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Following the discovery of WUPyV in Australia, the virus was detected in specimens from patients with respiratory tract disease on all continents suggesting a worldwide distribution [10,29–31]. So far, WUPyV-DNA was reported to be found in respiratory tract specimens (e.g. nasopharyngeal washes, tracheal secretion, BAL), serum, and faeces. The virus could not be detected in urine or from UV light-associated primary malignant lymphomas. Specimens from other malignant diseases have not been investigated. The use of tracheal secretion for diagnostics has been shown to lead to an underestimation of the rate of positive specimens compared to other respiratory materials for HBoV. This may be true for WUPyV, too.
The data available are mainly based on retrospective studies exclusively including symptomatic patients. The detection rate in respiratory samples from children with respiratory disease varies from 0.4% to 11.5%. The age of WUPyV infected patients ranged from a few weeks to 53 years, children <3 years of age were dominating. Infections were predominantly detected in late winter, spring, and early summer. High infection rates were reported for study populations preselected for lack of immunocompetence. HIV positive patients had detection rates of up to 35.7% in respiratory tract specimens and 8.3% in blood. The rates of co-infection with established respiratory viruses lay between 30.8% and 91.7%, commonly exceeding 50%. WUPyV was detectable in blood, possibly indicating its potential for systemic infections.
Le and co-workers presented evidence for viral persistence, Wattier et al. for nosocomial infections with WUPyV.
The real time protocols available allow the quantification of WUPyV. Quantification of viral loads in respiratory tract specimens revealed viral titers up to 1010 copies/ml, but low and medium viral loads were dominating. No correlation between viral load and the rate of co-infection or clinical diagnoses was observed.
Few studies included asymptomatic control groups [34,36,42–44]. The results were not concordant and reached from higher detection rates in the control group to higher detection rates among the group of patients with respiratory tract diseases.
Prospective studies have been published only recently. Van der Zalm and co-workers reported the detection of WUPyV in a cohort of 18 children. Their parents were contacted twice a week over a 6-months period (November to April) and asked for symptoms of respiratory tract disease in their children. Every two weeks respiratory tract specimens were collected, regardless of respiratory symptoms. 11.5% of the specimens of children with symptoms were WUPyV positive, but only 3.1% of specimens of healthy children, indicating at least an association of WUPyV with disease.
Emerging infectious diseases under this category were subcategorized into 1a, 1b and 1c. Subcategory 1a covers known pathogens that occur in new ecological niches/geographical areas. A few past examples belonging to this subcategory are the introduction and spread of West Nile virus in North America; chikungunya virus of the Central/East Africa genotype in Reunion Island, the Indian subcontinent and South East Asia; and dengue virus of different serotypes in the Pacific Islands and Central and South America.18,19,20,21,22,23 Factors that contributed to the occurrence of emerging infectious diseases in this subcategory include population growth; urbanization; environmental and anthropogenic driven ecological changes; increased volume and speed of international travel and commerce with rapid, massive movement of people, animals and commodities; and deterioration of public health infrastructure. Subcategory 1b includes known and unknown infectious agents that occur in new host ‘niches'. Infectious microbes/agents placed under this subcategory are better known as ‘opportunistic' pathogens that normally do not cause disease in immunocompetent human hosts but that can lead to serious diseases in immunocompromised individuals. The increased susceptibility of human hosts to infectious agents is largely due to the HIV/acquired immune deficiency syndrome pandemic, and to a lesser extent, due to immunosuppression resulting from cancer chemotherapy, anti-rejection treatments in transplant recipients, and drugs and monoclonal antibodies that are used to treat autoimmune and immune-mediated disorders. A notable example is the increased incidence of progressive multifocal leukoencephalopathy, a demyelinating disease of the central nervous system that is caused by the polyomavirus ‘JC' following the increased use of immunomodulatory therapies for anti-rejection regimens and for the treatment of autoimmune diseases.24,25,26 Subcategory 1c includes known and unknown infectious agents causing infections associated with iatrogenic modalities. Some examples of emerging infections under this subcategory include therapeutic epidural injection of steroids that are contaminated with Exserhilum rostratum and infectious agents transmitted from donor to recipients through organ transplantation, such as rabies virus, West Nile virus, Dandenong virus or Acanthamoeba.27,28,29,30,31
HPyV6, thought to be a skin-tropic polyomavirus, was initially described in 2010. Since then, subgenomic fragments of HPyV6 DNA have been detected in a variety of specimen types, including skin, respiratory secretion samples, and various tumor samples (for instance, squamous cell carcinoma, basal cell carcinoma, and multiple myeloma) [8, 32–34]. Although HPyVs had been identified several decades ago, there are still many basic questions to be clarified, for examples, how HPyVs interact with their hosts and how to spread throughout a population. For instance, BKPyV and JCPyV are shed in the urine and transmitted via the respiratory route. No data on the immune responses of healthy individuals to the HPyVs are available. It is unknown whether the HPyVs exist in human cells with low viral replication to establish a latent state, or how the cells restrict viral replication. The answers to these questions will require more information on the biology and epidemiology of the HPyVs.
The genomes and proteins of HPyV6, one of the novel HPyVs, show little sequence homology with previously reported HPyVs (BKPyV and JCPyV). Although HPyV6 encodes a conserved, potentially carcinogenic LTAg, previous studies have shown no association between HPyV6 and tumors. Furthermore, a phylogenetic analysis indicated that LTAg of HPyV6 (KM387421) is only distantly related to its homologues in other cancer-associated HPyVs. The HPyV6, WUPyV, and KIPyV strains formed a clade in the complete genome and VP1 amino acid phylogenies, whether HPyV6 also associate with respiratory infection which need more clinical and experimental evidences to support. HPyV6 has been detected in specimens from the human respiratory tract, but there are as yet insufficient epidemiological data to demonstrate a correlation between HPyV6 and respiratory disease. Because initial infections with most HPyVs occur in infancy, the prevalence of HPyV6 in NPAs from children was detected with real-time PCR.
