It has been recommended that treatment or prevention of a viral disease may be a superior method for diminishing of complications from influenza.84,85 Since viral infections might lead to secondary bacterial infection, it is prudent to vaccinate patients with the influenza vaccine to diminish the risk of OM in children and pneumonia in adults.62

It has also been published that live attenuated influenza vaccine is effective in reducing the incidence of all-cause AOM86–88 and pneumonia89 compared to placebo in children. In addition, the intranasal influenza vaccine can reduce OM by 44%.90 Moreover, studies have shown that a combined influenza/pneumococcal vaccine is efficient in the prevention of OM in children and pneumonia.91,92 However, the credit of protection was awarded to the influenza vaccine since studies have shown that pneumococcal vaccine has no benefit in the reduction of AOM.93,94 In addition, the pneumococcal polysaccharide vaccine showed no efficacy in the prevention of pneumonia in adults.95

Treatment of viral infection is anticipated to prevent bacterial superinfections. Currently, the only respiratory virus that is pharmacologically treatable is the influenza viruses (Type A and B).62 Neuraminidase inhibitors can potentially diminish the morbidity related to influenza.96 Oseltamivir can reduce the incidence of AOM in preschool children,97 and the reduction rate can be up to 44%.98 A meta-analysis review showed that oral oseltamivir reduces the rate of hospitalization by 25% and morbidity by 75%.99 In addition, its use can reduce the use of antibiotics by up to 50%,100,101 The same concept of protection applies to vaccines that prevent against RSV infections.62 The vaccine available for RSV is palivizumab (MedImmune, Gaithersburg, MD, USA), a humanized monoclonal antibody that perceives the fusion protein of RSV. The other monoclonal antibody that is under clinical trials is motavizumab (MedImmune), which has a higher affinity for RSV fusion protein than palivizumab and can prevent against medically attended lower respiratory tract infection.102

The rate of concurrent serious bacterial infections with viral illness is appreciable. Similar emphasis must be given to the prevention and treatment of viral illnesses, especially in young children. Furthermore, health care providers should emphasize to parents on the importance of clinical follow-up of infants and young children diagnosed with VRTI. Moreover, the introduction of MxA in the diagnosis of viral illnesses in children is promising.

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Although very common in the US and other countries, there is no nationwide syndromic surveillance system to electronically monitor medical records in Japan. Because of the low prevalence of electronic medical records and a restrictive privacy policy, we perform prescription surveillance nationwide for syndromic surveillance by monitoring the number of prescriptions for certain types of drugs such as anti-influenza medications.

There are approximately 45,000 pharmacies that deliver almost half of the prescribed drugs nationwide and almost all record prescriptions electronically. The prescription surveillance system was developed by the Infectious Disease Surveillance Center of the National Institute of Infectious Diseases in collaboration with EM Systems Co. Ltd. (Osaka, Japan), a leading provider of prescription surveillance used by pharmacies through the Application Server Provider (ASP) system. The ASP system is very useful for syndromic surveillance because data transfer is unnecessary. Thus, it can dramatically decrease costs and maintain a high level of confidentially. Its widespread use started in April 2009, and approximately 6,300 (13%) Japanese pharmacies actively participated in the program as of October 2011.

The ASP system tracks prescription information, but patient symptoms and diagnoses are not recorded. Categories of syndromic surveillance include the type of prescribed drugs. Currently, the syndromic surveillance system monitors several types of drugs, including those for relief of fever and pain due to common colds, as well as antiviral agents, anti-influenza medications (except amantadine), and anti-varicella zoster virus (VZV) drugs. The surveillance of the last two is also classified by age: <15, 16–64, and >65 years. Data collection and analysis are automatically performed every night, and the results are available on the home page of a secure internet site early the next morning.

Monitoring the usage of anti-influenza and anti-VZV drugs is particularly useful for early detection of outbreaks of infection because these drugs are used only to treat specific viral infections.

