplitude. The highest peak, with 62% of detection rate, was recorded on December 2013. HAdV circulation pattern shows no seasonality even if results suggest a higher activity of these viruses during cold periods. It should be pointed out that the cold periods (between December and February) experience some instability in Senegal with possibilities of shifting.

Between January 2012 to December of 2015 a total of 6381 samples were collected from patients meeting case definition for ILI at the different sentinel sites, and analyzed: 1213 (19%) from 2012, 1519 (23.8%) from 2013, 1930 (30.2%) from 2014 and 1719 (26.9%) during 2015 (Table 1). Patient ages ranged from 1 month to 95 years. The mean age was 10 years 11 months and median age was 4 years. Approximately the male/female ratio was 0.99 (3163 [49.6%] males and 3185 [49.9%] females). For 33 (0.52%) patients the sex was not documented. More than half of patients (51.7%; 3297/6381) were children of ≤ 5 years old followed by 5–10 years age group with 11.5% (731/6381) and 25–50 years age group with 10.9% (696/6381). Patients above 50 years old represented only 3.3% (210/6381) of enrolled patients and for 8.2% (526/6381) ages were not reported.

Tables 4 and 5 summarize the most important patient’s demographic and clinical information found in association with viral infection (total positive, single and multiple). Viral infection increases in subjects living in a humid environment (p = 0.041), and with a primary care in the nursery (p = 0.040). Children breastfeed after birth were at high risk of being infected by a single virus than being infected by multiple viruses (p = 0.001). Multiple infections were found to increase the risk for developing respiratory irritation (p = 0.001).

The statistical comparisons between individual viral infection and patient’s information are summarized in Table 6. RSV A/B and PeV were the most associated pathogens with patient’s demographic situation and the manifestations of ARTIs: RSV A/B infection increases with the increasing number of siblings and occurs more in children with a primary care in the nursery (p<0.05). In addition, RSV A/B was found to increase the need for oxygen support (p = 0.043), the duration of hospitalization in ICU (p = 0.017), and the risk of nosocomial infection (p = 0.031). However, PeV was statistically associated with asthma and anemia (p<0.05). Interestingly, PeV and PIVs group were the single respiratory agents found to increase the risk of death (p = 0.032 and p = 0.013 respectively). RV was found to be statistically associated with a prolonged duration of oxygenation need after hospitalization (p = 0.042), BoV increases the risk for developing pharyngitis (p = 0.003), and EV was statistically related to nosocomial infections (p = 0.049). Infection due to CoVs group was found to increase the risk of bronchiolitis (p = 0.009) and laryngitis (p = 0.017). Vomiting was reported in 20.00% of patients, of which 1/3 were infected by AdV. In all of these cases, co-infections with at least 1 additional virus were found. In addition, AdV was found to be statistically associated with gastroenteritis (p = 0.004). MPV A/B was the single virus not found to be associated with patient’s demographic characteristics and the manifestation of ARTIs. As mentioned above, only 6/372 cases were found to be infected by InfVs. Thus, no statistical comparisons of InfVs and the manifestation of ARTIs were taken into consideration.

Fig 2 summarizes the distribution of viral infection rates within the four age groups. Viral infection was more frequent in G1group (76.88% of the 372 tested samples). RV, RSVA/B, CoVs group and MPVA/B were predominant within patients from G1 and G3 groups, while BoV, InfVs group, and EV were most frequently detected in patients belonging to G1 and G4 age groups. PIVs infection was most commonly described in patients aged from 7 to 12 months (G3 group). AdV infection was age-dependent (p<0.05), and was more frequent in children below 2 years old (results not shown). PeV was more frequent in children less than 4 months of age.

Our literature review suggests that community-acquired adenoviral pneumonia in immunocompetent adult civilians presents as a non-specific febrile respiratory illness that progresses rapidly to respiratory failure and often requires mechanical ventilation. The laboratory and radiological findings are typical of viral infection but are also non-specific. Novel respiratory virus real-time RT-PCR testing enabled us to rapidly detect adenovirus as the cause of severe community-acquired pneumonia in our patient.

A 44-year-old Caucasian woman was admitted to our emergency department with a three-day history of a febrile illness associated with sore throat, dry cough, myalgia and diarrhea. One day prior to admission she had developed a widespread, non-pruritic, erythematous rash. Her medical history consisted of hypertension, for which she was taking atenolol, and several episodes of gout, for which she was taking allopurinol.

Her physical examination revealed that she was obese, had a body temperature of 39.0°C, a pulse rate of 112beats/minute and blood pressure of 145/90 mmHg. Her respiratory rate was 20 breaths/minute with oxygen saturation of 94% on room air. Her chest auscultation was unremarkable. She had a widespread, erythematous maculopapular rash with scattered petechiae on both legs. Examination of the oropharynx revealed erythema but no exudate.

Initial laboratory tests showed a white cell count of 9.2 × 109/L, a neutrophil count of 7.9 × 109/L, a lymphocyte count of 0.69 × 109/L, a platelet count of 254 × 109/L, a C-reactive protein concentration of 169 mg/L, an alanine aminotransferase level of 22 IU/mL, a creatinine phosphokinase (CPK) level of 950 IU/mL and a creatinine concentration of 73 μmol/L. Her HIV test was negative. Her anti-nuclear antibodies, rheumatoid factor and anti-neutrophil cytoplasmic antibodies were negative, and her complement components C3 and C4 and immunoglobulin levels were within the normal range. Her initial chest radiograph was unremarkable. She was commenced on intravenous ceftriaxone for presumed meningococcal disease.

Twenty-four hours following admission her condition rapidly deteriorated with acute respiratory failure and hypotension requiring admission to the intensive care unit for mechanical ventilation and vasopressor support. A repeat chest radiograph showed widespread interstitial infiltrates bilaterally (Figure 1). Her antibiotics were changed to imipenem and doxycycline to treat presumed bacterial pneumonia, and oseltamivir was empirically added to treat a possible 2009 pandemic influenza A (H1N1) infection.

Bacterial cultures of her blood and sputum, Legionella antigen testing of her urine, and a polymerase chain reaction (PCR) assay of her blood for Neisseria meningitidis and Streptococcus pneumoniae were all negative. Her nasopharyngeal and tracheal samples were negative for influenza A and B (including H1N1), respiratory syncytial virus (RSV) types A and B and parainfluenza virus (PIV) types 1 through 4, but they were positive for adenovirus DNA on the basis of PCR assay (using the hexon gene as the target for amplification), with a cycle threshold value of 18. Subsequent sequencing analysis performed at the respiratory Virus Reference Laboratory, London, revealed the isolate to belong to serotype 4.

The patient made an uncomplicated recovery without any specific antiviral therapy and was extubated on the fifth day of her admission. Antibiotics were stopped after a total of five days, and she was discharged to home on the ninth day of her admission. Further tests for immunodeficiency were negative.

We performed a literature search of MEDLINE for cases of community-acquired adenovirus pneumonia in immunocompetent adults. We used the search terms "adenovirus," "pneumonia," "immunocompetent," "adult" and "civilian." We excluded cases that involved military recruits, nosocomial cases and those cases in which bacterial pathogens were also implicated.

We identified 19 articles published between 1975 and 2008 describing 21 patients that matched our search terms. The demographic, laboratory, radiological and clinical details of these cases and our own are shown in Table 1.

Of the 21 cases retrieved in our literature search, 57% of the patients were men, and overall the patients' median age was 40 years (age range, 18 to 60 years). Where recorded, the commonest ethnic origin of patients was Caucasian (40%). Significant co-morbidity was uncommon among patients, but obesity was frequently noted as an examination finding.

The median duration of illness prior to admission to the hospital was five days. The following presenting symptoms were noted: fever (90%), cough (81%), dyspnea (70%), myalgia (57%), sore throat (29%), abdominal pain (14%) and diarrhea (10%). Common examination findings on presentation included abnormalities in chest auscultation (90%), pyrexia (89%) and hypoxia (66%). The presence of pharyngitis, conjunctivitis or rash was noted infrequently (19%, 19% and 5% respectively).

The median white cell count on admission to the hospital was 7.7 × 109 (range, 3.9 × 109 to 28 × 109), although neutrophilia was relatively common (33%). Lymphopenia and thrombocytopenia were noted in 52% and 19% of patients, respectively. Other frequently noted laboratory abnormalities were mildly elevated transaminases and elevated levels of CPK.

The chest radiograph at presentation was abnormal in 90% of patients. The most common pattern of abnormality was bilateral interstitial infiltrates (57%), although lobar consolidation was also noted reasonably frequently (24%).

Intubation and mechanical ventilation were required in 67% of patients and occurred at a median of one and half days following admission. Overall 24% of patients died. The median length of stay in the hospital was 21 days. Two patients received antiviral therapy with cidofovir, one of whom died.

Where recorded, the most common adenovirus serotypes identified were serotype 7 (24%), serotype 3 (19%), serotype 21 (14%) and serotype 4 (10%). The diagnosis was made most frequently on the basis of lower respiratory tract samples (principally bronchoscopic alveolar lavage fluid and lung biopsy tissue), and viral culture was the most common method of adenovirus detection (76%). There were no cases identified in the literature where molecular methods were used to diagnose adenovirus pneumonia.