HPyV6 displayed an overall prevalence of 1.7 % in NPA samples collected from children in a hospital in China, which is similar to its prevalence reported previously (0.5–2 %) [34, 35]. It has not been confirmed that HPyV6 infects humans via the respiratory tract, but the respiratory tract may be a possible route of transmission. In this study, HPyV6 was mainly detected in children less than 5 years of age, and the peak incidence occurred in spring. All 15 HPyV6-positive patients were coinfected with other respiratory viruses, of which IFVA and RSV were the most common. The HPyV6-positive patients were diagnosed with lower RTIs, 60 % had bronchopneumonia, and the most common symptoms were cough and fever.
Although known HPyVs cause disease in patients with immune-system imbalances, they do not seem to cause obvious illnesses in the great majority of infected individuals. However, BKPyV can induce nephropathy in kidney transplantation recipients and JCPyV causes progressive multifocal leukoencephalopathy. Progressively increasing or high viral loads are also associated with high-level viral replication and disease. For instance, progressive multifocal leukoencephalopathy and hemorrhagic cystitis are related to high viral loads of JCPyV and BKPyV, respectively. The present study cannot confirm that HPyV6 is the cause of RTIs in hospitalized children, because the viral loads of HPyV6 were low (1.38–182.42 copies/μl) and the coinfection rate with other respiratory viruses was high.
In previous reports, HPyVs were detected in the respiratory tract and skin but, to date, there has been insufficient evidence that HPyV6 is associated with any respiratory tract disease or skin disease. Whether HPyV6 induce any disease requires the analysis of further data.
Autism is a severe neurodevelopmental disorder affecting the paediatric population (60). Autism spectrum disorders (ASD) include disorders, such as psychomotor regression, language impairment and behavioural social withdrawal, placing patients with ASD in permanent need for healthcare and social support (61). Earlier reports have associated vaccination against MMR with the occurrence of ASD in children (62), thus leading in particularly low vaccination coverage. As a result, outbreaks regarding the vaccine preventable strains have reappeared throughout Europe (63–65), Asia (66,67) and the United States (68,69). Extensive research around the issue has emerged, soundly dissociating MMR vaccination from any ASD occurrence, even in high-risk populations (70–72). However, the loss of credibility of the MMR vaccine remains a concern. This can be partially explained by failure on behalf of the scientific community to effectively communicate: i) the limitations and bias of the original study of Wakefield et al (62) in 1998, ii) the mounting evidence supporting the lack of a causal relationship between MMR vaccine receipt and autism onset, as proven by large epidemiological studies (70–72) and iii) adverse effects of vaccination in the general setting of coincidental, rather than causal associations. Another contributing factor must be attributed to a powerful influence by the public media, such as television, newspapers and internet, regarding MMR vaccination, ultimately leading to a subsequent negative public health response. In the future, more effective communication strategies are required to reassure parents of vaccine safety and importance.
The discovery of novel respiratory viruses has the potential to diminish the diagnostic gap for respiratory tract infections. Creer and co-workers, who omitted the recently identified respiratory viruses in their study on adults, reported a diagnostic gap of 31%. The share of specimens from children negative for any respiratory pathogen investigated was 22% when hMPV, HBoV, and HCoV-NL63 were included. No study included the novel polyomaviruses or herpes viruses until now. Thus, a further reduction of the respiratory tract infections of unknown origin seems reasonable. However, a substantial gap is remaining leaving sufficient room for additional respiratory viruses to be discovered in the future.
So far it has not been finally proven, if WUPyV is a real causative agent for respiratory diseases. Association of virus detection with previously unexplained respiratory disease led to the tempting idea of WUPyV representing a new etiologic agent, particularly in cases where no other respiratory tract pathogen could be identified. Furthermore, the association of mouse pneumotropic polyomavirus with intestinal pneumonia and significant mortality indicates that polyomaviruses have the capacity to be respiratory tract pathogens.
Many studies correlating the detection of WUPyV with the incidence of respiratory symptoms argue for this hypothesis, but it remains difficult to prove as asymptomatic control groups have not been investigated sufficiently. Additionally, the studies including control groups report dissenting findings. However, in two studies asymptomatic individuals display a lower frequency of virus detection compared to symptomatic patients. One of these is the only prospective study available.
The difficulty to prove WUPyV to be a respiratory pathogen may be due to the fact that most studies available so far try to correlate the virus with respiratory tract disease in general. In case of WUPyV being the causative agent of a particular entity of respiratory tract disease, inclusion of patients displaying any kind of respiratory disease may bias the investigation. Focussing on a single entity of respiratory disease proved successful for HCoV-NL63, which was shown to cause croup.
No association of WUPyV viral loads and clinical symptoms could be observed so far, and co-infections with other viruses were described frequently. This weakens the hypothesis of WUPyV being a respiratory tract pathogen. However, collection of samples was not performed by means of a standardized protocol among the various groups or at a defined time point, after onset of symptoms. As viral loads decrease during the acute phase of infection, the combination of longitudinal data from different time points in one analysis may bias the correlation of viral loads and symptoms. Furthermore, detection of co-infection does not exclude pathogenic potential for WUPyV, as early childhood is characterized by subsequent episodes of respiratory infections. Co-detection of the declining pathogen responsible for the last episode of respiratory disease and the pathogen responsible for the present, acute disease has to be expected. Only determination of the viral load kinetics would allow defining the clinical impact of a detectable microorganism.
