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Ninety-three (4.8%) of the 1,926 nasopharyngeal aspirates obtained from patients of all age-groups were positive for HBoV. Our detection rate is similar to that stated in other reports [2, 3, 8]. Generally, HBoV is detected in fewer than 8% of respiratory specimens [1, 8-13]; however, higher detection rates ranging from 10.3% to 19% have been reported [5, 14, 15].
To investigate the epidemiological association of respiratory infection with viral load, HBoV-positive patients were categorized into low- and high-viral-load groups by using 1.0×106 copies/mL as a threshold value. The detection rate of HBoV infection was at its peak in the first year of life (rate of detection, 8.6% between the ages of 6 and 12 months), as in the cases of RSV or PIV infection. Most of the HBoV-positive patients aged less than 3 yr belonged to the high-viral-load group. The detection rate was lower (1.5%) in patients aged more than 10 yr, and these patients belonged exclusively to the low-viral-load group. HBoV is rarely detected in adults except in cases of immunosuppression [10, 16, 17]. The lower detection rate and viral load of HBoV in older patients may be attributed to immunity acquired from an infection at a younger age. A seroepidemiologic study of HBoV showed that 5.6-83.3% of children aged 6 months-3 yr were seropositive for HBoV. Lau et al. suggested that HBoV infection might develop only once because of the subsequent development of life-long immunity conferred by neutralizing antibodies produced in response to the infection.
The frequency of HBoV codetection with other respiratory viruses was 18.3% in the HBoV-positive samples and was lower than the previously published data [4, 20]. This difference in the codetection frequency is attributed to the different detection methods; molecular diagnostic methods were used for the detection of other respiratory viruses in the other studies whereas we used virus culturing. Among the 17 HBoV-positive patients who were also positive for infection with other viruses, 10 showed PIV infection. The high association of HBoV with PIV seems to be attributed to the high prevalence of PIV infection in 2006 (22.8% in May). Interestingly, almost all cases (except two) positive for both HBoV and another respiratory virus belonged to the low-viral-load group. As the virus culture was used for the detection of major respiratory viruses, the isolated virus could be the main causative agent of respiratory illness. Therefore, the presence of low copy number of HBoV, detected by molecular method, may indicate prolonged viral shedding or an asymptomatic infection. Recently, prolonged presence of HBoV in NPAs has been reported. These results suggest that single HBoV infection in the high-viral-load group may play an active role in respiratory infection. These findings are consistent with a Norwegian study that reported detection of HBoV alone and a high-viral-load were associated with respiratory tract infection. In that study, patients with a high-viral-load in NPAs developed viremia more frequently than the patients with a moderate-or a low-viral-load did. In our study, HBoV-positive patients in the high-viral-load group showed significantly higher pulse rates and respiratory rates than the in the low-viral-load group. These findings also support the idea that a high-viral-load may be associated with a respiratory infection.
Previous studies have reported that HBoV infection was more prevalent among individuals who had other respiratory viruses [10, 22]. In a study performed in Hong Kong, a higher detection rate of HBoV was observed in NPAs positive for common respiratory viruses than in those that were negative for the same. However, in our study, similar detection rate of HBoV was observed in the samples positive and negative for other respiratory viruses in the R-mix culture (Data are not shown).
Previous studies showed that cases of HBoV infection were found throughout the year with a peak incidence rate in the winter season [10, 13, 23]. However, in our study, cases of HBoV infection were detected most frequently during the spring season. This finding is similar to those of reports from Korea [4, 24]. This seasonal difference in the incidence of HBoV infection may be attributed to regional and temporal differences.
Bastien et al. suggested that risk factors for severe HBoV infection appear to be similar to those for RSV infection (prematurity, congenital heart disease, and asthma). Thirty percent (26/93) of the HBoV-positive patients had underlying conditions such as heart disease, asthma, allergy, preterm birth, and a history of convulsions; most of these patients (80.8%) showed a low-viral-load. Persistent HBoV shedding for more than 1 month is observed in both respiratory and fecal specimens obtained from patients with significant underlying diseases. Although these finding were not fully understood, it is postulated to be a result of underlying immunosuppression.
In summary, HBoV infection was more prevalent in young children. Patients positive for HBoV alone mainly constituted the high-viral-load group. Most of the HBoV-positive patients with infection caused by other respiratory viruses belonged to the low-viral-load group. These findings suggest that HBoV may be associated with a respiratory infection.
This present study is the first study reporting respiratory viruses' detection in ILI patients in Indonesia. During 2012, 1692 patients that meet ILI case definition criteria were enrolled. From 334 cases randomly selected, 175 (52.3%) were male and 159 (47.6%) were female. The median age was nine years with a range from 1 month to 79 years. Most of the cases selected were patients with age > 5 years old (60.7%).
Two hundred and fifty-six (76.6%) specimens contained one or more viruses. Single infection was detected in 138 (41.32%) cases and multiple infections were identified in 105 (31.4%) samples. The high detection rate of respiratory viruses from ILI cases in this study is similar to the previous studies in other countries including Cameroon (65.06%), Nanjing China (50.6%), and Cambodia (35.5%) [3, 14, 15]. However, this result is higher compared to the previous results which focused on hospitalized patients in Indonesia: 27% and 8.2% [23, 27]. The difference of viral detection rate among outpatient ILI cases and inpatient is most likely due to the time of infection. Hospitalized patients who suffer from lower respiratory tract infection might experience viral infection first which is then followed by bacterial infection later.
Of these positive specimens, influenza virus is the most detected virus (36.1%), followed by human rhinovirus and human adenovirus. Table 1 shows the distribution of respiratory viruses associated with ILI cases in 2012. The high prevalence of influenza virus detected from ILI cases in this recent study is concordant with several results from other countries [3, 15, 28]. Previous results in hospitalized suspected influenza patients in Indonesia demonstrated similar results as influenza is the most detected virus.
The difference in the prevalence of viruses identified in the ILI specimens among studies has been recognized. Time of samples collection and the composition of age in the study population are assumed to influence the proportion of virus identified. Previous results between studies in temperate countries showed the variation of viruses detected based on the seasons [18, 29]. The seasonality of respiratory viruses in tropical region is relatively undefined clearly except for RSV and influenza seasonality which associated with rainy seasons. Therefore, further investigation is needed to determine the seasonality of other respiratory viruses in the tropic region.