All the 21 patients who got HAdVs infections presented fever in between 38.2 to 40.0 degrees, seven (33.3%) of 21 patients had radiographic evidence of pneumonia, one patient (4.8%) had bronchitis, and others 13 patients (61.9%) had only upper respiratory tract infection symptoms such as cough and runny nose (Table 2). Among them, HAdV-55 infections (2 cases) and HAdV-7 infections (4 of 6 cases) seems led to patients of severe symptoms (pneumonia), while HAdV-3 and HAdV-4 infection caused minor symptoms (symptoms of upper respiratory tract infection or bronchitis), with only one HAdV-3 infection causing pneumonia. It is worth noting that the two patients infected with the undefined HAdV type appeared to have only mild symptoms such as fever and cough, and both patients affected by this recombinant virus are infants (below 1 years old), while patients infected with other HAdVs are all teenagers or adults.

Our study characterized the virome in fecal samples of pediatric HFMD patients during a 2012 widespread outbreak in Thailand. We used high-throughput next-generation sequencing to better understand the viruses present in HFMD patients who tested negative for enterovirus species A EV-71 and CV-A16/A6, predominant causes of HFMD and the main focus of many existing diagnostic assays [26, 27]. Analysis of the sequences either assembled into contigs or as singletons revealed a complexity of the virus population. Although previous examination of these samples did not initially detect enteroviruses using PCR-based assays, the present study utilizing deep sequencing enabled the detection of enterovirus sequences as well as other enteric virus at concentration as low as a few genome copies, providing a more systematic analysis of the prevalence of the viral genome.

The majority of viral reads identified here belonged to the family Picornaviridae, and were dominated by several members of the genus Enterovirus, namely HRV-C, CV-A21, and CV-A10. In addition to the commonly identified EV71 and CV-A16, other enteroviruses have been reported to cause HFMD outbreaks in different countries including CV-A10 and CV-A6 in Finland and France in 2010 and in China during 2008–2012. CV-A21 was least reported to cause HFMD with only 42 detections throughout the 36 years of surveillance in the United States. However, a study in China cited a high incidence rate of CV-A21 infection in adults with RTI. Another rarely detected virus found was EV68, originally isolated in the USA in 1962 from patients with RTI and has been detected sporadically thereafter. EV68 is unusual among EV in that it shares phenotypic properties of both enterovirus and rhinovirus. Reports suggested that this virus was associated with RTI including pneumonia and bronchiolitis with greater severity in infant and school-aged children. Between 2008 and 2010, an increasing number of EV68 clusters in cases of RTI have been reported worldwide [85–88]. Outbreaks have also been reported in the USA in 2014 when EV68 infection affected more than a thousand people, mostly young children and resulting in at least 12 deaths. The association of EV68 infection and diseases other than RTI remains uncertain. Studies have linked the virus with infection of the central nervous system including acute flaccid paralysis and fatal meningomyeloencephalitis. In addition, by using un-biased metagenomic analysis, our results not only expanded the number of identified types of enterovirus, both common and rare, but also revealed possible circulation of multiple enterovirus types during the outbreak.

Nearly completed genome of HRV species C was detected in the virome. Although the ability for HRV to replicate in the gastrointestinal tract is unknown, the detection of HRV in fecal samples has been reported [42, 43]. The amount of HRV RNA detected in feces could be as abundant as enteroviruses and feces-derived HRV can retain infectivity in cell culture [91, 92]. No role for HRV has been postulated for HFMD and the virus is typically present in respiratory secretion and associated with asthma exacerbation. Nucleic acids from HRV and other typical respiratory viruses such as RSV in feces may reflect inactivated particles in swallowed respiratory secretions or actual replication in the digestive tract possibly aided by adaptive mutations.

The second most abundant viral sequences identified belonged to astroviruses. The classic HAstV species consisting of eight serotypes has been associated with diarrhea particularly in immunodeficient patients. Astroviruses detected here belonged to recently described species MLB1 and MLB2. MLB1 was initially sequenced from the fecal samples of a child with diarrhea and serological survey revealed it to be a common childhood infection. A study of children in India failed to associate MLB1 with diarrhea. Meanwhile, MLB2 was also initially detected in fecal samples from Indian children with diarrhea and in the plasma of a child with undifferentiated fever, suggesting replication beyond the digestive tract. Other astroviruses in human and animal tissues have also indicated likely neurological involvement [99–101], therefore a wide range of diseases may be associated with astrovirus infections.

Viral metagenomic studies of human fecal samples have been reported [102–105] and had suggest evidence of frequent co-infections with known enteric viruses from the family Picornaviridae, Astroviridae, and Parvoviridae [106, 107]. Our study found enterovirus, cardiovirus, astrovirus, rotavirus, norovirus, adenovirus, bocavirus, and picobirnavirus in stools from pediatric HFMD patients. Several of these viruses are known to cause gastrointestinal diseases. Although co-infections with different viruses and correlates of disease severity are ongoing, it is conceivable that viral co-infections may aggravate clinical manifestation of an otherwise mild virus infection, which could result in compromised gut function and upregulated immune response leading to increased risk of morbidity. For example, during an HFMD outbreak in Sarawak, Malaysia in 1997, a subgroup of adenovirus and enterovirus were isolated from three fatal cases. It is therefore conceivable that co-infection could precipitate or aggravate HFMD symptoms. Future studies to examine viral profile in individual clinical samples are warranted to clarify the possible role of co-infection with disease severity.

Since a significant fraction of HFMD cases remains negative for the viral enterovirus pathogens and a direct or aggravating role for other viruses is conceivable, our study illustrates an overview of the virus community in stools from pediatric HFMD patients using unbiased sequencing approach. Our finding shows that such EV71, CV-A16/A6 PCR-negative children shed multiple types of picornaviruses and other enteric viruses in their feces including enterovirus, cardiovirus, astrovirus, rotavirus, norovirus, adenovirus, bocavirus, and picobirnavirus. Such overview information may help clinicians figure out the contribution of different types of picornaviruses as well as other pathogens to HFMD, with potential public health implications on the disease control.

The analysis of pooled samples and the deep sequencing method used presented limitations in the interpretation of the results. First, it was not possible to equate viral read numbers with the number of children infected. Second, comparison of the viral loads of viruses with different genome types (e.g. ssRNA, dsRNA, dsDNA) was not possible since the relative efficiency of converting their genomes into next-generation sequencing-compatible DNA may vary. Although stool samples used in this study were convenient samples collected for our previous study, inclusion of other clinical specimens such as throat swab, vesicular fluid, and skin lesions would be ideal. Stool samples may harbor unknown inhibitors of PCR and present nucleic acids of non-viral origin, thus complicating analysis. Nevertheless, our findings highlight the potential advantage of next generation sequencing to detect viruses from clinical specimens, which may be present below the limit of detection by conventional PCR assay. Assessing the role, if any, of these viruses in HFMD will require the study of larger populations, including epidemiologically matched healthy controls, and the analysis of individual, rather than pooled, clinical samples.

During 2011–2013, 4617 cases of respiratory infections were identified by the surveillance project; 45 were identified as adenovirus-positive. After virus isolation, 21 adenovirus isolates were obtained from sporadic and outbreak cases of respiratory infection: 2 cases from 2011, 2 cases from 2012, and 17 cases from 2013 (Table 2). Penton base, hexon, and fiber genes from virus isolates were successfully amplified. By the phylogenetic analysis of 21 Beijing HAdV strains and 22 HAdV type strains representing 7 species, three species (species HAdV-B, HAdV-C, and HAdV-E) were identified, including HAdV-3 (species HAdV-B; 10 strains: 1 in 2011, 1 in 2012, and 8 in 2013), HAdV-4 (species HAdV-E; 1 strain in 2013), HAdV-7 (species HAdV-B; 6 strains: 1 in 2011 and 5 in 2013), HAdV-55 (species HAdV-B; 2 strains in 2013), and a undefined HAdV type with HAdV-1 (accession number: AF534906) like penton base gene and HAdV-2 (accession number: AC_000007) like hexon gene and fiber gene (species HAdV-C; 2 strains: 1 in 2012 and 1 in 2013) were identified (Fig. 1).

Based on the nucleotide alignments of their penton base, hexon, and fiber gene sequences, the HAdV-3, HAdV-4, HAdV-7, and HAdV-55 strains, isolated in Beijing during 2011–2013 were more conserved, with nearly 100% nucleotide identity to the corresponding type of HAdV. In penton base, hexon, and fiber genes, the identities between HAdV-7 strains from 2011 and 2013 were about 99.9%-100% with 0–3 nt substitutions, and HAdV-3 strains from 2011 to 2013 also had the highest sequence similarity (99.8–100%, 0–3 nt substitutions). While strain BJ04 (isolated in 2012) and BJ09 (isolated in 2013) that belong to the undefined HAdV type showed high identities in penton base gene (99.7%, 5 nt substitutions) and fiber gene (99.9%, 2 nt substitutions), but shared only 98.9% nucleotide sequence identity with 33 nt substitutions in hexon gene, indicating slight variation. According to the chronological distribution of the viruses, 9 representative HAdV strains [HAdV-3 (3), HAdV-4 (1), HAdV-7 (2), HAdV-55 (1), and the undefined HAdV type (2)] were selected to deposit in the GenBank nucleotide sequence database (penton base: KP270906- KP270914; hexon: KM458622- KM458630; fiber: KP270915- KP270923), and also for further analysis (Table 2).