The large T-antigen of WUPyV contains binding sites for the RB protein and p53. Thus, tumourigenic potential cannot be excluded. However, the association of WUPyV with other diseases, particularly tumour diseases has not been sufficiently investigated yet.
Taken together, the currently available data neither prove nor deny WUPyV to be a respiratory tract pathogen. Several scenarios may describe the role of the virus best. WUPyV may be
part of the endogenous viral flora without pathogenic potential;an opportunistic pathogen with pathogenic potential in the respiratory tract under conditions still to be defined;a pacemaker for secondary infections;a viral pathogen using the respiratory tract only as an entry route to reach the final target cell; infection of this cell type is the basis of a disease not related to the respiratory tract.
To reach a final conclusion more powerful, controlled studies need to be performed prospectively. However, retrospective analysis of age- and time-matched control populations also permits conclusions if the data collected at the time are related to respiratory illness. Ideally, prospective studies would be carried out in a multicenter-design including control groups concentrating on the correlation of WUPyV with single entities of respiratory tract diseases. Criteria for inclusion of specimens have to be comparable, as well as the time point and the protocol for extraction of specimens. All known or suspected respiratory tract pathogens should be included and follow-up investigations should be performed. The detection method of choice would be quantitative PCR to allow discrimination between colonisation and productive infection. Additionally, serological investigations should be included to show that the patients seroconvert in response to an infection. The study participants should be evaluated before enrolment. Beside investigation of respiratory tract disease, the cell type hosting the persisting virus has to be identified and a possible association with tumour diseases has to be investigated.
Several studies have reported the widespread distribution of MWPyV in different areas of the world, including Africa, the USA, Asia, Italy, Australia, and South America [5, 11–15]. In this study, we used a qPCR assay to rapidly and accurately screen for MWPyV in NPA, serum, and fecal samples collected from different populations in China. Thirteen (1.47%) of the samples from children with ARIs and three (1.72%) from children with diarrhea were positive for MWPyV, whereas no serum (n = 200) was positive for MWPyV. In brief, the analysis of 1261 clinical samples only detected MWPyV in respiratory and fecal specimens from children, suggesting that the establishment of the primary infection occurs at an early age, and that the gastrointestinal and respiratory tracts are sites of viral persistence. This indicates a possible fecal–oral route of transmission during early childhood.
To verify the specificity of the primers and probe used in this study, other viruses (WUPyV, KIPyV, JCPyV, BKPyV, and HPyV7) were also tested, but no fluorescent signal was detected. We excluded any false-positive results by sequencing all the PCR products. It is noteworthy that all the 72-bp PCR products from positive samples shared 100% identity with the corresponding genomic regions of MWPyV, HPyV10, and MXPyV, accessed from GenBank. The sequences of the complete VP1 genes confirmed that NPA samples BJ289, BJ531, BJ538, and BJ541 contained MWPyV, and revealed that the viruses in the last three samples contained 5–10 single-base mutations. In total, 16 MWPyV-positive samples were detected in this study, and the cycle threshold (Ct) values for most of these were high (average Ct = 36.62, range 31.42–38.61), suggesting that the viral load was low. It is possible that the PCR failed to amplify the complete VP1 gene sequences from the other 12 positive samples.
Most ARIs are caused by viruses. In this study, the prevalence of MWPyV in NPA was 1.47%. This is similar to findings in Australia (1.5%), but differs from a study in Mexico, which reported a detection rate of 0.74% in nasal washes from children suffering ARI. In a study by Rockett et al., MWPyV was the most prevalent polyomavirus (56%) detected in respiratory specimens from children with ARI. These findings are supported by other studies that frequently detected MWPyV in respiratory samples from acutely ill patients and babies with upper respiratory symptoms [13, 17]. Possible explanations for these discrepancies may be the use of different detection assays, differently defined cut-off values, different geographic regions examined, and the varying characteristics of the study populations.
MXPyV, MWPyV, and HPyV10 are presumably human tropic, and appear to be largely confined to the gastrointestinal tract. For this reason, we tested fecal specimens from children with a diagnosis of viral diarrhea. We detected MWPyV in 1.72% of fecal specimens from these children, which is lower than previously reported rates of 2.3% and 5.9% in children with diarrhea in St Louis, USA, and Australia [5, 17]. These discrepancies are probably attributable to the different periods at which the samples were collected and the different detection methods used. The detection of MWPyV in the feces of children with diarrhea indicated that MWPyV may not be a strong candidate for gastroenteritis in children because of the co-infection with rotavirus.. WUPyV, KIPyV, SV40, BKV, JCV, MXPyV, and MCV have also been detected in human feces [6, 18–21], although their primary sites of pathology are elsewhere in the human body.
Seroepidemiology plays an important role in establishing the link between HPyVs and disease and in understanding the dynamics of infection. In this study, MWPyV was undetectable in the blood of healthy adults, consistent with other published data, and MWPyV was not previously detected in the blood of autoimmune children, as has also been observed for the MXPyV, JCPyV, and BKPyV. However, in contrast to our findings, the seroprevalence of MWPyV in adulthood was 42% in a large sample from Italy, 66% in a sample from Colorado, 84% in 1614 serum samples from the Czech Republic, and almost 100% in samples from New Hampshire, USA. These contrasting data may be attributable to the relatively small number of blood samples we analyzed (n = 200) or a different MWPyV may have been isolated from the human samples, to which have affected the antibody binding during serological analysis. Moreover, MWPyV DNA is more likely to be detected in blood during the primary infection than during viral reactivation. In a study by Nicol et al., MWPyV was cleared or the virus was not reactivated with age, as has been reported for TSPyV, which may also explain why we detected no MWPyV-positive sera.