The prevalence of viruses is assumed to be associated with the age of patients. Regarding the age group in this study which is mostly more than 5 years old, the prevalence of RSV was low as RSV is mostly detected in children under one year old. The prevalence of adenovirus and RSV A infections increased with age. This finding is similar to other researches. Influenza was the most prevalent viral infection under-one-year-old group and above-five-year-old group. In the one-to-five-year-old group, adenovirus was the most prevalent. Figure 2 also shows the number of respiratory virus infections by each age group.
The frequency of multiple infections (31,4%) in all ages was significantly higher than single infection (45,2%) (Z = 5,069, P < 0,0001, 95% CI 0,2647–0,3636). This number is higher than the previous study in China (10,8%). The viral coinfection in children under five years old in this study is slightly higher than the older one (52.3%). This result is the same as several other studies [18, 20, 22]. From the coinfection detected, the most frequent combination of viruses was ADV and HRV. The frequency and combination of multiple viruses detected are presented in Table 2.
Indonesia has five major islands with Java Island as the main island where 5 of 13 sentinels located. The total number of influenza-like illness cases in the first quarter of the year was greater than the rest of the year, as shown in Table 3. Respiratory viruses were more frequently detected in the early months of the year in all regions in Indonesia. However, the virus detection rate in each month was not significantly different. The increased respiratory infections during this period may be related to the rainy season in Indonesia which usually starts from October to April, annually. The humidity and temperature are assumed to be major factors, as described before [19, 31]. Respiratory viruses use aerosol as a mode of transmission. In the high humidity atmosphere as in tropical countries like Indonesia, the aerosol that contains respiratory viruses will survive longer.
Our recent study has several limitations. First, fewer patients <1 year old were enrolled. Specimens from young children tend to be hard to collect. There may have been selection bias, as surveillance staff may have avoided collecting swabs from this age group. Second, we only sampled patients visiting public health centers. Our findings may be limited to patients at private clinics or those who do not seek healthcare. Third, our study did not analyse the relationship among viruses, coinfection, and clinical features (severity). Further studies are encouraged to determine the type of viruses and coinfections that contribute to disease severity.
Lower respiratory tract infection (LRTI) remains one of the major causes of mortality and morbidity in children under five years globally. Viruses have already been recognized as important etiologies of respiratory infections with influenza virus which is considered as the main contributor. The epidemiology and public health impact of influenza infections are relatively well described as many studies and surveillance have been conducted in part of pandemic preparedness [2–5]. Most of the countries in the world, including Indonesia, have developed influenza surveillance, influenza-like illness (ILI) surveillance, and severe acute respiratory illness (SARI), which form the network under WHO through Global Influenza Surveillance and Response Systems (GISRS) [4–8]. This network improves influenza disease control by providing support on influenza vaccine recommendation, laboratory diagnostic tools, antiviral, and public health risk assessment. As influenza virus contributed only less than 30 percent of viral respiratory infections, there is an urge to investigate the contribution of other respiratory viruses for improving respiratory disease control program.
Recent advancement of molecular technology supports the investigation and characterization of several respiratory viruses. The molecular technology improves the capability to study respiratory viruses, which are previously identified: rhinovirus, adenovirus, respiratory syncytial viruses, parainfluenza virus, and also the new emerging viruses/strain viruses: MERS coronavirus, human metapneumovirus, and human rhinovirus strain C. Multiple detection platforms, which recently have been developed, allows relatively inexpensive and timely detection of several viruses [9–11]. The detection of multiple respiratory viruses will accommodate the efforts to determine the epidemiology of noninfluenza respiratory viruses in the community, which will further help the respiratory disease control program including the use of antimicrobial agents.
Previous results of the investigation on acute respiratory infection patients in several countries showed the difference in the prevalence of respiratory viruses among studies [3, 13–15]. Study design including the case definition, study population, time of the study, and diagnostic tools being used have been considered as factors that influenced the variation. Each virus has different seasonality circulation and an age-related prevalence that can lead to a specific pattern of virus cocirculation in many studies [17–19]. Moreover, the occurrence of virus coinfection in which two or more viruses are detected in a single patient has been described in recent studies using multiple pathogen detection platforms [20–22].
There are limited studies on viral pathogens of respiratory tract infection in low-middle income countries including Indonesia. Previous studies have been conducted mostly focused on specific viral pathogens, especially influenza [6, 23, 24]. The prevalence of noninfluenza respiratory infections is relatively unknown. Therefore, this study has an objective to investigate the prevalence of viral etiologies from ILI cases in Indonesia.
As HBoV has been first identified in respiratory samples, it has been suggested as a respiratory tract infection agent. The majority of the following studies in fact detected HBoV in children with respiratory tract infections. Clinical symptoms mostly described in conjunction with an HBoV infection are wheezing, fever, bronchiolitis and pneumonia. Studies including asymptomatic controls showed that HBoV is also detectable in these controls but with a lower incidence. For example, HBoV was detected in 17 % of children hospitalized because of respiratory infection, while only 5 % of the surveyed asymptomatic children were HBoV positive. This supports the assumption that HBoV in fact could be assigned to the respiratory viruses.
In contrast to other studies, in the study of Longtin et al. 43 % of asymptomatic children tested positive for HBoV. Most of those children underwent myringotomies, adenoidectomies or tonsillectomies. Thus, Lu et al. suggested that HBoV may be present in tonsillar lymphocytes. They tested DNA extracts of lymphocytes from nasopharyngeal tonsils or adenoids and palatine/lingual tonsils. 32.3 % of the tested extracts were HBoV positive, indicating that HBoV establishes latent or persistent infection.
Coinfections with other viruses are frequently observed in HBoV infections and often occur in more than 50 % of the tested samples. Two recent studies report that the viral load of HBoV was significantly higher in children with monoinfections than in children with coinfections. The high rate of coinfections with other viruses may then be explained by the persistence of HBoV in the respiratory tract. DNA quantification in HBoV positive samples revealed that the viral load of 42.5 % of the positive patients was > 1.0 × 105 DNA copies/mL, suggesting that below this cut-off HBoV may be a persistant virus or a bystander.