Adenoviruses, first isolated in the 1950s from explanted adenoid tissue, are double-stranded nonenveloped DNA viruses that naturally infect many vertebrates, including humans and nonhuman primates. The human adenoviruses in the Mastadenovirus genus, comprised of all mammalian adenoviruses, are classified into 7 species A-G, and at least 51 different serotypes (and 5 proposed types, HAdV-52 to HAdV-56) have been described to date,. Adenoviruses are the cause of an estimated 5–10% of febrile illnesses in children worldwide. Some serotypes, such as human adenovirus type 14 (HAdV-14), have been associated with severe and potentially fatal outbreaks of pneumonia in residential facilities and military bases. Adenoviruses have also been associated with other clinical syndromes including conjunctivitis, hepatitis, and diarrhea. In nonhuman primates, most epidemiologic studies of adenoviruses have focused on their identification in fecal samples from asymptomatic animals,,. Overt respiratory disease associated with simian adenoviruses has also been observed. Although adenoviruses are significant pathogens, genetically modified strains are being actively explored as potential vectors for vaccines and gene therapy.

Infection by adenoviruses has generally been thought to be species-specific. Human adenoviruses do not usually replicate in monkey cells in the absence of helper viruses, and do not productively infect rodents (and vice versa). Studies of sera from animal handlers and zoo workers exposed to chimpanzees in captivity fail to detect antibodies to chimpanzee adenoviruses,. However, recent serological surveys have found antibodies to New World and Old World monkey adenoviruses in donor human sera from regions where the monkeys are endemic,. In addition, phylogenetic analyses of adenoviruses from greater apes reveal that they fall precisely into “human” adenoviral species B, C, and E. The high degree of sequence relatedness within members of each species suggests that at least some adenoviral strains may be capable of infecting both nonhuman primates and humans.

Beginning in May of 2009, a deadly outbreak of fulminant pneumonia and hepatitis occurred in a closed colony of New World titi monkeys of the Callicebus genus at the California National Primate Research Center (CNPRC). Routine microbiological testing for an infectious etiology was negative. We previously developed the Virochip (University of California, San Francisco) as a broad-spectrum surveillance assay for identifying viral causes of unknown acute and chronic illnesses,,,,,,. The Virochip, a pan-viral microarray containing ∼19,000 probes derived from all viral species in GenBank (n∼2500),, has been previously successful in detection of novel outbreak viruses such as the SARS coronavirus, and the 2009 pandemic H1N1 influenza virus. Here we apply the Virochip to identify a novel and highly divergent adenovirus as the cause of the titi monkey outbreak. In addition, we present clinical and serological evidence that this virus may have infected a researcher at the CNPRC and a family member, thus demonstrating for the first time the potential for cross-species infection by adenoviruses.

Respiratory problems are common in pig herds worldwide and can be associated with significant production losses. They often have a multifactorial background and can be associated with a number of different factors such as environment, management, production system, animal genetics, etc., in addition to different pathogens. Major viral pathogens associated with respiratory disease are PRRSV, PRV, and swine AIV, which are all known to induce respiratory disease and lesions. Other viruses such as paramyxovirus (PMV), porcine cytomegalovirus (PCMV), PCV2, PRCV, and TTSuV are considered minor pathogens that could play a role through co-infection or in combination with other outer factors. In this study, none of the viruses considered major pathogens were detected, which was partly expected, as Sweden has been declared free from PRRSV and PRV. Viruses of minor relevance (TTSuV1 and PCV2) were identified, but only in one pig (R8). In contrast, five SPF pigs were positive for TTSuV1, one of which was also co-infected with TTSuV2.

In general, there was no major difference regarding viruses detected in the two different groups, but the variation was at the individual level. The viruses that differed between the two groups were porcine lymphotrophic herpes virus 1, adeno-associated virus, unclassified circovirus, and porcine sapelovirus A and sapovirus, which were only present in conventionally reared pigs, while TTSuV2 only was detected in one SPF pig. The fact that many of the viruses were found in both groups was not surprising, as many studies have shown a high co-infection rate of several viruses in both healthy pigs and in pigs with different disease complexes. Many respiratory viruses are also known to be ubiquitous in pig populations. It is possible that viral load could play a role in the development of clinical signs, but also that co-infections could have a synergistic effect. For example, enhanced respiratory disease development has been seen in co-infection situations with PRRSV and viruses such as PRCV, swine AIV, and PCV2. The exact mechanisms behind this are not always clear, but it is known that suppression of the immune system and alteration of cytokine responses can be important. Some viruses can also affect macrophage function. Considering that different viruses affect the host in different ways, the order that viruses infect their hosts could be important for particular outcomes, hence making them very complicated to study. Furthermore, bacteria are also known to induce respiratory disease, either alone or through viral-bacterial/bacterial-bacterial interactions. In addition, migration of parasites through the lungs may also aggravate signs of respiratory diseases.

Looking at the specific viruses, it was clear that viruses of certain families were well represented in most of the pigs investigated. For example, adenoviruses were present in all pigs, and 16 out of 18 pigs were positive for picornaviruses. Porcine adenovirus is ubiquitous throughout the world; although the virus has been isolated in connection to disease investigations, it is not considered a major pathogen but rather is believed to often result in subclinical infection. There are three species of porcine adenovirus recognised: porcine mastadenovirus A (porcine adenovirus 1–3), porcine mastadenovirus B (porcine adenovirus 4), and porcine mastadenovirus C (porcine adenovirus 5). Of these, porcine adenovirus 3 and 4 were identified in the different samples from this study. A total of 94% of the pigs were positive for porcine adenovirus 3 and 63% for porcine adenovirus 4. No sequence reads were classified as porcine adenovirus 5. There is a lack of complete genomes of adenoviruses available, making the classification of the reads in this kind of dataset difficult. It is likely, for example, that many of the reads referred to as “other mastadenovirus” in Table 1 could be porcine adenovirus 4 but have been classified as “other mastadenovirus” because only the fibre, pVIII, E1B 55K, and DNA repeat region have been previously sequenced. The dataset may also contain more divergent porcine adenovirus/es that are not classified in any of the present genera.

Different parvoviruses are also known to be present, sometimes at high prevalence, in pigs worldwide. We have previously, using metagenomics, discovered porcine bocavirus 1 in Swedish pigs; PBoV3 has also been shown to be present in Sweden. The pathogenicity of these viruses is not well known, and they have been detected in both healthy pigs and in pigs suffering from both respiratory and enteric diseases. In our study, there was no major difference in the detection rate of these viruses between the two groups. In four of the samples, an additional parvovirus, porcine parvovirus 7, was identified. This virus was first detected in 2016 from a rectal swab collected from an adult pig in the US and showed a very low identity to any known parvovirus. The virus in that study was detected in different sample types and at a detection rate of 8.6%. Since that first discovery, this virus has also been identified in China. The non-structural protein of the PPV7 characterised in Sweden showed a high degree of similarity (93.1–94.6%) to the US and China isolates, while the capsid was more divergent, with a nucleotide identity of 84.7–86.8%. The potential role of this virus in connection to disease has yet to be determined.

Picornaviruses were also detected to a high degree in the investigated pigs, and altogether four different picornaviruses were identified: pasivirus A, posavirus 1, teschovirus, and porcine sapelovirus A. Picornaviruses can cause subclinical infection, as well as diseases ranging from those with mild symptoms such as fever to more severe diseases. In two of the conventionally reared pigs, a high number of teschoviral reads were identified, enabling the assembly of two complete genomes. Porcine teschovirus is endemic in pigs worldwide, and there are 13 known serotypes. Often the infection is subclinical, but PTV-1 is associated with encephalomyelitis (Teschen disease). PTV2, PTV3, and PTV5 have been associated with a milder version called Talfan disease. The viruses identified in these two pigs showed were most similar to PTV-10, which has not been associated with disease, and whether they belong to the serotype 10 remains to be determined.

In some instances, the number of reads that matched to a specific virus was very low, sometimes less than 10 reads (marked with a lighter green in Table 1). It should be noted that although these low read numbers could be due to low abundance viruses or amplification bias, other reasons such as contamination within the sequencing run cannot be ruled out.

In conclusion, we observed a variable co-infection rate in the individual pigs in the two studied groups. The difference was seen on an individual level rather than on a group level. Thus, no specific virus could explain the respiratory disease of these pigs, but the results obtained provide important information on the viruses circulating in pig populations.

Here we present evidence supporting infection of common marmosets with TMAdV, a novel adenovirus previously associated with acute respiratory illness in humans and rapidly fatal hepatitis and pneumonia in titi monkeys. Although we were unable to re-isolate the virus from blood or tissues from TMAdV-infected marmosets, nasal cultures were positive for TMAdV by real-time qRT-PCR for up to 15 days post-inoculation, and TMAdV inoculation produced both clinically apparent disease and antibody responses in inoculated animals. Thus, River’s modifications to Koch’s postulates, which recognize the additional serological evidence of antibody production in response to a viral infection, have been fulfilled,. Experimental infection by nasal inoculation at a physiologic dose of 104 TCID50 resulted in an acute, self-resolving “flu-like” illness starting around days 5 to 10 post-inoculation and was characterized by low-grade fever, reduced activity, decreased stool production, and anorexia; repeated episodes of sneezing were observed in 1 animal. This was accompanied by an increase in virus-specific neutralizing antibody titers in serum in all 3 inoculated animals, confirming an acute, productive yet self-limited infection by TMAdV in marmosets. The sole animal with pre-existing neutralizing antibody to TMAdV was a surprising finding given that we did not anticipate that infection by TMAdV or a serologically cross-reactive New World monkey adenovirus was prevalent among marmoset populations. Further investigation into this possibility is ongoing. The general lack of significant histologic lesions consistent with active adenovirus infection in inoculated animals (Table 2) may be a function of the delay between appearance of clinical signs and necropsy. Taken together, these results demonstrate that TMAdV is infectious and causes disease in marmosets, and clearly documents the ability of adenoviruses to cross species barriers in closely related hosts.