Factors such as age, sex, the time of year when multiple viruses circulate, and a history of immunosuppression are associated with an increased chance of viral co-infection. In this study, 14 of the 16 MWPyV-positive patients were co-infected with other viruses, and four of these had multiple infections. The primers and probes used in this study are listed in Additional file 1: Table S1 [28–30]. Influenza A virus and HCoV were the respiratory viruses most frequently detected in the MWPyV-positive patients, which is consistent with their frequent detection with WUPyV and KIPyV in respiratory samples. The detection rate of Influenza A virus was 69.2%, followed by HCo-OC43, and then PIV3, the predominant subtype among PIVs. A comparison of the clinical symptoms experienced by the patients with and without co-infections indicated that co-infection with MWPyV did not affect the severity of the illness. 11 MWPyV-positive children with ARIs were diagnosed with bronchopneumonia, and their commonest symptoms were fever and cough. The symptoms of the three MWPyV-positive children with diarrhea who were co-infected with Rotavirus included diarrhea and fever. Our data suggest that primary MWPyV infections occur in childhood, predominantly sporadically. However, it is possible that MWPyV is shed within both the gastrointestinal and respiratory tracts. Understanding these factors will help us prevent the transmission of MWPyV infections. To our knowledge, this is the first report of the epidemiological and clinical profiles of MWPyV in children hospitalized with ARIs or diarrhea in China, based on a real-time qPCR specific for MWPyV.
Real-time PCR is an effective tool for the detection of MWPyV in different types of samples. MWPyV infection mainly occurs in young children, and fecal–oral transmission is a possible route of its transmission.
The detection rate for HPyV6 by real-time PCR assay was 1.7 % in 887 NPA samples collected from hospitalized children with RTI. An association between HPyV6 and respiratory diseases could not been revealed due to the high coinfection rate and the low HPyV6 viral load.
MCPyV is a newly-discovered small, human DNA virus, which causes a widespread, previously unrecognised, human infection in adulthood and childhood (92,93). It is classified in the family Polyomaviridae, a group of non-enveloped, double-stranded DNA viruses with icosahedral symmetry, which can infect a variety of vertebrates, including humans and can cause malignant tumours upon their inoculation into heterologous hosts (94). First described in January 2008, the prototype sequence of MCPyV has a 5,387 base pair genome and contains the early region, which encodes the large tumor (LT) antigen and the small tumour (sT) antigen and the alternative tumor antigen open reading frame (ALTO), the late region, which encodes VP 1, VP 2 and VP 3 and a non-coding regulatory region. Of note, the genome of MCPyV has been detected in approximately 80% of Merkel cell carcinoma (MCC) cases (95). Although MCC is a relatively rare, highly aggressive, human skin cancer of neuroendocrine origin, its worldwide incidence has increased over the past twenty years from 500 to 1,500 cases per year. In the majority of MCC cases, MCPyV is integrated into the host genome in a monoclonal manner and the viral T antigen has truncating mutations, which render the T antigen unable to initiate the DNA replication required to propagate the virus (94).
Recent serological data using enzyme-linked immunosorbent assay (ELISA) techniques have suggested that MCPyV infection is common in childhood and occurs during early childhood, after the disappearance of specific maternal antibodies against MCPyV (93). MCPyV DNA has been detected in nasopharyngeal aspirate samples collected from children, indicating the presence of MCPyV in the upper respiratory tract of children (96,97). MCPyV is acquired in childhood through close contact involving saliva and the skin (93). As to date, the mode of MCPyV transmission has not been fully elucidated, the precise role of the respiratory secretions in MCPyV transmission in childhood requires further investigation. The respiratory tract system is involved as a unique reservoir of MCPyV in children and respiratory secretions seem to play a significant role in MCPyV transmission in childhood. Moreover, future studies are required in order to fully elucidate the potential implications of MCPyV infection in neoplasms in children.
Hepatitis B is found in virtually every region of the globe. Of the more than 2 billion people who are or have been infected, 350 to 400 million are carriers of the chronic disease; the remainder undergo spontaneous recovery and production of protective antibodies. Nearly 100% of infected infants (that is, those born to HBV-infected mothers) become chronically infected. The risk of developing a chronic infection decreases with age.
At least 30% of those with chronic HBV infection experience significant morbidity or mortality, including cirrhosis and hepatocellular carcinoma. Most people do not know they are infected until they present with symptoms of advanced liver disease, which means that infected individuals can spread the infection unknowingly, sometimes for many years. Although oral antiviral therapies are effective at stopping HBV replication, they do not cure the disease. Therefore, therapy is usually lifelong. Treatment is also complicated by the development of drug resistance and side effects. A vaccine against HBV is safe and effective in 90 to 95% of people; however, the individuals who are most at risk of becoming infected are often those with limited access to the vaccine, such as marginalized populations or people living in resource-limited countries.
There is substantial evidence that an individual's likelihood of recovering from an acute HBV infection or developing severe sequelae from infection is influenced, in part, by genes [39–45]. Candidate gene and genome-wide association studies have identified variants associated with HBV-related disease progression or hepatocellular carcinoma in various populations [46–52]. Treatment response to interferon (IFN)-α has been associated in some, but not all, studies with IFNλ3 polymorphisms. Finally, specific gene variants (HLA and non-HLA alleles) have been associated with vaccine response and non-response [54–57].
Examples of past emerging infectious diseases under this category are antimicrobial resistant microorganisms (e.g., Mycobacterium tuberculosis, Plasmodium falciparum, Staphylococcus aureus) and pandemic influenza due to a new subtype or strain of influenza A virus (e.g., influenza virus A/California/04/2009(H1N1)).9,32,33,34,35 Factors that contribute to the emergence of these novel phenotype pathogens are the abuse of antimicrobial drugs, ecological and host-driven microbial mixing, microbial mutations, genetic drift or re-assortment and environmental selection. Accidental or potentially intentional release of laboratory manipulated strains resulting in epidemics is included in this category.