Of the 172 that tested positive for respiratory agents, there were 24 (14.0%) cases with mixed infections of two or three viruses. No patient was infected with more than three viruses. Twenty-one cases were infected with two viruses, with the most frequent mixture being adenovirus and influenza A virus (n = 5). Other frequent viral mixtures were adenovirus and rhinovirus A (n =3) and influenza A virus and coronavirus OC43 (n =3) (Table 3). Adenovirus was present in 62.5% (n = 15) of mixed infections which was almost a half (46.9%) of all detections for this virus. Respiratory syncytial virus B was the only aetiology that was not detected in a mixed infection.

This study reported a high prevalence of respiratory viruses in children ≤ 5 years using a custom assay and an FTD assay. Good concordance was observed for all the viruses between both assays except for RSVA/B. However larger numbers of positive samples need to be tested for thorough evaluation of less prevalent viruses. The custom primer and probe mix was much more economical than the commercial FTD kit. Our study suggests that this custom multiplex real-time RT-PCR can be used for simultaneous and rapid detection of multiple viruses in resource limited settings. This will help prevent unnecessary use of antibiotics and permit timely initiation of supportive therapy/antiviral drugs if available.

Nucleic acids were extracted from all samples using Microlab Nimbus IVD (Seegene Inc.), and RNAs were used for cDNA synthesis using cDNA Synthesis Premix (Seegene Inc.). The samples were tested by using Anyplex II RV16 detection kit (Seegene Inc.) according to the manufacturer's instructions. The assay was used to detect Flu-A, Flu-B, RSV A, RSVB, AdV, HMPV, HCoV-229E, HCoV-NL63, HCoV-OC43, PIV-1, PIV-2, PIV-3, PIV-4, HRV, HEV, and HBoV. Reaction mixtures for virus detection were divided into two panels: A and B. Each panel was used to detect 8 viruses with appropriate controls. Two types of DNA and 14 types of RNA viruses were amplified and detected by using CFX 96 Real-Time PCR Thermal cycler (Bio-Rad). Seegene Viewer software was used to analyze the amplification results. The study was approved by the Research and Ethical Committee of King Abdul-Aziz Medical City, Riyadh.

The Heidelberg University Hospital is a tertiary referral center. The department of hematology comprises four inpatient wards for adult patients—two wards for normal and high-dose chemotherapy, one intermediate care unit and one transplant unit—as well as several outpatient clinics and a day hospital where chemotherapy on an outpatient basis is administered. Most of the patients treated suffer from malignant lymphoma, multiple myeloma, and acute leukemia. Each year about 200–250 autologous and 100–120 allogeneic transplantations are performed. The majority of allogeneic transplant patients receive peripheral blood stem cells after a reduced conditioning regimen.

As part of the standard operation procedures and in order to inform for infection control measures, from autumn to spring all hematological patients are tested on development of respiratory symptoms for infection with influenza virus, parainfluenza virus and RSV (standard screening); during the summer months only patients with very severe or not otherwise explicable symptoms are tested due to the lower prevalence of respiratory virus infections in that season. With significantly increasing numbers of respiratory infections, standard screening was escalated in February 2013 and subsequently all patients were screened for infection with influenza virus, parainfluenza virus and RSV on admission to one of the wards regardless of respiratory symptoms as well as tested again once respiratory symptoms developed (intensified screening). Additional screening for other community acquired viruses such as rhino-, adeno- or coronavirus was not performed. All patients with proven viral infection were retested until clearance of virus. Several patients were lost to follow-up regarding their viral shedding. Furthermore, symptomatic patients were isolated while awaiting the laboratory result. Infected patients were isolated in single rooms or isolated in cohorts.