In vivo infection of marmosets with a physiologic dose of TMAdV was unable to replicate the rapidly fatal pneumonia and hepatitis syndrome seen in some of the titi monkeys infected with the virus during the pneumonia outbreak at the CNPRC (Table 3), and necropsy tissues including lung, intestine, liver, were negative for TMAdV, albeit after complete resolution of clinical signs. Although the mild bronchitis seen in one TMAdV-infected marmoset (CJ29019) is consistent with acute respiratory infection, the enteritis and/or colitis seen in all 3 inoculated marmosets is most likely a manifestation of mild marmoset wasting syndrome, which is extremely common even in healthy marmoset populations, especially given the absence of intranuclear inclusion bodies and negative immunofluorescence results (Fig. 4). The difference in clinical outcome from TMAdV infection may also be related to anatomical differences between titi monkeys and common marmosets. In titi monkeys, enlargement of the larynx and the presence of laryngeal air sacs have been described, which may have predisposed the animals to aspiration pneumonia secondary to laryngeal swelling from viral infection. It is possible that evidence of more severe and invasive infection would have been observed by increasing the infectious dose or numbers of experimentally infected animals, or by serial sacrifice experiments. However, naturally acquired infections by adenoviruses are typically mild and fatal outcomes are rare in immunocompetent individuals. Notably, clinical signs in more than one-third of titi monkeys documented to be infected by TMAdV during the previously reported pneumonia outbreak by antibody testing were also mild or absent.

Similar to the 3 inoculated marmosets in the present study, infection by TMAdV in a human researcher at the CNPRC and household member was associated with self-limited “flu-like” respiratory illness. The observed peak levels of neutralizing antibodies (1∶16 to 1∶32) in response to TMAdV infection in marmosets from the current study, as well as in rhesus monkeys, titi monkeys, and humans with mild/absent clinical signs or symptoms during the CNPRC outbreak, were significantly decreased relative to those in titi monkeys with fulminant pneumonia (up to >1∶512). Levels of neutralizing antibody titers may thus correlate with disease severity, as seen for other respiratory diseases such as SARS, and this association may be related to the phenomenon of immune hyperactivity and “cytokine storm” in critically ill individuals.

Necropsy tissues tested negative for TMAdV by both PCR and immunofluorescence, and TMAdV was also absent in blood throughout the course of infection. The absence of significant viremia in TMAdV-infected marmosets is not surprising given that TMAdV viremia was rarely found even in titi monkeys with fatal pneumonia and hepatitis (Table 3). These results indicate that the respiratory tract, and not blood, may be the primary site of infection and replication by TMAdV. Indeed, in our current study, we were able to detect TMAdV in marmosets up to 2 weeks after post-inoculation in nasal swabs but not in blood.

Another potential explanation for the differences in TMAdV infection between marmosets or humans (acute, self-respiratory illness) and some titi monkeys (fulminant pneumonia) may have been attenuation of the virulence of the serially passaged inoculated strain by the lone 388P→R amino acid change in the adenoviral E1B-55K protein (Fig. 1; Table S1). Previously, we noted that adaptation of TMAdV to cell culture required 6 passages for robust growth in Old World monkey (rhesus and African green monkey) and human A549 cells. The present study has now uncovered the molecular basis for that adaptation. We hypothesize that this single 388P→R coding alteration during passaging may have promoted growth and adaptation of TMAdV in culture, yet may have attenuated the infectivity and/or virulence of the virus in vivo. The clinical significance, if any, of this alteration is unclear; the original proline residue at that position can be found in simian, bovine, porcine, and tree shrew, but not human adenoviruses (data not shown), but the 388P→R change or an arginine at that position has not been noted in any sequenced adenovirus to date. Experiments to investigate this role of this amino acid change in infectivity are now underway.

The different manifestations of TMAdV-associated disease between naturally-infected titi monkeys and experimentally-infected marmosets are also not surprising given that they are members of two different families of nonhuman primates. For many viral infections, such as monkey herpesvirus B infection in humans and macaques, the spectrum and severity of the disease vary significantly between different, albeit related, hosts. In this study, productive infection of marmosets by TMAdV is documented by the clinical signs of illness beginning at days 5-10, specific neutralizing antibody responses, and detection of the virus in nasal swabs up to 2 weeks post-inoculation. These results along with additional data from recent literature,,, now firmly establish that infection by adenoviruses can indeed cross taxonomic barriers within different nonhuman primate and human hosts. Additional research is needed to develop the common marmoset as a nonhuman primate model for adenovirus disease, especially from viral strains such as TMAdV with the potential to cause both fulminant illness and cross-species infection.

The genus Cosavirus consists of five species (Cosavirus A, B, and D to F), which have been associated with gastroenteritis in children (39). Six near-complete human cosavirus (HCoSV) genomes were identified: 1 from children less than 3 years old (HP49), 3 from those between 3 and <20 years old (HP6A and HP6B, HP57), and 2 from pools of individuals between 20 and <60 years old (HP44, HP24). Some of these pools had direct or indirect contact with bats (HP6, HP24, and HP44), while others had no contact with bats (HP49 and HP57). Phylogenetic analysis (Fig. 4C) showed that cosaviruses from HP6B, HP49, and HP57 formed a clade with two other strains from Australia and Nigeria (HCoSV/E1/AUS and HCoSV/NG385/NGA) in species HCoSV E. Meanwhile the strains in HP6A, HP24, and HP44 clustered with HCoSV in species A, D, and B, respectively. Therefore, it seems that humans in Cameroon host a diverse range of cosaviruses.

In hindsight, only one individual at the CNPRC reported becoming ill during the titi monkey outbreak, the researcher in closest, daily contact with the animals. Symptoms began near the onset of the outbreak, although whether they began prior to or after identification of the index case is unclear. The researcher, with a past medical history of multiple sclerosis, initially developed symptoms of a viral upper respiratory infection (URI), including fever, chills, headache, and sore throat, followed by a dry cough and “burning sensation in the lungs” that was exacerbated by a deep breath or coughing. The researcher endorsed a history of recurrent upper respiratory infections, and did not regard the illness as related to the titi monkey outbreak. Although symptoms persisted for 4 weeks, at no time did the researcher seek medical care, and no antibiotics were taken during the illness.

We carried out contact tracing to identify family members and other individuals in close contact with the researcher. Interestingly, two family members in the household also developed flu-like symptoms about 1–2 weeks after the researcher initially became sick. Their symptoms – fever, cough and muscle aches – appeared milder than those of the researcher and completely resolved within 2 weeks. Neither individual sought medical care for these symptoms, and notably, neither had ever visited the CNPRC.

The genus Parechovirus is comprised of two species, Parechovirus A (human parechovirus [HPeV]) and Parechovirus B (Ljungan virus, isolated from bank voles) (33). HPeV is subdivided into 19 types (HPeV1 to -19). HPeV is associated with mild gastrointestinal or respiratory illness; however, severe disease conditions, such as meningitis/encephalitis, acute flaccid paralysis, and neonatal sepsis, may occur (34–36). Here, three (nearly) complete HPeVs were identified in pools HP2, HP46, and HP48 with sequence lengths of 7,142 bp, 7,202 bp, and 7,219 bp, respectively, collected from children less than 3 years old (age group A). In terms of bat contact status, they were in pools of those either in indirect contact with bats (HP2 and HP48) or without contact (HP46). They were all distantly related to each other, with HPeV-CMRHP46 and HPeV-CMRHP48 having the highest identity (76% and 86% nt and aa identity, respectively). Phylogenetically, HPeVs in HP46 and in HP48 fell into a clade of type 1 HPeVs (Fig. 4B). The HPeV in HP46 clustered together with HPeV1/Harris strain with 76% nt identity, while CMRHP48 clustered closely with Japanese and Norwegian strains A1086-99 and NO-3694 (84 to 90% nt identity). Furthermore, HPeV-CMRHP2 clustered distantly with type 16 HPeVs from China and Bangladesh with only 70 to 71% nt identity. Considering the 75% identity demarcation for HPeV types (37, 38), this strain potentially represents a novel type.

Enterovirus is a genus in the family Picornaviridae, consisting of four human enterovirus species. Enteroviruses can cause many illnesses, including paralysis, meningitis, and cardiomyopathy, although most infections are asymptomatic or cause less severe conditions, such as colds and fever. A number of reports have described enterovirus infections linked to swimming pools.