In the last few years, a number of viruses have been found in association with enteric disease in cats. Many of them have been discovered serendipitously, using advanced molecular techniques to screen feline stool samples. The epidemiology of these newly discovered viruses is still largely unexplored, but several pieces of evidence suggest their possible role as primary causative pathogens or synergistic agents in feline gastrointestinal disease. In order to obtain a complete picture, each novel enteric virus should be included in the panel of pathogens for routine testing of cases of feline enteritis. Furthermore, large structured epidemiological studies and experimental infections might help to clarify any possible association with enteric diseases.
Interestingly, some of the viruses considered in this review have also been identified in the canine fecal virome, suggesting the possibility of inter-species circulation between the two carnivore species. Cats and dogs may harbor NoVs of the same genogroups and genotypes, GIV.2 and GVI.2. Binding of GVI.2 and GVI NoVs in dog tissues has been demonstrated to be mediated by the presence of the H and A antigens of the histo-blood group antigen (HBGA) family. Accordingly, it has been hypothesized that dogs and cats share a similar pattern of HBGAs as the attachment factor for NoV infections. Meanwhile, novel carnivore protoparvoviruses identical to each other in their capsid gene (>99.9% nt identity) have been found in stool and respiratory samples either in cats or dogs. Since a few aa mutations in the VP2 can modify the host range of FPV and CPV-2, it has been hypothesized that the novel carnivore protoparvovirus 2 has recently crossed the species barrier from a yet unidentified source, with a recent bottleneck event in the evolution of this virus in domestic carnivores.
The global distribution of cats and their close contacts with humans represents an additional reason to better understand the composition of their enteric virome. Historical evidence suggest that some feline viruses are potentially zoonotic. Infection of young children by rotavirus strains of feline origin has been documented in Italy and more recently, in Germany. The discovery of GIV.2 NoVs in cats and dogs genetically closest related to human GIV.1 NoVs has raised public health concerns about potential interspecies transmission between humans and pets. This eventuality has been demonstrated in a serosurvey performed in Italy on a collection of human serum samples, in which specific IgG antibodies against VLPs based on the lion GIV.2 NoV have been detected with prevalence ranging from 6.8% to 15.1% among different age groups. Furthermore, in a study conducted in Portugal, the presence of antibodies to GVI.2 NoV were found in 22.3% of the veterinarians and 5.8% of the control group, revealing for the small animal veterinarians an increased risk for exposure to this virus. Besides NoVs, the zoonotic potential is also suspected for other caliciviruses as the novel 2117-like vesiviruses (VeVs), firstly identified in dog stool samples in Italy. IgG antibodies against the canine 2117-like VeVs have been detected in 7.8% of Italian human sera and, more recently, the RNA of a 2117-like VeV was detected in the feces of a clinically healthy cat. Accordingly, understanding the ecology of novel enteric viruses in cats will be helpful also to assess more precisely if and to which extent pets may pose a risk of infection for humans.
Between 2012 and 2013, we tested by multiplex real-time RT–PCR [21–23] a total of 1,454 specimens from pediatric respiratory infections. Out of these, 57 remained negative after the analysis with different molecular assays. In order to shed light on the etiology of these infections, these 57 specimens were subjected to target-independent HTS analysis, along with 70 age-matched specimens from a control group. Similarly to the 57 specimens from the respiratory infections, the specimens from the control group resulted negative in the aforementioned molecular assays.
Upon HTS analysis we further identified known viral respiratory pathogens (HRSV, HRV, EV, HPIV, HCoV, HMPV and Influenza B virus). Altogether, at least one respiratory viral pathogen could be identified in 35 out of the 57 NPA specimens from the group of respiratory infection. By contrast, in the control group, only 2 samples contained contigs attributed to viral respiratory pathogens (HMPV and HRV). Moreover, the HMPV contigs detected in one of these samples, hoarded the vast majority of the reads assigned to any eukaryotic virus (99.1%). The remaining viral entities were anelloviruses and papillomaviruses common to both groups.
Identification of respiratory viruses by HTS was confirmed by contig-specific RT–PCR analysis. The HMPV and HRV identified in the control group occurred in two cases that showed no symptoms of respiratory disease. Asymptomatic infections by HRV have been previously reported and are more frequent in young children. Although asymptomatic infections by HMPV can occur at the pediatric stage, they are more frequent in immunocompetent adult individuals [31–33].
Apart from common respiratory viral pathogens, Human parechovirus 1 (HPeV-1) and Human polyomavirus 4 (WU polyomavirus) were identified by HTS analysis. Both viruses were identified in specimens from the group of respiratory infection (1 sample each); none in the control group. The detection of HPeV-1 is not surprising as this virus is a frequent cause of infection in childhood, where it causes mild gastrointestinal and respiratory disease. Human polyomavirus 4 (WU polyomavirus) was originally detected in respiratory secretions of a pediatric patient diagnosed with pneumonia of unknown origin, and from patients with acute respiratory co-infections. Nonetheless, a subsequent larger study found no link between WU polyomavirus and acute respiratory disease. In our study, WU polyomavirus was identified in one case of respiratory infection. Although no other respiratory virus was identified in this sample, WU polyomavirus presents low prevalence in cases of respiratory infection and high rates of co-infection with other common respiratory viral pathogens, and further studies are needed to ascertain its clinical significance [35–37].