A total of 128 patients were recruited over the period of January to December 2008. The median age of the patients was 12 months (IQR: 6-24 months) and the number of males was 81(63.3%). Sixty one (47.7%) had severe pneumonia with the rest diagnosed as very severe pneumonia. At least one respiratory virus was detected in 33 (25.7%, 95%CI: 18.5% to 34.2%) children. Multiple viral infections were detected in 2 patients. One had an RSV and PIV 1 co-infection whiles the other had a combination of RSV, Influenza B and Adenovirus co-infection. Bacteria was isolated from only 12 (9.4%, 95%CI: 4.9 to 15.8%) patients with 10 of these being Staphylococcus aureus. The other two were Klebsiella species and Coliform. RSV and Staphylococcus aureus coinfection was also identified in two patients.
Six (20.0%) out of 30 children less than six months of age were found to be positive for viral infection (Table 2). The highest number of children infected were between 6 months and 2 years, the proportions infected for the various age groups were however not significantly different.
Clinical presentation of patients were compared to general pathogens identified (Table 3) and the individual viral pathogens (Table 4). Generally, there were no detectable significant differences in the clinical presentation as well as the gender and duration of illness of patients for the various pathogens isolated.
The study also investigated the seasonal variation of viruses. RSV infections were detected throughout the year however the peak infection rate occurred during the minor rainy seasons (October) (Figure 1). Adenovirus infections on the other hand had two main peaks occurring in the month of April and October.
MVC and BPV, the two other members of the genus bocavirus are also known to cause gastrointestinal infections in dogs and calves, respectively. Several studies detected HBoV in stool samples from children with acute gastrointestinal illness, but the role of HBoV infection in the gastrointestinal tract is still unclear. A study on the role of HBoV in gastroenteritis outbreaks in day care facilities detected HBoV in 4.6 % of 307 stool samples. Coinfections with Norovirus were frequent. Another study on hospitalized children with acute gastroenteritis also reports a high coinfection rate with other gastroenteritic viruses. Both reports could not link HBoV to gastroenteritis in children, indicating that HBoV may not be a causative agent. The gastrointestinal epithelium may instead be the place of HBoV replication.
Besides HBoV detection in respiratory samples and feces, HBoV DNA was also found in serum/whole blood and one study reports detection in urine. As there are no currently established methods to detect HBoV particles, it remains unclear if the detection of HBoV in serum indicates viremia or if HBoV targets blood cells. Parvovirus B19 infects erythroid progenitor cells in the bone marrow, but HBoV DNA was not detected in bone marrow of HIV (human immunodeficiency virus)-infected and HIV-uninfected individuals, while parvovirus B19 was detected in both groups.
Viral agents play an important role in acute lower respiratory infections and may herald the onset of pneumonia caused by secondary bacterial infections. Although information on the causes of respiratory illness in tropical countries is very scanty, available data indicates about one -third of the cases of respiratory tract infections are due to viruses.
The present study identified viruses in 25.8% of patients hospitalized for ALRTI with RSV being the most predominant. The overall prevalence is comparable to previous studies done in other developing countries and the predominance of RSV is in accordance with the assertion that this virus is the single most frequent lower respiratory tract pathogen in infants and young children worldwide.
Adenoviruses, the second highest viruses detected in this study have been reported to be responsible for 5-10% of lower respiratory tract infections with the highest rate occurring in younger children. Our study similarly recorded a 10.2% detection rate of Adenoviruses however there was no statistical difference in the age specific prevalence. This could possibly be due to the few number of younger children (less than one year) enrolled in this study.
This study generally recorded higher cases of tachypnoea, chest recessions and very severe pneumonia in RSV compared to Adenovirus infected patients but the differences were not statistically significant. Although similar clinical presentations have been found to be associated with RSV, our small sample size could account for our inability to detect these differences.
Among patients who were poorly fed, those without viral or bacterial infections were found to be higher (p = 0.03). Poor feeding and malnutrition has generally been associated with less risk for respiratory viral infection in developing countries however the possibility of increased mortality could occur especially when bacterial infections are involved. Further case controlled studies are therefore needed in developing countries to investigate this observation
There have been fewer studies of PIV infections in developing countries and most of them do not differentiate the subtypes of the viruses. Our study recorded a 3.1% prevalence of PIV infections with the predominant type being PIV-3. Similar hospital based studies have been reported in other developing countries. PIV infections have been strongly associated with croup. The present study recorded no case of croup among PIV patients.
Viral associated bacteremia was detected in two patients (1.7%). Low rate of secondary bacterial infections has generally been reported in developing countries with the isolation rate varying between 2 and 10%. Studies in some developed countries however reported the converse probably due to the higher sensitivity of the bacteria identification techniques used. The blood culture identification system used in this study may be limited in the detection of bacteria associated with lower respiratory tract infection. Perhaps the detection rate could have increased if the present study had identified bacterial pathogens from nasopharyngeal aspirates or washings as reported by Hishinki et al..
The isolation of Staphylococcus aureus in an RSV positive patient in the present study was similar to studies reported by Berman et al., and Cherian et al.,. In their case, the bacterium was associated with fatality in the children.
The present study observed that all subjects enrolled in this study were treated empirically with antibiotics in accordance with the paediatric emergency treatment protocol for managing severe and very severe pneumonia. Similar occurrences have been reported where patients were treated unsuccessfully with multiple antibiotics. Policy makers may therefore consider reviewing the clinical algorithm for patient management as the economic cost both to the health service and the patient's household derived from the use of antibiotics could be enormous.
The period of this study experienced major rainfalls in the months of May to July and minor rainfalls in the month of September to October. The month of October recorded the highest detection of viral infections (57.6%; 19/33) with the commonest being RSV. This phenomenon has been reported in other developing countries. The occurrence of RSV infections in the rainy and cold season could be due to overcrowding as a result of populations staying more to their homes. More studies are however needed to define the seasonality of respiratory viruses in tropical countries.
Our study had some limitations which include underestimation of the overall prevalence of respiratory viruses since we did not test for coronaviruses, human metapneumovirus, bocaviruses and rhinoviruses which have also been reported in hospitalized patients. Also the negative results recorded for Influenza A and PIV 2 and the low prevalence of influenza B in our samples could be due to the low sensitivity of the assay for these viruses. The limit of detections of Influenza A, Influenza B and PIV 2 are 120 copies/μl, 480 copies/μl and 400 copies/μl respectively as such viral copies below these limits of detections could be missed by our assay.