The first enterovirus swimming pool-related outbreak occurred in 1987, at a municipal pool in Colorado, USA. Twenty-six children presented with fever along with at least one additional symptom such as malaise, headache, stomachache, nausea, or diarrhea. It was found that the pool chlorination system was operating improperly, with chlorine levels close to zero. Stool specimens collected from the children affected were tested for common enteric bacterial pathogens (Salmonella, Shigella, Aeromonas, and Campylobacter), but not for viruses. Enterovirus was suggested as a likely etiological agent based on clinical manifestations, course of disease, incubation time, and the exclusion of likely bacterial pathogens.

An enterovirus outbreak occurred in Ireland in 1992, with 46 cases experiencing vomiting, diarrhea, and headache after attending an outdoor swimming pool in a small seaside village. One subject had vomited into the pool, and echovirus 30 was isolated from this case and from six other cases. Chlorine levels were found to comply with health standards, but were inadequate to contain the risk of infection from vomitus.

Another echovirus 30 outbreak occurred in Rome, Italy, in late 1997. Children from two schools showed clinical manifestations after swimming in a pool. Twenty children had meningitis-like symptoms (fever, headache, and vomiting), and six of them were hospitalized. Other 48 children had respiratory symptoms consistent with enterovirus infection. Echovirus 30 was isolated from the cerebrospinal fluid and stools of the hospitalized children. Based on the epidemiological characteristics, it was hypothesized that person-to-person transmission occurred both at the swimming pool and in a number of classrooms. Data on chlorination at the time of the outbreak were not available. Virological analysis of pool water was performed one month after the outbreak, but yielded no positive results.

In South Africa, an outbreak involving 90 children occurred following a summer camp in 2001. Camp activities included swimming and other aquatic sports. Symptoms included mainly headaches, sore eyes, and/or abdominal discomfort, with one case of vomiting. Four children were hospitalized for meningitis. Echovirus 3 was detected in cerebrospinal fluid and stool samples from symptomatic and asymptomatic children. The presence of viruses in the pool was not investigated. Water contamination was confirmed through a total coliform count.

In Germany, 215 cases of aseptic meningitis were recorded from July to October 2001. Swimming in a public, nature-like pond was identified as a risk factor for disease. Up to 1500 people visited the pond each day during the summer holidays. Echovirus 3 and 30 were detected in cerebrospinal fluid samples taken from some of the patients. An echovirus 30 sequence obtained from one water sample collected from the pond showed a high level of genetic similarity (99% nucleotide homology) with sequences obtained from patient isolates.

In August 2003, an outbreak of meningitis occurred among campers staying at a campground in Connecticut, USA. A total of 12 cases of aseptic meningitis, four hospitalized patients and 24 cases of enterovirus-like illness with symptoms such as headache, neck stiffness, photophobia, sore throat, chills, or exanthema were identified. Echovirus serotype 9 was detected in cerebrospinal fluid samples from three of the patients. The spread of the virus was associated with swimming in a crowded pool, which had low chlorine levels. As a result, the pool water was intermittently contaminated with enterovirus.

In this study, viral surveillance of the sewage collected from 10 wastewater treatment plants throughout Taiwan was performed from July 2012 to December 2013. During this 18-month survey period, 300 raw sewage specimens were examined to detect the presence of viruses. The results showed that coxsackievirus type B, echoviruses, adenoviruses, and mammalian orthoreoviruses were isolated from the sewage specimens with a positive rate of 54.3%, but no poliovirus was found. Among these, enteroviruses (34.3%) and MRV (35.1%) predominated, followed by adenovirus (30.6%). Based on data provided by the WHO, at least 30% of concentrated sewage from grab samples can be expected to test positive for NPEV. Therefore, the high rate of non-polio enteroviruses in the environment can be considered proof that the environmental samples were processed and analyzed appropriately to preserve virus infectivity. This study was undertaken to supplement poliovirus surveillance in Taiwan by monitoring the possible presence of wild-type (WPV) or vaccine-derived (cVDPV) poliovirus in wastewater with a view to obtaining further evidence supporting the maintenance of Taiwan's polio-free status.

The frequencies of detection of different enteroviruses differed by geographical area and year, but overall, coxsackievirus type B strains were isolated more often than echoviruses. These findings are similar to the results of some studies on sewage surveillance in other countries [42–45]. Among coxsackievirus B strains, CVB2, CVB3, and CVB4 were the most prevalent between July 2012 and 2013 in Taiwan; however, CVB1 and CVB6 were not isolated in this study. According to a surveillance report by Taiwan's Centers of Disease Control (Taiwan CDC), among the coxsackievirus B strains, CVB3 and CVB4 were more frequent in 2012, whereas CVB2 and CVB4 predominated in 2013. This phenomenon was in agreement with the results of this study that showed the majority subtype of coxsackievirus B in 2012 was CVB3, which shifted to CVB2 and CVB4 in 2013. The results indicated that the environmental virus strains reflect the viruses circulating in the population and highlight the potential risk of viruses spreading via wastewater. In addition, the rare isolation of coxsackievirus type A in our study might be related to the lower susceptibility of the cells used to isolate the viruses or the lower resistance of the viruses in the environment and the isolation process.

Besides the enteroviruses, adenoviruses and MRV were also identified by environmental surveillance in this study. Previous studies have reported adenoviruses and MRV in contaminated surface water and wastewater [46, 47]. Adenoviruses are non-enveloped, double strand DNA virus from the family Adenoviridae and are classified into species A to G with more than 57 identified genotypes. It has been shown that adenoviruses of species B (Ad 3, 7, 11& 14), species C (Ad 1, 2 & 5), and species E (Ad 4) are associated with acute respiratory disease [48–51]; adenoviruses of species F (Ad 40 & 41) are related to acute gastroenteritis in infants and children; species D (Ad 8, 19, 37, 54) is thought to cause epidemic keratoconjunctivitis; and species B (Ad 11, 21) has been linked to hemorrhagic cystitis [52–55]. According to data on clinical adenovirus isolation in Taiwan, Ad3, Ad7, and Ad4 were found mainly during outbreaks in southern Taiwan between 1999 and 2001, and Ad3 circulated in northern Taiwan between 2004 and 2005 [56, 57]. Recently, we reported that Ad3 was the dominant strain in southern Taiwan from 2002 to 2011, and a high incidence of co-infection with Ad2 was identified.

The unexpected identification of MRV by PanEV RT-PCR may be explained by the sequence similarity between the primers for enterovirus 5′-NTR and L1 gene of MRV, as MRV was formerly classified as ECHO 10. We found MRV was positive for PanEV antibody stain, but failed to be identified by CODEHOP PCR of enteroviruses. This may be attributed to the cross-reactivity of the PanEV Blend antibody toward reoviruses, as noted by the manufacturer in the instructions provided with the kit. According to our results, a high positive rate of MRV was found in our sewage specimens. MRV belongs to the Orthorovirus genus, Spinareovirinae subfamily, Reovirus family, and is also commonly termed reovirus. MRV are non-enveloped viruses that contain 10 segmented double-stranded RNA genomes, including three large (L1-3), three medium (M1-3), and four small (S1-4) segments. It can be classified into three major serotypes: Type 1 Lang (T1L), Type 2 Jones (T2J), and Type 3 Dearing (T3D), which commonly cause asymptomatic infections or mild respiratory tract illness and enteritis in infants and children [37, 59, 60]. Other studies have reported a seropositive rate of more than 70% in 4-year-old children [27, 61]. Recently, a few novel MRV viruses were found in humans, such as, novel Type 2 MRV (MRV2TOU05), which seems to be closely related to porcine and human strains first isolated from 2 children with acute necrotizing encephalopathy in France; the mother of one patient also had influenza-like symptoms, and specific antibodies against MRV2TOU05 were detected. Another novel Type 3 MRV was isolated from a child in the United States with meningitis. The virus also showed systemic spread and was found to produce lethal encephalitis in newborn mice after peroral inoculation. Besides the novel MRV-infected pediatric cases, another novel MRV (Kampar virus) was identified from a throat swab of a 54-year-old patient with high fever, acute respiratory disease, and vomiting. Based on epidemiological tracing, there is a high probability that Kampar virus originated from fruit bats and is capable of causing human to human transmission according to the results of serological studies. In previous studies, reoviruses were commonly found in environmental water sources, and human fecal contamination has been suggested as the source of the virus [64, 65]. Our PCR sequencing data of isolated MRV showed that mammalian orthoreoviruses Types 1, 2, and 3 were present in the environment. Sequence analysis also showed that MRV1 and MRV2 persistently circulated in Taiwan and most MRV isolated in Taiwan were closely related to the reference strains isolated from patients with severe acute respiratory syndrome or meningitis. These results suggest that the reoviruses isolated from sewage may have the potential to infect humans. However, no cases of MRV infection have been reported in Taiwan to date. Since the identification of MRV requires additional molecular analysis, it may be missed by routine viral identification. In addition, this study identified MRV from L20B cells, which are not normally used in routine settings. The etiologic agent remains unknown in many cases of encephalitis (32%-75%). MRV may be a potential risk factor with public health implications. Thus, L20B cell should be tested for routine viral isolation and PCR test is needed to identify MRV when MRV is suspected in human subjects.