Thus, out of 57, 21 (36.84%) remain unexplained from a virological standpoint, as no known respiratory or novel virus was identified. Analysis by BLASTn only returned matches to anelloviruses, papillomaviruses, and Human PoSCV5-like circular virus. In spite of thorough, in-depth analysis of those contigs with no match as well as of the unmapped reads, no viral match in the GenBank database was obtained. Anelloviruses are frequently detected in most tissues and organs, including the respiratory tract of healthy individuals, and there is no association to any disease in humans. Papillomaviruses are very common worldwide, and most infections are asymptomatic and resolve spontaneously. Apart from the common warts and the various types of cancer (including an oropharyngeal form) associated to infection by HPV, low-risk types 6 and 11 are the predominant cause of respiratory papillomatosis, a disease in which noncancerous tumours grow in the air passages of the respiratory tract. However, other than oropharyngeal cancer and respiratory papillomatosis, there is no evidence of an association between papillomavirus infection and respiratory disease. In addition to the lack of evidence for an association with respiratory disease, in our study anelloviruses and HPV were also identified in the control group, strengthening the conclusion that their presence was not related to the respiratory disease. Human PoSCV5-like circular virus was recently identified in respiratory secretions from an unexplained human case of febrile illness, although its association with disease was not determined. Small, circular, single stranded, REP-encoding, DNA (CRESS-DNA) viruses have been increasingly identified by metagenomic HTS techniques in environmental samples and in a variety of vertebrates as well as various invertebrates [42–45]. In humans, they have been reported in feces of healthy individuals, and in samples from unexplained cases of encephalitis and diarrhea, pericarditis, acute central nervous system infections, as well as in NPAs from children with respiratory infections. However, neither of these studies could establish a direct association with disease. Thus, it is not clear what is the value of finding contigs that match this virus in the specimens from the group of respiratory infection. Further studies are needed to determine their role (if any) in human respiratory disease.
Our findings agree with previous studies with similar design and tools. Xu et al. employed HTS to analyse a set of respiratory specimens taken from children with community-acquired pneumonia that had returned negative results in a commercial respiratory viral panel detection assay. They also identified HPIV, Torque teno virus, Torque teno minivirus, and WU polyomavirus. Zhou et al. studied cell-cultured supernatants with apparent cytopathic effect that had been prepared from undiagnosed respiratory specimens and identified a high prevalence of EV accompanied by HRV, HRSV, HPIV, AdV, Influenza C virus, Herpesvirus 1 and Dengue virus. Taboada et al. studied nasal washings from children with respiratory infections previously found negative for common bacterial and viral respiratory pathogens by PCR, where they identified at least one known respiratory virus (including HRSV, HCoV, and HRV) in the vast majority of the specimens. Neither of these studies revealed the presence of any putative novel virus on the undiagnosed respiratory infections subject of study, and the large majority of the cases could be attributed to known viral respiratory pathogens. These studies suggest that all respiratory viral pathogens of clinical relevance during the pediatric stage have already been identified. In our study, we analysed 57 cases that, out of a total of 1,454, were negative in all pathogen-specific PCR assays. In consequence, only 3.9% (57/1,454) of all respiratory specimens collected during the entire epidemic season remained without a known etiology of infection after using pathogen-targeted diagnostic techniques. While target-independent HTS analysis allowed us to come to a specific diagnostic in more than half of the 57 undiagnosed infections, all the additionally resolved cases were produced by known respiratory pathogens. From these results, we can conclude that the current pathogen-specific techniques should be able to diagnose the vast majority of the respiratory infections. In consequence, we can conclude that the risk of overlooking a novel unknown viral respiratory pathogen in the pediatric population is very low when using target-specific diagnostic methods and that there is very little value in using target-independent assays. Routine virological surveillance based on target-specific techniques, such as real-time (RT)PCR or target enrichment HTS approaches (which use probes specifically designed against the viral genomic sequence of interest in order to enrich specifically for sequencing libraries derived from said virus), is appropriate and constitute a first-line diagnostic tool.
The question about the etiology behind those cases of respiratory infection that remain negative for all routine diagnostic assays have been previously addressed by several groups [24,25,50–52]. Although in our study we used an overall approach which was similar to such previous studies, we introduced several improvements to our specific study design in order to address different limitations of the previous works. The identification of viral reads in clinical specimens remains controversial because it does not necessary imply that such viruses are responsible for the symptoms observed. Many viruses can cause asymptomatic or subclinical infections, or simply be present among the normal, healthy microbiota and replicate without any pathogenic consequences. This hinders the interpretation and understanding of the results unveiled by virus discovery studies based on HTS in regard to their clinical significance. In our study, we included a contrast study population (control group) formed by a prospective cohort of healthy individuals. Such healthy control group was matched at all the critical levels with the cohort of patients with unexplained infection of the respiratory tract: (i) The individuals of the control group were age-matched; (ii) They came from the same geographic area; (iii) Their samples were also taken systematically during the same time window covering the epidemic season of virus circulation. By including the control group, we were able to determine that the viruses detected in the clinical specimens taken from the patients with an infection of the respiratory tract were not circulating among the healthy population, providing evidence that such viruses were responsible for the respiratory disease observed in those cases. This would acquire special importance if a novel viral entity for which there is no previous information available is detected. In summary, the inclusion of the healthy control group allowed us to assess the clinical relevance of the viruses identified in the samples taken from the cases of unexplained respiratory infections. Whenever possible, any future viral discovery studies should include a matched healthy control group to which the viruses identified by HTS in the patients with clinical disease can be contrasted.
In addition to the inclusion of a matched healthy control group, we processed and sequenced in parallel negative controls consisting on sterile nuclease-free water (see the Materials and Methods section) and we confirmed the viral identifications made by HTS with specific RT–PCR assays. The objective of these procedures was to minimize the chances of reporting any false viral identification. It is worth highlighting also, that we performed a systematic sampling on our study groups: samples were collected from all the children arriving consecutively at the hospital during the period of the study, whose parents or legal guardians gave explicit written consent, and according to pre-established clinical and medical criteria (see the Materials and Methods section) with no further selection. By combining all the procedures discussed above, our study design constitutes a novel integrated approach that ensures a robust representativeness of the results, minimizes any possible bias, and provides a better understanding about the clinical implications of the viral identifications made by HTS analysis.