This is the first epidemiological study to assess risk factors for BRSV seroprevalence carried out in Brazil. Even though BRSV prevalence of 79.5% in the animals sampled was similar to that estimated, the prevalence in adult animals was higher than that expected, reaching 87% of samples. In calves, the seroprevalence was lower than that found in adult animals (62.8%) and could be even lower once VNT does not allow the distinction between antibodies from colostrum and natural infection. Thus, this study demonstrated that the prevalence of BRSV antibodies was higher in adult animals, as previously reported in other countries [13, 16].
Adult animals are associated with high seroprevalence of BRSV as consequence of a repeated exposure to the virus infection throughout their life and possibility of reinfections. Similarly, the highest antibody titers were associated with non-vaccinated adult cattle, probably due to the exposure to successive viral reinfections, which results in a booster effect on antibody titers. Other factor related to high antibody titers is recent BRSV infections, which can be confirmed only by paired serology, antibody screening in calves after the period of colostral antibody detection or viral detection by direct methods. As respiratory disease was not reported in half of the herds studied, it is indicative that BRSV infection can be subclinical. This is consistent with previous reports. Herds can remain free of clinical BRSV infection for many years even in areas of high prevalence of the virus.
The presence of other pathogens is also associated with the prevalence of BRSV [8, 11, 14, 16]. This information explains the association of BRSV serological prevalence with the prevalences of BoHV-1 and BVDV-1. The infection by these viral agents is also reported in Brazilian herds, with high prevalences [27, 28]. BVDV infection can cause impairment of the animal’s immune function and thereby decrease resistance to other infections. The synergistic effects of BVDV with other respiratory pathogens have been observed [29, 30]. Thus, health status of the herds may also be affected indirectly by BVDV control measures.
Dairy cattle herds in São Paulo State usually have poor biosecurity measures, such as the lack of quarantine of newly purchased animals, lack of diagnosis of respiratory diseases (particularly for BRSV) and vaccination is rarely performed against these viruses. Therefore, we hypothesized that risk factors for the seroprevalence of BoHV-1, BVDV-1 and BRSV in the studied population likely to overlap.
Despite the logistic regression not confirming “type of calves feeding” variable as a risk factor for high prevalence of BRSV, the Fisher’s exact test detected “natural suckling” as a protective factor. “Natural suckling” would be important as it may be able to reduce the risk of calves becoming infected by BRSV. Weaning can be stressful and results in impaired immune function, which may further exacerbate a BRSV exposure. Suckling reduces the occurrence of diarrhea, prevents the abnormal behavior of cross-suckling of other calves and improves animal health [31, 32]. Prior to the current study there have been no report about “natural suckling” and its relationship with BRSV seroprevalence or its role as a protective factor, therefore, based on the results presented, it has the potential to decrease seroprevalence to BRSV.
Similarities were observed among the results found at the present study and those previously obtained by others conducted in Brazil [18–20]. In Latin America countries, equivalents prevalences of BRSV have also been reported [12, 14–16], as well as difficulties in detecting the risk factors involved in the dissemination of the agent, even using different forms of sampling and analyzing a considerable number of variables. Thus, the dynamics of infection may differ even in a particular country or geographic area.
The high serological prevalence of BRSV found in this study shows the importance to know more about this infection since it is not considered important in the country, mainly due to the lack of diagnosis. The awareness of the risk factors involved in the BRSV dissemination can allow understanding its mechanisms, even though, as in other studies, these factors were not very clear. Thereby, further studies as a complement to the current one should be performed until concrete information has been found.
Influenza viruses are known to constantly evolve and cross species barriers. The genetic diversity of influenza viruses is ever increasing with more novel influenza subtypes being discovered periodically. The purpose of this review is to provide an up-to-date overview of ecology and evolution of influenza viruses including the novel influenza viruses in bats and cattle. In addition, we discussed the growing complexity of influenza virus–host interactions and highlighted the key research questions that need to be answered for a better understanding of the emergence of pandemic influenza viruses.
BPI-3V sometimes cause severe disease as a single agent and can predispose the animal to bacterial infections of the lung. Our results revealed high BPI-3V seroprevalence (47.1%) in the three explored municipalities that indicate most adult cattle have been exposed to this pathogen. These results agree with those by Carbonero et al., who found high seroprevalence values in cattle of Yucatan, Mexico. However, the results obtained in this study differ with those published by Betancur et al., who reported lower seroprevalence values (13.5%) in cattle from Monteria, Colombia. The high seroprevalence of BPI-3V found in this research is in agreement with the ubiquitous nature of the virus and with its worldwide distribution. In this research, the seroprevalence was higher in the age group of >24 months of age (Table-4). This age group was a significant risk factor for BPI-3V transmission (OR=3.5). Possibly, due to the presence of some stress factor in these animals that favors reinfections with or without respiratory signs. In adults, especially BPI-3V, it is subclinical unless it is part of concomitant infections with other viruses and bacteria such as Pasteurella multocida, Mannheimia haemolytica, Mycoplasma spp., and immunosuppressive factors. With regard to the clinical signs, conjunctivitis had a statistical association with the BPI-3V seroprevalence values, and regarding sex, female was a significant risk factor for BPI-3V infection (OR=3.6). This result differs with those published by Betancurt et al., who found no statistical association between BPI-3V infection and sex.
The results of the indirect ELISA revealed that the overall prevalence of BRSV in the Nineveh Governorate was 83.11%, with the highest prevalence in cattle that were aged >7 months-1.5 years (relative risk (RR)=2.12) (Table-1). BRSV prevalence was higher in imported animals, compared to animals of a local origin (RR=1.17) (Table-1), and in animals originating from large herds (100 animals), compared to those from small herds (40 animals) (RR=1.48) (Table-1). There was no significant difference (p<0.05) between the prevalence of the disease in male and female animals (Table-1). BRSV prevalence varied significantly (p<0.05) across the different geographical areas of the Nineveh Governorate with the samples collected from the northern region displaying the highest prevalence (RR=1.33) (Figure-1). In addition, samples collected in the winter displayed the highest prevalence of BRSV (RR=1.38) compared to those collected in the spring, summer, or fall (85.09%, 83.18%, and 75.18%, respectively) (Table-2).
The current study investigated the prevalence of HBoV in patients suffering from respiratory tract infections in Saudi Arabia. The presence of the major viral causes of the respiratory distress in HBoV positive cases was also screened. HBoV was detected in 18/80 of the examined patients (22.5%) with ages ranging from 2 months to 10 years, (Table 1–2). Clinical findings for HBoV-positive patients were indistinguishable from those for patients with other respiratory viruses. Previously, HBoV has been detected in samples from patients aged between 5 months and 2 years,. Ma et al, speculated that the antibody against HBoV derived from the mother might protect children under 5 months of age from HBoV infection, however, we detected HBoV in two cases below 5 months: in a 2-month-old and 4-month-old child (Table 1–2) that may indicate the possibility of HBoV infection in very young children.