In this report we provide an analysis of the environmental circulating viruses in Taiwan. Our results showed that Taiwan was poliovirus vaccine strain-free in the environment two years after the oral poliovirus vaccine was replaced by the inactivated poliovirus vaccine. Although our surveillance data were negative for poliovirus, long-term monitoring is still needed to allow prompt action should WPV ever be detected. In addition, we combined cell culture and RT-PCR to assay large volumes of sewage and identify the viruses, so that infectious viruses could be detected, thereby providing meaningful data that can be applied in public health risk assessments. This is the first study to report the prevalence of MRV in sewage in Taiwan. The observations made in this investigation highlight the potential risk of MRV infection in humans. This report also suggests that continuous periodic surveillance of environmental virus is necessary to prevent the outbreak of disease or reduce casualties. Finally, since MRV was frequently identified in our environmental specimens, it is imperative that human cases with suspected MPV infection be thoroughly evaluated.

Despite the frequent occurrence of Canine Adenovirus worldwide, in Turkey, the only notified cases of Canine Adenovirus (CAV) infection are those reported by Okuyan and Gür and Acar. The antibody prevalence of the infection is reported to vary between 30 and 82% worldwide. Gür and Acar reported that out of 94 dogs belonging to the Kangal, Turkish Greyhound, and Akbaş breeds, which were sampled in Konya and Eskişehir provinces, 82 (82.7%) were positive for CAV antibodies.

Although, in most cases, CAV-1 and CAV-2 infections are not difficult to discriminate clinically from each other, they have the same morphological features under the electron microscope and the same cytopathogenic effects on cell cultures. There have been reports that CAV-2 can also infect the intestinal tract, one of the major target organs for CAV-1 [2, 19]. Diagnosis of CAV infections is usually based on serological tests, virus isolation, and negative staining.

In this study, the blood samples which are inoculated into cells were examined by direct immunofluorescence test for virus isolation. Viruses were not isolated from blood samples by direct immunofluorescence test. The adenovirus replicates in the lymph tissue and then spreads into the bloodstream. The replication reaches peak levels in 3–6 days after infection. Viral load decreases rapidly with respect to antibody production and longer CAV-2 cannot be isolated after 9 days [9, 20]. In the present study, the reason for unavailability of virus isolation may be due to the time of sampling.

The CAV-2 is a highly contagious viral agent that is incriminated in canine respiratory tract disease, particularly in young dogs kept in a crowded environment, such as pet stores, boarding kennels, and veterinary hospitals. This classical syndrome is commonly referred to as kennel cough. Infection with CAV-2 is generally transient and seldom fatal, unless it is complicated with a secondary bacterial bronchopneumonia [21, 22].

As adenoviruses do not have a lipid envelope, they are very resistant to environmental conditions and maintain their viability for an extended period in the external environment. Animals that recover from adenoviral infection continue to shed the virus for an extended period of time, and it has been even reported that vaccinated animals also shed the virus. Therefore, the prevalence of CAV infection has been reported to be rather high in dog shelters that lack reliable vaccination records.

CAV-1, which causes infectious canine hepatitis (ICH), is eliminated from the body of infected animals by saliva, urine, and faeces and is transmitted to susceptible animals by direct contact with contaminated material. In young animals, CAV-1 causes serious clinical symptoms, including anorexia, ataxia, and paralysis, which may result in death; and compared with older animals, the clinical course of the infection is more severe in the young. In the present study, antibody prevalence was found to be higher in animals aged 2 years and above. In this study, out of the 60 animals below 2 years of age, 23 (38%) and out of the 128 animals aged 2 years and above, 80 (62.5%) were confirmed to be positive for CAV antibodies. In particular, puppies below the age of 1, even if equipped with passive immunity through maternal antibodies, are not able to be protected against CAV infection when exposed, and mortalities may occur. Clinical ICH infection is more severe in young canids, as compared with adults.

The prevalence of the disease being higher in older animals that survive could be attributed to the high mortality in the young. In this study, few animals below 1 year of age were able to be sampled. Of the very few that were sampled, only 1 (a 2-month-old puppy) was determined to be positive for CAV antibodies. It was considered that this puppy had been protected by maternal immunity in early life. The remaining animals either could not have been exposed to the virus during life or could have suffered from low antibody levels in early life. No information was able to be obtained on whether the other animals survived.

In a study conducted in jackals in California, Cypher et al. determined that the prevalence of CAV antibodies was higher in adult animals. Higher CAV antibody prevalence among older age classes may be a function of greater mortality among pups resulting in a lower proportion of seropositive survivors in younger age classes [25, 26].

In Turkey, dogs both in rural areas and in dog shelters are not able to be regularly vaccinated against CAV-1 and CAV-2. In the present study, dogs that were admitted to the Internal Medicine Clinic of Selcuk University, Faculty of Veterinary Medicine, with complaints including fever (above 40°C), coughing, nasal changes, mucopurulent conjunctivitis, listlessness, inappetence, weight loss, pain and sensitivity of the abdominal region, vomiting, and diarrhea were sampled. Of the 111 dogs, which were admitted to the clinic, 37 were owned animals tended to by their owners. Twelve owned animals which were declared to have been vaccinated according to anamnesis were found to be negative for CAV antibodies. No reliable information was able to be accessed for the vaccination status of the remaining animals. Similarly, no reliable information was available for the vaccination status of the 77 dogs, which were sampled at the dog shelters in Isparta and Burdur provinces. The blood parameters of the animals that were admitted to the Internal Medicine Clinic of Selcuk University, Faculty of Veterinary Medicine, revealed the presence of lymphocytosis in some of the animals (5.84–12.61 m/mm3, reference range for dogs 0.6–5.1 m/mm3) and leucopenia (2.2–4.6 m/mm3, reference range for dogs 6.0–17.0 m/mm3) and anemia (0.99–5.24 m/mm3, reference range for dogs 5.5–8.5 m/mm3) in some others. Based on these findings, viral infection was suspected and the animals were tested for CAV. Two of the dogs exhibited photophobia and corneal opacity. In these 2 animals, although the presence of CAV viral antigen was not detected, the presence of CAV antibodies was confirmed. It is considered that these 2 unvaccinated animals were exposed to the disease and managed to survive the infection. For, in general, 7–10 days after being exposed to CAV, the acute signs of infection are replaced by corneal edema, which presents with a blue and rather opalescent appearance of the cornea. This appearance, which is generally observed in the convalescence period, disappears spontaneously. In some cases of mild infection, no clinical symptom is observed other than corneal edema. Although, rarely, this clinical picture may also develop as a vaccination complication, this option was discarded as the 2 animals that presented with corneal edema were unvaccinated. This clinical picture, specifically referred to as “Hepatitis Blue Eye” is known to be caused by CAV-1. However, as early detection was not possible, the CAV antibodies were not able to be typed in the animals that presented with leukopenia and lymphopenia.

According to the vaccination schedule applied in Turkey, new-born puppies are vaccinated as from 2 months of age, 3 times at 21-day intervals with live multivalent vaccines (CAV-2, Distemper, Parvovirus, Parainfluenza, and Leptospira). Literature reports are available, which suggest that canine adenovirus antibodies produced against the two different CAV types provide cross-protection, owing to the antigenic similarity of CAV-1 and CAV-2.

In the present study, apart from the correlation between age and antibody prevalence, the correlation between sex and antibody prevalence was also investigated. Accordingly, it was determined that antibody prevalence was higher in females (41%) in comparison with males (36%). Due to the number of female animals sampled in this study being greater than the number of sampled males, it is considered that the difference observed in antibody prevalence for sex is not significant (P > 0.05). On the other hand, in previously conducted studies, the correlation of sex with antibody prevalence was neither not investigated nor found to be statistically insignificant. Therefore, a comparative assessment was not able to be made in this study.

Of the 188 animals sampled in the present study, the majority were unvaccinated dogs housed at shelters. In view of the animals that had been admitted to the internal medicine clinic of Selcuk University being vaccinated dogs, the antibody presence confirmed in these animals was considered as an indicator of the vaccination schedule having been properly applied in these animals. On the other hand, the antibody prevalence detected in the unvaccinated animals sampled at the dog shelters in Isparta and Burdur provinces was considered as an indicator of the presence of CAV infection in these dog shelters. This study clearly demonstrates the high prevalence of CAV infection in dogs, which live in groups and are not vaccinated on a regular basis. It is considered that regular vaccination would provide protection against the disease for a certain time period in dogs, and in particular in puppies, which live in groups under unfavorable conditions. Furthermore, as the transmission of the CAV occurs by environmental contamination (contact with infected faeces, urine, etc.), both the maintenance of hygiene conditions and the prevention of contact among dogs are of great importance in the control of the disease.

Adenoviruses are the enteric viruses most commonly associated with swimming pool-related outbreaks. Human adenoviruses (HAdVs) belong to the Adenoviridae family and are classified into seven species (A to G) and more than 90 types. HAdVs are of major public health importance and can result in a variety of clinical manifestations, including gastroenteritis, respiratory, ocular and urinary tract infections. Illnesses are common and ubiquitous with a worldwide distribution. HAdVs are highly stable in the environment and can survive for prolonged periods in water. Transmission in swimming pools can occur by ingestion, direct contact with contaminated water, or through the inhalation of aerosol.

A Brazilian study recently detected HAdVs in Acanthamoeba isolated from water samples collected from swimming pools. HAdVs were found in 62.5% (10/16) of amoebae with DNA copies up to 5.1 × 105 per milliliter, suggesting that Acanthamoeba may act as a reservoir and promote HAdV transmission through water.