The samples were processed within two hours and afterwards kept frozen at -20°C. Analysis was performed with nasopharyngeal samples via reverse transcription polymerase chain reaction (RT-PCR). A case of respiratory tract infection was based on a laboratory-confirmed infection with influenza virus, parainfluenza virus or RSV presenting with or without respiratory symptoms. URTI was defined as presence of respiratory symptoms (e.g. coughing, sneezing) without signs of lower respiratory tract disease, LRTI was defined as presence of respiratory symptoms plus radiographic (chest X-ray or chest CT scan) signs of lower respiratory tract disease, severe LRTI was defined as LRTI plus requirement of treatment on the intensive care unit or death. Nosocomial infections were defined as virus detection on day seven or later after admission to the ward, the remainders were defined as community-acquired infection. However, some of the community-acquired cases had been previously treated at the day hospital. Patient records and information were anonymized and de-identified prior to analysis. Ethical approval was obtained from the Ethical Committee of the University of Heidelberg.

In the patients in Group I, those with LRIs were more likely to have uncontrolled underlying malignancies (p=0.038) and receive re-induction or palliative chemotherapy (p=0.006) than those with URIs (Table 4). Among respiratory symptoms, sputum (p=0.028) and dyspnea (p=0.001) were more frequently accompanied by LRIs. More patients with LRIs experienced co-infections than those with URIs (p=0.029). Among the five patients with LRIs and co-infections, four experienced invasive pulmonary aspergillosis: two of whom had concomitant RSV infection, another had concomitant adenovirus infection, and the other had HMPV infection. The other experienced C. difficile infection with concomitant parainfluenza virus infection. The two patients with URIs and co-infections experienced chickenpox with concomitant rhinovirus infection, and S. epidermidis bacteremia with concomitant RSV infection, respectively. The rate of rhinovirus infection was significantly higher in patients with URIs than in those with LRIs (p=0.021). Other viral infections showed no significant association with LRIs (Table 1). There were no independent risk factors for LRI in multivariate analysis (data are not shown). Of 14 patients with parainfluenza virus infection, five (35.7%) received ribavirin treatment and one (7.1%) also received IVIG. Of 10 patients with RSV infections, eight (80.0%) received ribavirin treatment and three (30.0%) also received IVIG. Patients with LRIs were more likely to receive oxygen therapy (p<0.001) than those with URIs and mortality was higher in patients with LRIs compared to those with URIs, although the difference was not statistically significant (p=0.101). All of the fatalities in both groups were caused by uncontrolled underlying malignancies.

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Standard infection control measures included isolation of patients who were tested positive for one of the respiratory viruses until two consecutive negative swabs were obtained. Cohort isolation of patients infected with the same virus was possible. Contact patients were isolated until a negative swab was obtained at the end of the estimated incubation period. Masks were to be worn in all patient rooms on the transplant unit, on the intermediate care ward as well as when in contact with infected patients. Rigorous hand hygiene was implemented.

During the period of intensified screening, infection control measures were intensified to isolation of all newly admitted patients as well as all patients who developed respiratory symptoms until a negative swab was obtained. The number of visitors allowed was reduced to two visitors per person and children under the age of twelve were no longer admitted to the wards. Masks were obligatory on all the wards.

Neither visitors nor medical or nursing staff were screened for presence of respiratory viruses. However, visitors with respiratory symptoms were not admissible to the wards and medical or nursing staff experiencing respiratory symptoms was discouraged from having direct patient contact.

The tracheal aspirate specimens were first treated with an equal volume of Sputasol solution (Oxoid, Basingstok, UK) in a 37 °C water bath for liquefaction. The total RNA/DNA was then extracted from the liquefaction sample using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) per the manufacturer's instructions. The nucleic acids was stored in aliquots at −80 °C until use.