The rate of HBoV in respiratory tract infections has been reported to be 1.5 to 19.3%,. Real-time PCR was used in the current study to screen HBoV due to its high diagnostic sensitivity that could be responsible for the higher rate of HBoV infection in Saudi Arabia than the widely accepted upper limit of infection rates worldwide. Meanwhile, a recent study showed 21.5% prevalence among children.
The evidence of HBoV as the main initiator of the disease in the infected cases is still uncertain because of its high co-infection rate with other pathogens, and it remains unclear whether HBoV is the sole etiologic agent or just a concomitant virus bystander. In previous studies, none of the nasal swabs obtained from healthy children yielded a positive HBoV test. This suggests that HBoV is not a frequent commensal virus inhabiting the respiratory tract,. HBoV infections are frequently present in concomitant with other viruses and often occur in more than 50% of the tested samples. In the current study, only one case was found to be infected only by HBoV as a single virus entity while most of isolates (17/18) showed coinfection with other viral pathogens. The most frequently detected co-pathogens were RSV (13/18; 72.2%), IAV (12/18 cases, 66.66%), respiratory adenovirus (6/18 cases, 33.33%) while only 1/18 (5.5%) case was coinfected with PIV-3 and none was coninfected with PIV-1 (Table 2). It is assumed that the rate and frequency of coinfections may be higher if more viruses were screened. Consistent with other studies,, the prevalence rate of bocavirus was higher in children under 2 years of age (Table 1).
Partial NP-1 gene sequence of the eighteen detected HBoV strains were obtained in our study. Multisequence analysis showed complete identity (100%) between each other, and phylogenetic analysis demonstrated that they belonged to HBoV1 (data not shown). Blast analysis revealed complete homology to the published sequence of HBoV1. Furthermore, the phylogenetic analysis results of three selected sequences showed that the Saudi HBoV1 strains obtained from respiratory samples belonged to group I human bocaviruses (Fig. 1).
To the best of our knowledge, this is the first report of HBoV1 in Saudi Arabia. Continuous surveillance and genome sequence analysis are needed to obtain more information on the genotypic variation and molecular evolution of HBoV in the country.
To the best of our knowledge, this is the first study demonstrating SAFV in adenoid tissues. Out of 70 adenoid tissue specimens from children, 11 (15.7%) were positive for SAFV. Noteworthy, all children underwent elective adenoidectomy and did not display symptoms indicative of an infection of the upper or lower respiratory tract at time or within two weeks preceding surgery. Although the number of SAFV RNA-positive throat swabs was low, SAFV was only detectable in throat swabs from children whose adenoids also contained SAFV RNA.
In literature, there are only very few reports describing analysis of SAFV in specimens from asymptomatic individuals. Zhang and colleagues collected 352 throat swabs from asymptomatic children, aged 4 to 78 months. In contrast to our results, these authors did not detect any SAFV positives among the asymptomatic children while 25/1829 (1.37%) swabs from children with respiratory symptoms were positive. The authors applied the same RT-PCR protocol as we did. Except for that particular study sampling oropharyngeal specimens, as we did, all other information on healthy children is derived from analysis of feces. Drexler and colleagues did not detect SAFV-RNA in stool samples from 39 control children while the detection rate in children with diarrhea was 0.7% (6/844). In contrast to these two studies [12, 27] reporting absence of SAFV in asymptomatic children, there are three reports describing a similar or an even higher prevalence of SAFV in feces from healthy children compared to children with gastrointestinal illness (2.8% vs. 1.5%; 2.8% vs. 2.6%) or nonpolio acute flaccid paralysis (12.2% vs. 8.8%). Our results demonstrating presence of SAFV in non-diseased individuals might be consistent with the results of these three studies, although the body site where the specimens were collected was quite different.
Surprisingly, detection of SAFV seems to be more frequent in adenoid tissue (15.7%) than in non-tissue specimens from the respiratory tract. In a recent study, SAFV was detected in 9.7% of throat swabs from 226 Taiwanese-children with respiratory symptoms. In a larger cohort of 1,525 children with acute respiratory infections in Japan, SAFV2 was found in 3.5% of nasopharyngeal swabs. In another study from Bejing including 1,558 children, SAFV was detected in 0.6% of nasopharyngeal aspirates from 506 patients with upper respiratory tract infection and in 0.4% of 1,032 patients with lower respiratory tract infections. The hypothesis of a higher SAFV prevalence in adenoid tissue than in non-tissue specimens from the respiratory tract might be underpinned by our results detecting SAFV in 15.7% of adenoid tissue samples vs. 4.4% in throat swabs, although the difference was not statistical significant (p = 0.059), most probably due to the low numbers analyzed. However, there is one study reporting a rather higher rate of SAFV-positive nasopharyngeal swabs (9/37 = 33.3%) taken from children with exudative tonsillitis than we found in the adenoid tissue.
Intriguingly, in our study testing healthy children, SAFV was only detected in throat swabs from individuals also positive for SAFV in their adenoids (2/8). Noteworthy, viral concentration in the swabs was rather low. However, comparison with results from other studies is not possible due to lack of information in literature about SAFV RNA load.
In our study, three samples could probably belong to SAFV2. According to current knowledge, genotype 2 appears to be the most prevalent one in Central Europe. In a large study performed in Denmark, all 38 SAFV-positive feces (2.8%) belonged to that type.
Besides SAFV, a high rate of common respiratory viruses was detected in every case of the SAFV-positive adenoid tissue sample. Enterovirus and Human parechovirus deserve a special mention regarding their belonging to the Picornaviridae family, as does SAFV. Overall, Enterovirus was the most frequent co-detected virus (9/11 (81.8%)), followed by Human bocavirus (7/11 (63.6%)). In the SAFV-negative tissues, both viruses were less frequently found, i.e., Enterovirus in 31/59 (52.54%) and Human bocavirus in 34/59 (57.62%) samples. However, the differences between the SAFV-positive and negative group are not statistically significant. Notwithstanding, the high prevalence of various viruses in adenoid tissue is surprising. One may speculate as to whether children with adenoid hypertrophy requiring adenoidectomy underwent a greater number of infections resulting in a higher rate of tissue-associated viral nucleic acid than children without adenoid hypertrophy. Alternatively, storage of viruses or viral genomes in adenoids might be a general phenomenon in childhood. The finding of DNA and RNA of multiple viruses, at least of fragments, supports the notion of a longer than previously anticipated persistence of viral nucleic acids in adenoids. Sato et al. speculated on a normal viral flora and a chronicity of selected respiratory viruses. Alternatively, viral presence in adenoid tissue might play a role with respect to the immune response. Interestingly, in contrast to adenoid tissue (Table 2), we rarely found nucleic acid from multiple viruses (> 2) in nasopharyngeal swabs (Table 3).