The first swimming pool-related outbreak, published in 1953, described a 1951 outbreak in Greeley, Colorado, which thus came to be known as the “Greeley epidemic”. The outbreak, involving 206 cases, caused a combination of symptoms, such as acute conjunctivitis, pharyngitis, muscle pain, and fever. Between 25% and 50% of children swimming in the pool were affected. The transmission apparently occurred either by contact with contaminated objects, such as toys, or while swimming in a pool. The water was heavily chlorinated, with residual chlorine being close to 0.4 parts per million. No definite pathogen was identified at the time of publication. Serum samples from Greeley patients were later tested and showed a specific neutralizing antibody response to HAdV type 3.

In 1954, an epidemic of pharyngeal-conjunctival fever occurred in Washington, D.C., with symptoms similar to those of the Greeley epidemic. Over 300 cases were documented, with acute respiratory illness characterized by one or more of the following symptoms: fever, pharyngitis, and conjunctivitis. Cases occurred in all age groups, but predominantly in children. Adenovirus type 3 was isolated in 80 of 300 patients from eye, throat washings and stools. The disease occurred at different sites: in a children’s summer day camp, in an orphanage, and in two residential neighborhoods. The suspected, but never confirmed, source of the epidemic was a swimming pool, even if cases due to direct contact were also recorded in houses and hospitals. The pool, chlorinated by hand, showed a low level of residual chlorine. Bell and collaborators were the first to suggest the term pharyngoconjunctival fever for this disease.

In August 1955, 112 cases of pharyngoconjunctival fever occurred in Toronto, linked to an indoor swimming pool. Seventy-four of the cases were children who had swum in the pool, while the others had swum in pools elsewhere, or had had direct contact with a case at home. Only one case had no history of either swimming or direct contact. Most of the children had pharyngitis, fever, malaise, and muscle pain. Conjunctivitis was absent or minimal in children, but was the main cause of discomfort in adults.

Another outbreak of pharyngoconjunctival fever was documented in August and September 1959 in Saitama Prefecture, Japan, among students of a primary and a middle school. Epidemiological investigations suggested that the outbreak was mainly due to the contamination of a swimming pool used by the students of both schools. A total of 358 students were affected: 248 primary school students (attack rate, 20.6%) and 110 middle school students (attack rate, 19.2%). Laboratory findings suggested that the epidemic was due to HAdVs 3 and 7.

Foy and coworkers described an outbreak of pharyngoconjunctival fever (45 cases) in two swimming teams in Washington in 1966. Adenovirus type 3 was the etiological agent. Most of the infected children had fever, pharyngitis, conjunctivitis, and diarrhea. In adults, symptoms were milder, with a high incidence of conjunctivitis. The attack rate was 65% and 67% for the two teams, respectively. Children had swum in the early morning, when the chlorinator of the pool was switched off, to avoid eye irritation with chlorine. Within one week, 25 of the 36 exposed swimmers became ill. Children swimming in the afternoon, when chlorination was still working, did not get sick. The infection was shown to spread in families having index cases (20 infected contacts). Analyses of water were done approximately 14 days after the presumed exposure. For this reason, attempts to isolate the virus from the water failed.

An outbreak of acute conjunctivitis due to HAdV type 7 occurred in Kansas, USA in 1973: 44 cases and one hospitalization were documented. Eye symptoms predominated (red or pink eyes, swollen eyes), but a variety of other signs were also noted (mainly fever, headache, and nausea). A school swimming pool was identified as the source of infection. Chlorine concentration was low due to an equipment failure. The epidemic was easily controlled by raising the pool’s chlorine level. Unfortunately, tests for viruses and bacterial indicators were carried out after super-chlorination and consequently results were negative.

In 1977, two outbreaks associated with swimming pools occurred in Georgia, USA. The first, due to HAdV type 3, involved at least 105 cases, with patients showing different symptoms, including sore throat, fever, headache, anorexia and conjunctivitis. In this case, a private swimming pool was the source of infection. A temporary malfunction in the water filtration system of the pool associated with inadequate chlorine levels was recorded. Both waterborne and person-to-person transmission occurred. In the second outbreak, HAdV type 4 was recognized as the etiological agent of pharyngoconjunctival fever in 72 persons. An insufficient amount of chlorine was found in the water of the pool. To stop the spread of infection, the pool was closed during the summer and adequately chlorinated. Adenovirus was recovered from the water sampled from the pool.

In Oklahoma, USA, an outbreak of pharyngitis caused by HAdV type 7a was recorded in 1982 among 77 children attending a swimming pool. Symptoms included conjunctivitis, fever, sore throat, headache, and abdominal pain. Two cases were hospitalized with dehydration from persistent vomiting. A malfunction of the automatic pool chlorinator was identified as the cause of the outbreak. In fact, during the two weeks preceding the epidemic, its failure forced the pool operator to manually add chlorine to the pool.

Another outbreak was recorded in 1995, in Greece, where 80 athletes under 18 years of age presented with fever, conjunctivitis, sore throat, weakness, and abdominal pain, after swimming in a pool. Seven athletes were hospitalized. Virological analyses on clinical samples were not performed and the illness was attributed to HAdV on the basis of clinical symptoms alone. Water samples from both the pool and the distribution system were tested for HAdV, enterovirus, and hepatitis A virus by molecular methods. The water of the pool tested positive for HAdV and negative for the other viruses, demonstrating its role as the source of infection. Samples from the water system were negative for all viruses tested. Chlorine levels were found to be low, probably due to a malfunctioning of the pool chlorination system.

Five HAdV outbreaks associated with swimming were recorded in the 2000s.

In the year 2000, an outbreak of pharyngoconjunctival fever occurred in North Queensland, Australia, where, after a school camp, 34 children aged 4–12, got sick. In addition to primary cases acquired at the camp (N° = 25), nine other cases were acquired within the households. The school camp had a large saltwater swimming pool. Adenovirus 3 was isolated from eye and throat swabs. A PCR analysis of water samples for HAdV did not yield positive results. It was, however, demonstrated that the pool was not properly maintained, and that the level of residual chlorine was inadequate.

An outbreak of pharyngoconjunctival fever affecting 59 children under 15 was recorded in a municipality of Northern Spain in July 2008. Forty-three cases were recognized as primary cases, all of whom attended a municipal swimming pool. The remaining 15 children were secondary cases, which had been in close contact with a primary case. Adenovirus type 4 was detected in pharyngeal swabs. Electrical system failures causing the intermittent breakdown of the pool’s bromine dosing pumps and the slowing down of water circulation were assumed to have been the cause of the outbreak. Swimming was only allowed after the disinfection system was restored and appropriate concentrations of bromine were reached. Due to logistic problems, no water samples were taken from the swimming pool for virological analysis.

In 2011, children (4–9 years old) who had attended a swimming training center in Eastern China showed symptoms of pharyngoconjunctival fever. Adenovirus type 3 was recognized as the etiological agent. A total of 134 cases were confirmed from among 900 amateur swimmers, with an incidence of 14.9%. Fourteen hospital admissions were documented. Fever, tonsillitis, sore throat, headache, sneezing, cough, conjunctivitis, fatigue, and diarrhea occurred among the bathers. The low level of residual chlorine in the water, along with excessive crowding in the pool were suggested as having caused the epidemic.

In the same year, in a primary school in Taiwan, an outbreak of HAdV infection occurred among 373 students, with four hospitalizations. Most of the students attended a swimming course in two swimming facilities outside the school and presented with fever and symptoms of upper respiratory tract infection. Other symptoms included diarrhea, vomiting, skin eruptions and conjunctivitis. Throat swabs of affected students were tested for influenza virus, adenovirus, respiratory syncytial virus, coronavirus, metapneumovirus, parainfluenza types 1–4, and herpes simplex virus. Samples were found positive only for HAdV type 7. Water samples were not obtained from any of the facilities for virological analysis.

In 2013, an outbreak of pharyngoconjunctival fever involved 55 people (49 students and six staff) at a university in Beijing, China. Fifty patients (91%) attending the same swimming pool two weeks before the onset of symptoms were considered primary cases. The other five subjects (9%) who had not swum in the pool were defined as secondary cases (person-to-person transmission). Human AdV type 4 was identified from both eye and throat swabs of the patients and from concentrated swimming pool water samples. Gene sequences obtained from the water samples exhibited a 100% match with the sequences obtained from swab samples. Control measures included the emptying and closing of the pool, and the disinfection with a high dose of sodium hypochlorite (500 mg/L).

Adenoviruses (AdVs) are non-enveloped icosahedral double-stranded DNA viruses that infect a number of vertebrate hosts, including humans and nonhuman primates. The genus Mastadenovirus within the Adenoviridae family includes 7 human adenoviral species A-G (HAdV-A through HAdV-G) and 1 simian adenoviral species A (SAdV-A). In humans, infections by adenoviruses cause conjunctivitis, gastroenteritis, hepatitis, myocarditis, and acute respiratory illness, ranging from the “common cold” syndrome to fatal outbreaks of pneumonia,. However, existing animal models of adenovirus infection to date have been primarily confined to rodents,, and no nonhuman primate (NHP) model has been established to study adenoviruses that infect humans and/or NHPs.