A total of 13 different systemic antibacterials were used as empiric treatment in patients with viral pneumonia without bacterial coinfection for a total of 466 DOT. Vancomycin (50.7 %), cefepime (40.3 %), azithromycin (40.3 %), meropenem (23.9 %), and linezolid (20.9 %) were the most frequently used empiric antibacterials in patients with viral pneumonia without bacterial coinfection (Fig. 3). The most common regimens used in viral pneumonia without bacterial coinfection were vancomycin plus cefepime (28.4 %) and vancomycin plus meropenem (13.4 %). A total of 44 (65.7 %) patients with viral pneumonia without bacterial coinfection received empiric MRSA coverage with vancomycin or linezolid. Empiric antibacterial therapy was continued for a median of 4.1 days (interquartile range, 2.5–6.1 days) in viral pneumonia without bacterial coinfection, with most (69 %) being days on intravenous antibacterials.

Total antibacterial exposure differed between the long-course and short-course groups at 2116 and 484 DOT/1000PD, respectively (Fig. 3). Patients with mixed viral and bacterial infections received a total of 780 DOT/1000PD of systemic antibacterials. Median total antibacterial DOT/1000PD was also significantly higher in the long-course group compared with the short-course group (12.2 vs. 6.4; P <0.001) and the mixed-infection group (12.2 vs. 6.3; P <0.001). The most common antibacterials used were similar between groups: cefepime (long-course group: 73.1 %; short-course group: 50 %; mixed-infection group: 58.2 %), meropenem (long-course group: 37.3 %; short-course group: 32.1 %; mixed-infection group: 43.0 %), and linezolid (long-course group; 31.3 %; short-course group: 25 %; mixed-infection group: 41.7 %). Vancomycin was more commonly used in the long-course group compared with the mixed-infection group (80.6 vs. 59.5 %; P = 0.007) but not compared with the short-course group (80.6 vs. 57.1 %; P = 0.081). Azithromycin use was less prevalent in the mixed-infection group compared with the long-course group (48.1 % vs. 67.2 % of patients; P = 0.029) and the short-course group (48.1 vs. 71.4 %; P = 0.047).

Among the 117 episodes of single respiratory virus infections, arthralgia was more frequently observed in influenza A infections than in non-influenza infections (66.1% [39/59] vs. 46.6% [27/58], P = 0.033); for these 2 types of infections, the other examined symptoms, including sore throat, rhinorrhea, cough, purulent sputum, wheezing, dyspnea, and headache, were detected at similar frequencies.

Of 55 cases of LRTIs, coinfection with bacterial pathogens by sputum culture or blood culture was found in 3 (8.8%) of 34 patients who tested positive for respiratory viruses and in 2 (9.5%) of 21 patients who tested negative for respiratory viruses. Four of 6 cases of influenza A LRTI had received oseltamivir. Two patients died of pneumonia and the worsening of an underlying malignancy; 1 of these patients tested positive for hMPV, and the other patient tested negative for a respiratory virus. Four (16.7%) out of 24 patients vaccinated for influenza vaccine in 2012–2013 (A/California/7/2009 (H1N1)-like virus A/Victoria/361/2011 (H3N2)-like virus B/Wisconsin/1/2010-like virus) and 61 (25.1%) out of 243 unvaccinated patients had influenza A infections (P = 0.358).

The need for informed consent was waived in view of the observational nature of the study with no interventions performed. The protocol and standardized clinical form, including the waiver of informed consent, were approved by the Asan Medical Center Institutional Review Board (IRB number: 2010-0079).