Lin et al. conducted a study which supports the hypothesis that SAFV might play a role in the pathogenesis of upper respiratory infections. Yet, the authors critically noted that it would need to be checked by looking for the virus in asymptomatic patients in further studies. Although our study focused on asymptomatic individuals, the results of our study do not contribute to the answer of the question about the true clinical significance of SAFV detection. However, our study at least demonstrated that SAFV, and in addition a spectrum of other respiratory viruses, can be detected at a relatively high rate in the adenoids of asymptomatic children. Additional studies with enlarged sample size including both adolescents and adults as well as attempts for SAFV isolation in cell culture are needed to clarify the duration period of SAFV persistence in tissue as well as the virus´ ability for replication in asymptomatic individuals.
The GenBank accession numbers for the three SAFV nucleotide sequences were as follows: MK182597, MK182598, and MK182599.
Bovine respiratory syncytial virus (BRSV) is an economically significant pathogen in cattle production, as it is one of the most important causes of lower respiratory tract infections in calves. In dairy cattle, BRSV infection usually occurs in young calves aged between 2 weeks and 9 months. Adult animals with subclinical infection are the main source of infection, since reinfections are common in the herds [1, 4, 5].
BRSV, bovine herpesvirus 1 (BoHV-1), bovine viral diarrhea virus (BVDV) and bovine parainfluenza type-3 (PI-3) are considered primary agents involved in the bovine respiratory complex. Additionally, secondary infection by Pasteurella multocida, Histophilus somni and mycoplasmas contribute to the aggravation of the disease. Clinical signs are characterized by respiratory symptoms, initially with moderated intensity, such as nasal and ocular discharges which can be aggravated leading to pneumonia. However, mainly in calves, an acute and severe onset is also observed, due to maternal antibodies not effectively protect against BRSV infection.
Considering the high prevalence of the disease, several studies determined risk factors involved in the epidemiology of BRSV. In Europe, risk factors were mainly attributed to herd size, herd density, purchasing of new animals, geographic location of the farms, herd type and concomitant BVDV infection [7–11]. Similar studies have also been performed in some Latin American countries and they showed that most of the animals probably have already been exposed to the virus with consequent high BRSV prevalence in cattle herds. In these countries, herd size, age group, presence of bordering farms, herd type and geographic location of the farms were the main risk factors associated with BRSV infection [12–16].
In Brazil, BRSV was first diagnosed in calves in the state of Rio Grande do Sul and some studies have shown that BRSV infection is widespread in Southern and Southeastern Brazil, with high serological prevalence rates [18–20]. Nevertheless, research has not been conducted in order to verify possible risk factors involved in BRSV epidemiology. Due to this, the current study aimed to determine antibody prevalence against BRSV and investigate some risk factors associated with BRSV seroprevalence in herds of an important milk producing region in São Paulo State, Brazil.
Influenza is among the major infectious disease problems affecting animal and human health globally. Several human influenza pandemics have been recorded since 1590 AD, with the most significant of those being the “Spanish flu” of 1918, often referred to as the “mother of all pandemics”. Spanish flu pandemic is believed to have affected approximately 25–30 percent of the world’s population and caused more than 50–60 million human deaths globally. Influenza infections in humans occur either as epidemic (seasonal or interpandemic) influenza caused by influenza A and B viruses, or as sporadic pandemic influenza caused by influenza A viruses. Study of influenza pandemics has been of great interest to epidemiologists. Influenza epidemics and pandemics have been repeatedly occurring for centuries, but to date the ability to predict a pandemic has not been achieved.
A total of 1,926 samples of patients with respiratory sym-ptoms were included in the present study. Ninety-three (4.8%) samples were found to be positive for HBoV by PCR and subsequent sequencing. Other respiratory viruses detected during the study period were as follows: influenza A virus (IFA), 56 patients (2.9%); influenza B virus (IFB), 50 patients (2.6%); parainfluenza viruses (PIV), 142 patients (7.4%); respiratory syncytial virus (RSV), 97 patients (5.0%); and adenovirus (ADV), 40 patients (2.1%). HBoV was more prevalent in men (73.1%) than in women (P=0.005).
HBoV was detected in patients ranging from 3 months to 65.6 yr (mean=36.2 months, median=19 months) with a peak prevalence between the ages of 6 and 12 months (8.6%, 22/257). Children aged 5 yr or less constituted 92.5% (86/93) of the HBoV-positive patients (Table 1).
HBoV was detected in the samples obtained throughout the course of the study with the detection rate being the highest in June (16/129, 12.4%), followed by August (9/110, 8.2%) and May (13/162, 8.0%). From April to June 2006, 43% of the HBoV-positive cases were observed (Fig. 1).
All the 93 HBoV-positive cases detected by conventional PCR were also confirmed by real-time PCR performed using the TaqMan probe. The viral load detected in the HBoV-positive samples by using real-time PCR was in the range of 1.3×103-4.6×109 copies/mL (median, 1.82×105 copies/mL). The HBoV-positive cases were categorized in 2 groups: low-viral-load group (viral load ≤1.0×106 copies/mL, N=58) and high-viral-load group (viral load >1.0×106 copies/mL, N=35) (Table 1). The HBoV-positive patients aged less than 3 yr had a significantly higher viral load than that in the patients aged more than 3 yr (P=0.001).
The other respiratory viruses found in 17 (18.3%) of the total HBoV-positive samples were as follows: IFA, 1 sample; IFB, 1 sample; PIV, 10 samples; RSV, 4 samples; and ADV, 1 sample. Most of the cases (88.2%, 15/17) belonged to the low-viral-load group. RSV was isolated from the remaining 2 samples with HBoV copy numbers of 1.09×106 copies/mL and 2.65×108 copies/mL.