While HTS was superior in the overall rate of detection of pathogens, that was not due to the presence in the cohort of unknown pathogens, but mostly on the underperformance of molecular methods (real-time RT–PCR) against targeted pathogens. All the viruses additionally identified by HTS in our study were well-known respiratory pathogens, and no novel viruses were detected. Altogether, our results show that already known viral respiratory pathogens play a main etiologic role behind the unexplained cases of respiratory infection in the pediatric population. This is a very significant finding, because if extrapolated to other clinical syndromes and specimens, it might allow us to quantitatively assess the risk. Under that new paradigm, “Agnostic” technologies would still have a role in pathogen detection under outbreak and event situations where a true “unknown unknown” is suspected, but “Targeted” approaches would become desirable for Next-generation sequencing-based microbial diagnostic. This paradigm might be desirable from a regulatory standpoint for those diagnostic laboratories seeking to incorporate these technologies, since “Targeted” approaches allow for specific and reproducible library enrichment and thus they are easier to assess and validate.
Picornaviruses are positive-sense, single-stranded RNA viruses with icosahedral capsids. They infect various animals and human, causing various respiratory, cardiac, hepatic, neurological, mucocutaneous and systemic diseases [1, 2]. Based on genotypic and serological characterization, the family Picornaviridae is currently divided into 29 genera with at least 50 species. Among the various picornaviruses belonging to nine genera that are able to infect humans, poliovirus and human enterovirus A71 are best known for their neurotropism and ability to cause mass epidemics with high morbidities and mortalities [3, 4]. Picornaviruses are also known for their potential for mutations and recombination, which may allow the generation of new variants to emerge [5–10].
Emerging infectious diseases like avian influenza and coronaviruses have highlighted the impact of animal viruses after overcoming the inter-species barrier [11–15]. As a result, there has been growing interest to understand the diversity and evolution of animal and zoonotic viruses. For picornaviruses, numerous novel human and animal picornaviruses have been discovered in the past decade [1, 16–27]. We have also discovered a novel picornavirus, canine picodicistrovirus (CPDV), with two internal ribosome entry site (IRES) elements, which represents a unique feature among Picornaviridae. Moreover, novel picronaviruses were identified in previously unknown animal hosts such as cats, bats and camels [29–31], reflecting our slim knowledge on the diversity and host range of picornaviruses. The discovery and characterization of novel picornaviruses is important for better understanding of their evolution, pathogenicity and emergence potential.
Although rodents can be infected by several picornaviruses, the picornaviral diversity is probably underestimated, given the enormous species diversity of rodents. Moreover, little is known about the pathogenicity of the recently discovered rodent pricornaviruses, such as rodent stool-associated picornavirus (rosavirus) A1, mouse stool-associated picornavirus (mosavirus) A1, Norway rat hunnivirus and rat-borne virus (rabovirus A) [32, 33]. In this report, we explored the diversity of picornaviruses among rodents in China and discovered two potentially novel picornaviruses, “Rosavirus B” and “Rosavirus C”. While rosavirus B was detected in the street rat, Norway rats, rosavirus C was detected in five different wild rat species, suggesting potential interspecies transmission. Their complete genome sequences were determined, which showed that “Rosavirus B” and “Rosavirus C” represent two novel picornavirus species distinct from Rosavirus A. Rosavirus C isolated from cell culture causes multisystemic diseases in a mouse model, with histopathological changes and positive viral antigen expression in lungs and liver of infected mice.
Acute viral infections such as influenza also have profound impacts on global health. In contrast to the yearly epidemics caused by seasonal influenza, a pandemic can occur when a new virus emerges in a naive population and is readily transmitted from person to person. The US Centers for Disease Control (CDC) estimates that the H1N1 2009 pandemic resulted in 41 to 84 million infections, 183,000 to 378,000 hospitalizations, and nearly 285,000 deaths worldwide. Although the morbidity and mortality of that pandemic were lower than feared, public health professionals continuously monitor for the emergence of more virulent strains.
As an airborne infection, influenza is transmitted easily and quickly, and its effects can be acute, although there is wide variability in response to infection. Much of the heterogeneity in the severity of seasonal influenza infections has been attributed to the degree of acquired immunity in the population affected, patient co-morbidities and the virulence of the strain. Also, influenza epidemics and pandemics are often caused by the introduction of novel viruses for which most people have limited acquired immunity. The emergence of new strains, and the lack of cross-protection by existing vaccines, does not leave much time for vaccine development. In pandemics, including the H1N1 2009 influenza pandemic, healthy young individuals with no co-morbidities have comprised a significant proportion of fatal and severe cases. These pandemics have provided an opportunity to evaluate the host innate immune response among populations without underlying background immunity.
Research has identified genetic factors associated with severity of illness due to influenza [63–65] and death from severe influenza. Genetic information about immune response to influenza could inform vaccine development and distribution, and disease treatment strategies. Several candidate gene studies suggest that variations in HLA class 1 and other genes contribute to differences in antibody response to influenza vaccines. Ongoing experience with vaccine use has provided opportunities to learn about the potential role of genetics in vaccine safety and efficacy.