Between October 4 and 28, 2011, 50 patients were included in the present study who either presented at a single clinic with a chief complaint of respiratory symptoms or fever or were suspected of having respiratory tract infections after being identified through the syndromic prescription surveillance system. In Japan, a rapid diagnosis kit suitable for use at outpatient clinics is currently available, and the costs are covered by the national health insurance program. The tests allow for rapid detection of infections caused by the influenza virus, RSV, and adenovirus. A total of 18 pharyngeal swabs to screen for adenovirus infections and 32 nasal swabs to screen for RSV and influenza viral infections (rapid RSV) were collected. Viruses were extracted from the swabs using immunochromatography (IC) kits with approximately 500 μL of a mucolytic agent provided by the manufacturer. After the assay, approximately 200 μL of the agent remained in the IC-kit tubes. This medical waste was transferred to universal transport medium (359C; Copan Italia S.p.A, Brescia, Italy) and analyzed using real-time polymerase chain reaction (PCR) and Hyper-PCR, which is a faster technique compared with the previously available PCR applications. Thus, we used Hyper-PCR for the applicable pathogens. The CycleavePCR respiratory infection-pathogenic virus detection kit (Takara Bio, Shiga, Japan) was used to detect 11 types of viruses: human RSV types A and B, human parainfluenza virus types 1–3, human metapneumovirus, influenza A and B viruses, human adenovirus, human bocavirus, and human rhinovirus. The Thermal Cycler Dice Real Time System II MRQ (Takara Bio) was used to detect and identify the 11 types of viruses detected by the CycleavePCR kit. Hyper-PCR was performed using the One Step SYBR High Speed RT-PCR Kit (Hyper-PCR) (Takara) to detect RSV types A, B, human parainfluenza virus types 1, 3, human rhinovirus, enterovirus, and influenza A (H1N1) 2009 (primers are listed in Table 1) using the Hyper-PCR MK IV PCR system (Trust Medical, Hyogo, Japan). The accuracy of the Hyper-PCR methods was confirmed by comparison with other conventional PCR methods. Conventional PCR was used to detect M. pneumoniae. In addition, the presence of coronavirus infection was tested in patients from whom no infectious agents were detected.

Respiratory virus infections have a major impact on health. Acute respiratory illnesses, mostly caused by viruses, are the most common illness experienced by otherwise healthy children and adults worldwide. Upper respiratory tract infections (URIs) such as common cold are exceedingly prevalent in infants and young children and continue to be common in older children and adults. Infants and young children may have 3–8 episodes of cold per year; those who attend daycare centers may have many more episodes per year [1–4]. URI can lead to complications such as acute otitis media, asthma exacerbation, and lower respiratory tract infections (LRIs). While LRIs such as pneumonia, bronchitis, and bronchiolitis occur much less frequently, they cause higher morbidity and some mortality, thus they are associated with high impact and greater healthcare costs. Approximately one third of children develop an LRI in the first year of life; LRI incidence decreases to 5%–10% during early school year, and 5% during preadolescent to healthy adult years [5, 6].

Common respiratory viruses include influenza A and B, respiratory syncytial virus A and B, parainfluenza virus types 1–3, adenovirus, rhinovirus, human metapneumovirus, and coronavirus types OC43 and 229E. Less common respiratory viruses include parainfluenza virus type 4, influenza virus C, and specific types of enteroviruses. The significance of more recently discovered viruses such as human bocavirus, coronavirus NL63, and HKU1 has yet to be elucidated [7, 8].

Clinical presentations of respiratory virus infections overlap among those caused by various viruses. In addition, clinical manifestations may mimic those of diseases caused by bacteria. Therefore, antibiotics are most often used in these infections, most of them unnecessarily. Furthermore, LRIs often require hospitalization for management such as intravenous antibiotics and symptomatic and supportive treatment. Specific antiviral treatment for respiratory virus infections is only available for influenza. Respiratory viral diagnosis is an integral part of patient management. Accurate diagnosis of specific respiratory virus infection not only improves the knowledge of disease the patient has but also can affect patient management and help prevent secondary spread of the infection. Rapid viral diagnosis may result in discontinuation of unnecessary antibiotics, initiation of antiviral drug for influenza, reduction of costs related to reduction of unnecessary investigations, and shortened hospital stay [9–11].

This paper describes up-to-date information on laboratory methods presently available in the diagnostic virology laboratories and those upcoming for detection of respiratory viruses.