Patients positive for HBoV alone had a higher viral load than that in the patients who were positive for both HBoV and another respiratory virus (median 3.78×105 copies/mL vs. 1.94×104 copies/mL, P=0.014). A high-viral-load was almost exclusively seen in the HBoV-positive patients alone (94.3%, 33/35) (Fig. 2).
The high-viral-load group had a significantly higher pulse rate and respiratory rate than the corresponding rates in the low-viral-load group (P=0.007 and P=0.0231, respectively; Table 2). Although the duration of hospital stay was not significantly different between the 2 groups (Table 2), in cases of patients with less than 10 days of hospital stay, the high-viral-load group had a longer hospital stay than the low-viral-load group did (5.2±1.5 days vs. 4.1±1.4 days, P=0.009). Most of the other clinical characteristics had no significant correlation with the viral load of HBoV. The HBoV-positive patients presented with cough (80.6%), sputum (63.4%), fever (62.4%), rhinorrhea (50.5%), crackle (44.1%), wheezing (33.3%), diarrhea (14.0%), and dyspnea (6.5%). No significant difference was observed in the HBoV viral loads in the cases of upper and lower respiratory tract infections (P=0.077). Twenty-one out of 26 patients with underlying conditions had low-viral-loads.
Clinical examination of the HBoV-positive patients showed pneumonia, bronchiolitis, bronchitis, croup, asthma, sinusitis, and pharyngotonsilitis (Fig. 3). HBoV was detected in 8.4% (N=48) and 5.6% (N=45) of the samples obtained from patients with and without pneumonia, respectively (N=572 and 805, respectively; P=0.049). However, no significant difference was observed in the viral load between the patients with and without pneumonia (P>0.05).
Statistical analysis was performed using IBM SPSS Statistics for Windows, version 19 (IBM Corp., Armonk, N.Y., USA). The two-sided Chi-square test and Fisher’s exact test were used to assess the difference in BRSV prevalence and various risk factors in the different cattle groups. The rate of relative ratio (RR) between BRSV risk factors was calculated at 95% significance using Epi-Info TM 7, version 7 (CDC, Atlanta, GA, USA).
A total of 99 viruses were detected in 84/222 specimens from a total of 79/183 patients and 4/5 National External Quality Assurance Scheme (NEQAS) controls; immunofluorescence did not detect the parainfluenza virus type 2 virus in one of the NEQAS specimens. Viruses were detected in all of the specimen types processed. The molecular strip detected virus in: 16/36 (44.4%) broncho-alveolar lavages, 62/120 (51.6%) nasopharyngeal secretions, 11/35 (31.4%) sputa and 10/31 (32.2%) combined throat and nasal swabs. Immunofluorescence detected virus in: 6/36 (16.6%) broncho-alveolar lavages, 23/120 (19.1%) nasopharyngeal secretions, 1/35 (2.8%) sputa and 1/31 (3.2%) combined throat and nasal swabs.
The median age of male and female patients where virus was detected was 3 y (range 2 weeks – 79 years) and 4 y (5 weeks – 81 years) respectively. Sixteen viruses were detected in 14/27 (51.8%) specimens, confirming a respiratory virus in 12 out of 24 (50%) patients investigated in general practice. Seventy-nine viruses were detected in 70/191 (36.6%) specimens, confirming a respiratory virus in 67 out of 159 (42.1%) patients investigated in hospital. Of the 16 viruses detected in specimens from the community, PCR detected all 16 in contrast to a single identification, influenza A (H3), by immunofluorescence.
A total of 70 children (41 males and 29 females), ranging from 0.8 to 12 years of age, were included in the prospective study. The median age at adenoidectomy was 3 years (IQR 2.5 years). All parents gave written informed consent. The study was approved by the ethics committee of the University of Bonn (044/11) in written form.
In 2005, Allander et al., reported the discovery of a previously undescribed human parvovirus in respiratory secretions from children with respiratory tract disease in Sweden. Phylogenetic analysis showed that this virus belonged to the genus Bocavirus (subfamily, Parvovirinae; family, Parvoviridae) and was most closely related to bovine parvovirus (BP) and minute virus of canines (MVC). The virus was thus named "human bocavirus" (HBoV).
Human bocaviruses (HBoV) contain 3 open reading frames (ORFs) encoding a nonstructural protein (NS1, NP1) and two capsid proteins VP1 and VP2, respectively. The genomic organization of HBoV closely resembles that of bovine parvovirus type 1.
HBoV has been reported in respiratory samples from children with acute respiratory tract disease in various parts of the world (including Australia, North America, Europe, Asia, and Africa), suggesting that the virus is circulating worldwide. Pneumonia, acute bronchitis, bronchiolitis, are the main manifestations of HBoV infection.
HBoV seems to be a new member of the community-acquired respiratory viruses such as respiratory syncytial virus, adenovirus, influenza virus, parainfluenza virus, and rhinovirus, which cause common respiratory tract infections in the community. Because of its very high copy numbers in respiratory tract secretions, aerosol and contact transmission are likely effective, as they are for other respiratory viruses.
HBoV has been detected also in nasopharyngeal, serum, fecal and urine samples obtained mainly from young children around the age of 2 years predominantly during the winter season. HBoV was detected in two pediatric patients after organ transplantation, in human immunodeficiency virus-infected pediatric patients, and immunosuppressed adult patients.
Diagnosis of HBoV infection is based on the PCR amplification of viral genome fragments present in human respiratory, serum, stool and urine samples. A great number of different PCR techniques employing varying sets of primers specific for the viral genes NP1, NS1, and VP1, VP2 have been described. In addition to the detection of viral genomes by PCR, recent reports describe the detection of HBoV-specific IgG and IgM-antibodies against HBoV VP2 in serum samples using western blot or immunofluorescence assays. Furthermore, a standardized ELISA for the quantitative determination of HBoV-specific antibodies has been established by. The aim of the work was determination of HBoV in respiratory specimens (NPA) of infants by qualitative PCR and determination of acute HBoV infection by estimation of IgM antibodies in serum by ELISA.