We previously identified a novel adenovirus, titi monkey adenovirus (TMAdV) in association with a fatal outbreak of pneumonia and hepatitis in a closed colony of captive New World titi monkeys (Callicebus cupreus), with evidence for potential cross-species transmission to a human researcher and family member with concurrent acute respiratory symptoms. The origin and natural host reservoir for TMAdV remained unknown, although neutralizing antibodies to TMAdV were found in 23 titi monkeys demonstrating clinical signs, 14 titi monkeys exposed to animals demonstrating clinical signs, 1 rhesus macaque (Macaca mulatta), and the 2 humans. Studies by other groups have also revealed the widespread presence of closely related adenoviruses in both human and nonhuman primates,,, and large-scale serological surveys have detected antibodies to monkey adenoviruses in humans living in endemic regions,. In addition, human adenovirus species E and G each contain only one member isolated from humans,, with the remaining members all isolated from monkeys or apes. Collectively, these data have raised concerns regarding the potential of adenoviruses as sources for emerging zoonotic disease in humans.

To further investigate the pathogenicity of the novel adenovirus TMAdV, we sought to develop an in vivo animal model of infection and disease for TMAdV. While TMAdV was originally discovered in a closed colony of captive titi monkeys (Callicebus cupreus) at the California National Primate Research Center (CNPRC), we recognized that in vivo testing in this monkey species was contraindicated due to the significant devastation to the colony caused by the virus. Since TMAdV was able to be successfully propagated in the marmoset lymphocyte cell line B95a in vitro (Table 1), we instead elected to pursue in vivo testing of TMAdV infection in the common marmoset (Callithrix jacchus).

The common marmoset is a New World primate that is small, easily handled, and highly susceptible to infectious agents, and thus a suitable nonhuman primate model for infectious disease,. Marmosets have been successfully used to characterize a number of emerging viral diseases, including filovirus- and arenavirus-induced hemorrhagic fevers, encephalitis, and severe acute respiratory syndrome (SARS). Since titi monkeys (Family Pithecidae, subfamily Callicebiniae) and marmoset monkeys (Family Cebidae, subfamily Callitrichinae) are classified into separate families, the use of the marmoset for in vivo testing of TMAdV infection also afforded the opportunity to directly test the capacity of TMAdV, and, by extension, adenoviruses in general, to cross the species barrier and cause productive infection in a related yet taxonomically distinct secondary host.

In HFMD virome, 2 contigs of 5 reads (195 nt and 257 nt) and 1 singlet (158 nt) were identified as BK polyomavirus (BKV) with approximately 99% nucleotide similarity to the previous sequences. BKV is a non-enveloped virus with a circle dsDNA genome that belongs to the Papovaviridae family. BKV has been detected in a wide range of tissue types and can persist without causing symptoms. BKV can establish latent infection of the kidneys and causes neuropathy and nephropathy in immunosuppressed transplant patients [74, 75]. It has been suggested that the gastrointestinal tract is the latency site for BKV. In addition, BKV can cause colonic ulceration in kidney transplant patient under immunosuppressive drug. So far, route of transmission of BKV has not been clearly determined. However, accumulating evidence suggests that BKV can also be detected in the stool samples of hospitalized children and from gastroenteritis patients as well as healthy adults [78, 79].

Acute respiratory infections (ARI) are a major cause of morbidity and mortality in children under five, the elderly, and vulnerable patients. Upper respiratory tract infection incidence is estimated at 7–8 per year in children under 4, and at 2–4 in adults.

Most respiratory viruses belong to five different viral families (Para- and Ortho-myxoviridae, Picornaviridae, Coronaviridae, Adenoviridae), and include 14 viral species, defining what is called the “respiratory panel”. Some of them have a high potential of emergence and can cause pandemics. Although some viruses have been associated with particular diseases (respiratory syncytial virus and bronchiolitis, parainfluenza virus 3 and laryngitis, rhinovirus and common cold, influenza virus and flu syndrome), there is no evidence for a clinical specificity, and only the virological diagnosis provides an accurate identification of the ARI [4, 5].

Detection of respiratory viruses is of little interest in general practice, in that the infection does not present a risk of severity for the patient. However, virological confirmation of ARI is needed in severe clinical presentations, requiring hospitalization in intensive care units and occurring in vulnerable subjects [4, 6]. The goal of early virological diagnosis would be an optimization of patient care, which could lead to reduction in length of hospital stay, a saving of antibiotics, and complementary examinations.

Virological tests allow for the establishment of accurate diagnosis of infection, assessment of evolving risks (bacterial infection, acute respiratory distress syndrome), and the establishment of measures to limit its spread (isolation, wearing gloves and masks).

Pandemics of Severe Acute Respiratory Syndrome (SARS, 2002-2003) and influenza A-H1N1 (2009) lead to the development of molecular biological techniques applied to virological diagnosis, mainly based on PCR (polymerase chain reaction). Performances of molecular methods in respiratory virology are so significant that they have replaced conventional techniques (culture, detection of viral antigens) as a reference method [8–11].

Multiplex PCR techniques are particularly suited to medical diagnosis because they can detect multiple viral targets in the same time, avoiding the virologist a selection of viral targets to search. There are now many commercial kits for the detection of a range of 12 to 15 respiratory viruses and some intracellular bacteria [6, 7, 12, 13]. Molecular techniques (real-time PCR) also make it possible to achieve a semi quantification of the viral molecular material present in the sample, giving additional information about the respiratory viral load (interest in therapeutic monitoring and infection transmission risk). A normalized viral load can be obtained by adding a cell quantification step.

The primary site for replication of respiratory viruses is the ciliated airway epithelium. The sample must be taken as soon as possible after the onset of symptoms. This is usually a nasal swab or nasopharyngeal aspiration (especially realized in children under 2). These samples are easily accessible and especially adapted to upper ARI. If a rich cell collection appears to be an important prerequisite for the quality of respiratory viral diagnosis, there is currently no information on a possible cellularity threshold that would validate the result of the viral molecular detection.

The main objective of this work is the study of cellularity in 800 respiratory specimens previously characterized virologically. The results should help to define the concept of “cellular richness” and determine the factors that influence it.

The World Health Organization (WHO) Global Polio Eradication Initiative (GPEI) was established in 1988 and successfully prevented wild-type poliovirus (WPV) transmission in the Americas, the Western Pacific (WPR), and Europe (EUR) [1–3]. The Southeast Asia Region (SEAR), home to a quarter of the world's population, was also certified polio-free in March 2014. WHO certified Taiwan, along with the entire WPR, as polio-free in 2000 and Taiwan changed its immunization strategy from oral (OPV) to inactivated polio vaccine (IPV) in 2010. To date, WPV remains endemic in Afghanistan, Nigeria, and Pakistan. Numerous outbreaks in heretofore polio-free regions have been reported recently in China (2011), Somalia (2013), Ethiopia (2013), and Kenya (2013) caused by importation [5–7]. Besides WPV, cases of circulating vaccine-derived poliovirus (cVDPV) causing acute flaccid paralysis (AFP) have risen since 2000, and have been identified in eight countries in 2013 and in two countries in May 2014.

Normally, acute flaccid paralysis (AFP) surveillance is the gold standard for poliovirus surveillance in eradication initiatives; under certain circumstances, environment surveillance is also employed to monitor the circulation of poliovirus in populations in order to better understand its evolution and transmission [9–13]. For instance, although certified as polio-free in 2002, Israel isolated WPV in routine environmental sewage samples in early February 2013, and immediate steps were taken to implement national supplementary immunization with OPV to prevent its spread. Recently, the WHO included environmental poliovirus surveillance in a new strategic plan as part of its global eradication initiative to supplement AFP surveillance. In Taiwan, AFP surveillance has long been established for poliovirus surveillance of the population, but environmental surveillance is not routinely performed.

Besides poliovirus in populations, enteroviruses, adenoviruses, reoviruses, and noroviruses are often found in environmental raw sewage [16–19]. These groups of viruses can cause a broad range of asymptomatic to severe gasterointestinal or respiratory infections, or even more acute conditions such as meningitis and paralysis, thus constituting a considerable public health problem in the community. Among these fecal-oral viral pathogens, reovirus is usually the most abundant virus detected in environmental water [22, 23]. Mammalian orthoreovirus (MRV), which belongs to the family Reoviridae and the genus Orthoreovirus, are a common class of enteric viruses capable of infecting a broad range of mammalian species, including humans. Previous studies have indicated that reoviruses have a high endemic infection rate in humans and seroconversion was found in more than 70% of 4-year-old children. Although reovirus infection in humans usually induces mild respiratory or gastrointestinal symptoms, there are reports of human reovirus-associated neurological disease [26, 27]. Several studies also described the isolation of reovirus strains directly from cerebrospinal fluid (CSF) or neural tissues obtained from patients with meningitis or encephalitis [28–31]. In addition, immunocompromised, pediatric, and elderly populations may become susceptible to severe bacterial respiratory disease due to an initial reovirus infection [32, 33].

In response to the international threat of WPV importation and the changes to the national vaccination policy, we adopted the WHO guidelines for environmental surveillance of circulation in Taiwan. Two-phase Dextran 40/Polyethylene glycol (PEG) separation and cell culture were performed to monitor environmental viral circulation. We successfully isolated enteroviruses, adenoviruses, and mammalian orthoreoviruses, but no poliovirus was detected in sewage collected islandwide. Our results showed a high incidence of MRV, which may cause human disease, and thus further research is warranted.