Total RNA was extracted from the viral transport medium containing the flocked swab using NucliSENS® easyMAG® (BioMérieux, France) as per the manufacturer’s protocol. Reverse transcription was performed using the AccuPower® CycleScript RT PreMix (Bioneer, Korea) and random hexamer was used to synthesize cDNA. All cDNA samples were stored at −20°C until use. Multiplex PCR was performed using Seeplex® RV15 ACE Detection (Seegene, Korea). To detect influenza A virus, influenza B virus, adenovirus, coronavirus OC43, coronavirus 229E/NL63, respiratory syncytial virus (RSV) A, RSV B, rhinovirus, human metapneumovirus, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, parainfluenza virus type 4, bocavirus, and enterovirus, each sample was tested in three tubes reactions. Briefly, 4 µL 5X RV15 ACE primer mix, 10 µL 2X Multiplex master mix, 3 µL 8-MOP solution, and 3 µL of the sample’s cDNA were mixed in a tube. After denaturation at 94°C for 15 minutes, a PCR reaction was performed using 40 cycles at 94°C for 30 seconds, 60°C for 90 seconds, 72°C for 90 seconds, followed by extension at 72°C for 10 minutes. Among the samples tested, those positive for influenza A virus were selected and were tested to differentiate subtype using Seeplex® Influenza A/B Onestep Typing (Seegene, Korea). Briefly, 7.5 µL 8-MOP solution, 2.5 µL random hexamer, 10 µL 5X Flu A/B One-Step primer mix, 10 µL 5X OneStep RT-PCR buffer, 2 µL OneStep RT-PCR enzyme mix, 8 µL RNase-free water, and 10 µL sample RNA were mixed in the tube. Cycling conditions included a reverse transcription step at 50°C for 30 minutes and a denaturation step at 94°C for 15 minutes. The PCR reaction was performed using 45 cycles at 94°C for 30 seconds, 60°C for 90 seconds, 72°C for 60 seconds, followed by extension at 72°C for 10 minutes. Positive and negative controls were included in each PCR analysis. Electrophoresis of all PCR amplicons was performed on a 1% agarose gel.

The number of patients with subsequent MDRO colonization or infection was not significantly different between groups (Table 4). However, in instances of subsequent infection or colonization, where a single patient could have more than one organism, there was a higher rate of MDRO identification among isolates from the group that received long-course antibacterials compared with the group receiving short-course therapy (53.2 vs. 21.1 %; P = 0.027) (Table 4). VRE (35 %), coagulase-negative Staphylococcus sp. (15 %), Escherichia coli (10 %), Enterobacter cloacae (10 %), and Stenotrophomonas maltophilia (10 %) were the most commonly isolated MDROs in the long-course group. Other MDROs isolated from this group included Klebsiella pneumoniae, Serratia marcescens, S. pneumoniae, and Achromobacter sp. The three subsequent infecting MDROs in the short-course group were VRE, E. cloacae, and Corynebacterium sp. One patient in the long-course group and two patients in the mixed-infection group developed C. difficile infection 1 week after empiric antibacterial exposure.

In-hospital mortality was statistically higher for the mixed-infection group compared with the long-course therapy group (Table 4). Kaplan–Meier survival analysis showed that the mixed-infection group had the lowest overall survival, but these differences were not statistically significant (Fig. 4). ICU mortality was also significantly higher for patients in the mixed-infection group compared with the long-course therapy group. Patients receiving long-course therapy or those with mixed infection had statistically longer ICU length of stay compared with patients receiving short-course therapy. Hospital readmission rates were similar between groups at 30, 90, and 180 days after index hospitalization discharge.