Table 2 presents the results from the individual investigation of lactating cows in the 11 selected herds where paired BTM samples indicated new infection. Herd size, the number of lactating cows contributing to the BTM, the association between levels of antibodies detected in BTM and individual milk results from the lactating cows are presented. Indications of time since presence of BRSV based on the results of the antibody testing of serum of animals at different ages are also shown. This includes the samples of young stock. In eight of the herds, all the tested young stock was positive. In three of the herds, all the young stock up to a certain age was negative, and all animals above that age were positive. The time of exposure was therefore presumed to be between the age of the oldest negative and the youngest positive animal. The results indicated that 9 of the 11 herds had a recent infection (<17 months ago). For two of the herds (herds 3 and 4), the results indicated that BRSV had not been in the herd for the last five to seven years. The level of antibodies detected was also lowest for these herds. The percentage of positive animals contributing to the BTM was also lowest for herds 3 and 4, but the mean PP of the positive animals was high (114 and 86, respectively).
Bovine corona virus (BCV) and bovine respiratory syncytial virus (BRSV) are two worldwide distributed viruses. BCV causes diarrhoea in calves, winter dysentery in adults and various degrees of respiratory symptoms. BRSV is regarded as one of the most important causes of respiratory tract disease, especially in young calves. An infection can cause respiratory distress, fever, anorexia and subcutaneous emphysema and can lead to secondary bacterial pneumonia and death. Outbreaks of BCV and BRSV occur mainly in autumn and winter. These infections are common in dairy herds; in a nationwide survey in England and Wales the prevalence of antibodies to these viruses in bulk tank milk (BTM) was 100%. Swedish studies have shown a prevalence of 70-100% for BCV and 41-89% for BRSV, with the higher prevalence in southern parts. In a more recent study in a high animal-density area in south-west Sweden, the prevalence in BTM was 100% for both BCV and BRSV.
Previous studies have shown that BRSV and BCV infections are effectively spread within the herd. It has also been shown that acquired antibodies remain detectable for years, even without reinfection, whereas maternal antibodies are only detectable for a few months. Spot samples from a few young animals can thus be used to reflect recent infections of BRSV and BCV in a herd, whereas bulk tank milk samples mirror the long-term history. Spot sampling has previously been described for bovine virus diarrea virus (BVDV).
Despite the importance of these viruses and the fact that they are widely spread, little is known about transmission routes and management risk factors. Introduction of new animals and indirect spread via people and equipment are believed to be important and airborne transmission has been shown to occur for BRSV, at least under experimental conditions. Studies have been carried out to determine the relationship between herd health, reproduction efficiency and milk production and seropositivity to other viruses, for example bovine viral diarrhoea virus and bovine leukemia virus. Similar studies for BRSV and BCV have, as far as we know, not been conducted and it is therefore difficult to quantify their effect on the farm efficiency and economy. The purpose of this study was to explore if there were any associations between antibody status to BCV and BRSV and disease incidence, reproduction and some herd characteristics in dairy herds. A secondary aim was to investigate if there were any difference in proportion antibody positive herds between two neighbouring areas.
The study protocol was approved by the medical ethics review board of the College of Medicine, Taif University and by the pediatric hospital ethics committee in accordance with the guidelines for the protection of human subjects. Informed written consents from the next of kin of the participants involved in the study were taken.
A total of 341 samples from patients diagnosed with influenza or influenza‐like illness were analyzed for 18 respiratory viruses using the ResPlex II assay. The number of samples positive for INFA, INFB, and other respiratory viruses was 227 (66.6%), 85 (24.9%), and 38 (11.1%), respectively. Double infections were detected in 29 samples (8.5%) and triple infections in 3 samples (0.9%) (Table 1). The combination of viruses most frequently identified was INFA and INFB in the double infection group (8/29 double‐positive samples) and INFA, RHV, and CVEV in the triple infection group (2/3 triple‐positive samples).
To examine if there were specific patterns of correlation between the detected viruses, all virus pairs in Table 1 were evaluated with Fisher's exact test for 2 × 2 tables. In addition, the detection rate of one virus in the background of another virus‐positive sample was compared with that in the background of all samples, to determine whether correlation was positive or negative. We identified four pairs of viruses with significant association (P < .05), where INFA‐INFB and INFB‐CVEV showed negative correlation and CVEV‐RHV and CVEV‐RSVB exhibited positive correlation (Table 2). However, influenza viruses were not significantly correlated with most of the other respiratory viruses. These results suggest that INFA and INFB would mutually interfere in each other's infection, whereas infection with influenza and other viruses would occur independently in most cases.
To investigate whether MDCK‐S, MDCK‐A, and LLC‐MK2D can selectively amplify influenza virus in the presence of other respiratory viruses, co‐infection samples containing influenza virus plus other viruses were inoculated and passaged in the three cell lines. From all samples with triple or double infection, MDCK‐S and MDCK‐A could selectively allow proliferation of influenza viruses while eliminating all contaminating viruses (Table 3). Particularly, the isolation rates of influenza viruses were significantly higher than those of co‐existing viruses in the groups of double infection 1 (INFA and OC43, P < .01), double infection 2 (INFA and CVEV, P < .01), and double infection 3 (INFA and RHV, P < .05). For LLC‐MK2D, influenza viruses were isolated from the groups of double infection 1 and double infection 3 without propagation of adventitious viruses; however, the differences in detection rates between influenza virus and others were not significant. In the double infection 2 group, an isolate of CVEV was obtained from four specimens (25%), although no influenza virus was amplified during cell passages.
To examine further whether MDCK‐S, MDCK‐A, and LLC‐MK2D can eliminate contaminating viruses, we passaged clinical specimens containing respiratory viruses other than influenza virus in the three cell lines. The ResPlex II assay did not detect any respiratory virus in the cell‐passaged samples from all cell lines (Table 4). CVEV, RHV, RSVA, RSVB, OC43, and NL63 were eliminated during cell passages irrespective of single or double infection.
We selected 341 clinical specimens from the patients diagnosed as having influenza or influenza‐like illness based on their clinical symptoms and/or the results of rapid tests for influenza. These patients visited the hospitals during the 2006/2007 influenza season (from January 2007 to May 2007), 2007/2008 influenza season (from December 2007 to April 2008), or 2008/2009 influenza season (from September 2008 to February 2009) in Japan. Nasal or pharyngeal samples were collected using UTM 360C kits (Copan Italia, Brescia, Italy) or transport medium consisting of minimum essential medium (MEM) supplemented with 0.5% gelatin, 100 units/mL of penicillin, and 100 µg/mL of streptomycin. These specimens were aliquoted and stored at −80°C until use. The study protocol was approved by the ethics committee of the National Institute of Infectious Diseases, Japan.