Considering the confirmed RVI diagnosis in half of the immune compromised children and adolescents with respiratory symptoms in this study, the introduction of multiplex PCR tests for RV detection in this population should be encouraged, especially for patients complaining of rhinorrhea or sputum prominent over a cough. Moreover, the PCR test should address patients with more severe immune suppression, e.g., those with relapsed or refractory underlying malignancies and co-infections, as they are prone to have severe RVI-related outcomes. In addition, infection control strategies to prevent RVI transmission within the hospital environment should be emphasized, considering the current scenario, in which effective anti-viral therapies have not been established for most RVI cases. Thus, early RVI detection by a PCR test may open a window of opportunity for early intervention and infection control.

Of 128 patients with identifiable respiratory viruses, univariate analysis revealed that patients with 1 of the following conditions were more likely to have non-influenza respiratory virus infections: immunocompromised state, chronic obstructive pulmonary disease (COPD), and chronic renal failure receiving dialysis (OR 5.4, 95% CI 1.2–25.5, P = 0.02). Multivariate analysis demonstrated that steroid use was an independent risk factor for rhinovirus infection (OR 15.3, 95% CI 1.5–154.7, P = 0.02), active malignancy was an independent risk factor for hMPV infection (OR 29.3, 95% CI 2.4–358.1, P = 0.008), and COPD was an independent risk factor for parainfluenza infection (OR 229.2, 95% CI 10.5–5020.8, P = 0.001).

While comparing the URTI and LRTI groups, factors found to be associated with LRTI by univariate analysis included old age (≥60 years), a high comorbidity index, congestive heart failure, COPD, malignancy, immunocompromised state, and detection of parainfluenza or EBV, whereas detection of influenza A was less frequently associated with LRTI. Codetection of respiratory virus was not associated with the development of LRTI. By multivariate analysis, only old age, immunocompromised state, and detection of parainfluenza remained 3 independent factors associated with LRTI (Table 3).

Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.

The authors declare that they have no competing interests.

A total of 356 nasopharyngeal aspirate and throat swab samples were collected from patients with SARI by a trained technician using a sterile nylon flocked swab and placed in viral transport medium (VTM), labelled and transported on ice at the earliest to Advanced research lab (ICMR Grade-1 Virology Lab) of SMS Medical college Jaipur for further processing and storage of the samples. The study was approved by the institutional ethics committee.

A total of 44 6-week-old female C57BL/6 mice maintained under specific-pathogen-free (SPF) conditions were divided into two groups of negative controls (n = 4 each) and four inoculation groups (n = 9 each). The inoculation groups were inoculated with 400 µL of 105 TCID50 or 102.5 TCID50 of the bat paramyxovirus B16-40 via intra-gastric administration routes, and 30 µl of 105 TCID50 or 102.5 TCID50 of the bat paramyxovirus B16-40 via the intranasal administration routes. Each inoculation was conducted twice with 1 week in between. Weight loss was monitored daily for up to 2 weeks. The lungs, liver, brain, and intestine were harvested at 7 or 14 days post-challenge, fixed with 4% paraformaldehyde in PBS, pH = 7.2, and submitted to BioLead Inc., Korea, for histopathological examination. Additionally, RNA was extracted from each tissue and from oral swabs and fecal samples collected daily for 7 days post-challenge. The extracted RNA was tested using RT-semi-nested PCR with the consensus paramyxovirus primers19. In addition, real time PCR was performed with newly designed primers and probes targeting regions of the membrane and fusion protein. Briefly, the designed primers were as follows:

forward primer: PVM-F5′-CCCAGGAGTATGGTTATCAAGTGAGG-3′; reverse primer: PVM-R 5′-TCCATTGGGCTCTCTTTGTTTGC-3′; Taqman probe: PVM-P 5′-FAM-CCCATCCCAGACCAGCCACCAGACCC-TAMRA-3′

Real time RT-PCR was performed as follows: Reverse transcription at 45 °C for 10 min, followed by PCR −95 °C for 5 min, cycling 40 times at 95 °C for 10 sec, and 60 °C for 30 sec. Using the 10-fold diluted virus (bat paramyxovirus B16-40), standard curves were performed for every reaction.