We used a high throughput sequencing strategy to search for novel agents that were present in respiratory tract infections of unknown etiology. The focus of this study was on individual clinical specimens that still lacked a diagnosis after analysis with an extensive panel of diagnostic assays for known respiratory viruses. In one such patient sample, novel sequences with limited homology to known polyomaviruses were detected. Complete genome sequencing and phylogenetic analysis revealed that the new virus clearly had the genomic organization typical of polyomaviruses but was divergent from all previously described polyomaviruses. In keeping with the two-letter virus names for human polyomaviruses, we have named this novel polyomavirus WU virus. Overall, the predicted amino acid sequences of WU virus proteins were most similar to the newly described KI virus (Table 1). Outside of KI, WU shared only ∼15%–49% identity to its closest relatives (Table 1).
Detailed analysis of the viral DNA sequence and genomic organization confirmed the novelty of WU virus. At all loci, WU virus was most similar to KI virus, but the degree of divergence between WU and KI was greater than the divergence between SV40 and BK, indicating that WU and KI are clearly distinct viruses (Figure 2). Based on the phylogenetic analysis, it appears that WU and KI define a novel branch within the Polyomaviridae family (Figure 2). Relative to the established polyomaviruses, some analyses suggested that the WU/KI branch might be more closely related to the primate polyomaviruses, while other features of the WU genome suggested that it might be more similar to murine polyomavirus. For example, neighbor-joining phylogenetic analysis suggested that the predicted STAg, LTAg, and VP1 open reading frames of both KI and WU were most closely related to SV40, JC, BK, and baboon polyomaviruses. Analysis of the VP2/VP3 region was more equivocal, as the proteins were too divergent to reliably assess. The apparent absence of the C-terminal “host range” domain in the LTAg and the agnoprotein open reading frame, both of which are present in the known primate polyomaviruses, suggested that WU virus was more similar to murine polyomavirus than the primate polyomaviruses by these criteria. While the evolutionary history of this virus is not clear at the moment, the totality of the analysis indicates that WU is clearly a unique virus.
We detected WU in 37 out of 1,245 (3.0%) patient specimens in Brisbane (excluding the original case) and in six out of 890 (0.7%) patient specimens tested in St. Louis. As the positive specimens were all collected from 2003 through 2006, it appears that WU is currently circulating, and its presence in both North America and Australia suggests that the virus is geographically widespread in the human population. The age range of patients that tested positive for WU virus spanned from 4 months to 53 years. The majority (86%) of the cases were found in children 3 years of age and under. Of the four positive specimens from adult patients (S1, S6, B1, and B3 in Table 2), three clearly had altered immune status. One patient was HIV-positive, one was immunosuppressed due to treatment for Wegener granulomatosis, and one was pregnant. The fourth adult patient (S1), while not obviously immunosuppressed, also suffered from liver cirrhosis, hypertension, type 2 diabetes, and co-infection with herpes simplex virus, and required mechanical ventilation. In addition, there were two other positive patients older than 3 years of age: a 6-year-old child who had previously been a bone marrow transplant recipient (Table 2, B27) and a 6-year-old child diagnosed with acute lymphoblastic leukemia (Table 2, B9). While preliminary, the age distribution of the positive cases in this study combined with the established paradigms for BK and JC virus suggest a model where acute infection with WU virus may occur relatively early in life and result in a latent infection. Immunosuppression or other insults such as viral infection could then lead to reactivation of WU virus in older individuals.
The patients who yielded positive specimens suffered from a wide range of respiratory syndromes, including bronchiolitis, croup, and pneumonia as well as other clinical maladies (Table 2). Detection of WU virus sequences in these patients is merely the first step in assessing the potential etiologic role of WU virus in acute respiratory tract disease. It is not yet known whether WU is infectious or whether it is capable of replication in the respiratory tract. One possibility is that WU is not involved at all in respiratory disease, but rather is simply transmitted by the respiratory route. The human polyomaviruses BK and JC are hypothesized to be transmitted by the respiratory route before taking up residency primarily in the kidneys. Latency in the kidneys of BK and JC is believed to be the reason that both viruses are excreted in the urine of up to 20% of asymptomatic individuals. In this study, using the same PCR assays that were effective in respiratory secretions, we did not detect WU in any of the 727 urine samples we tested. The lack of detection of WU virus in the urine may reflect sensitivity issues, a bias in the cohorts tested, or simply that WU is unlike BK and JC viruses and is not secreted in the urine. A similar tissue profile to that of WU virus has been reported in initial studies of KI virus. Future experiments will aim to determine the tissue tropism of WU and whether any tissue reservoirs for WU virus exist.
In the literature, there is one animal polyomavirus that has been found extensively in lung tissue. Infection of suckling mice with the mouse pneumotropic polyomavirus (MPPV) causes interstitial pneumonia and significant mortality. MPPV also differs from other polyomaviruses in that besides the kidneys, it can also be detected in the lungs, liver, spleen, and blood of suckling mice. Thus, there is precedence for an animal polyomavirus causing respiratory disease, suggesting at least the possibility that WU virus could be similarly pathogenic in humans.
One striking observation from these studies is the relatively high frequency of co-infection detected in the respiratory secretions: 72% overall (100% in the St. Louis cohort and 68% in the Brisbane cohort). Although more extensive studies are necessary to confirm the generality of this observation, this raises several intriguing non-mutually exclusive possibilities to consider: 1) WU may be an opportunistic pathogen; 2) WU infection may predispose or facilitate secondary infection by other respiratory viruses; and 3) WU may be a part of the endogenous viral flora that is reactivated by inflammation or some other aspect of viral infection. Recent studies of the prevalence of the newly identified human bocavirus have also reported higher levels of co-infection than previously described for other viruses found in the respiratory tract, with co-infection rates as high as 50% reported. In addition, five of six samples positive for KI virus were reported to be co-infected with other known respiratory viruses. As detection methods improve in sensitivity and more comprehensive efforts are made to examine the diversity of viruses found in the respiratory tract, a greater appreciation for the rates of dual or multi-infection is gradually emerging. For example, the use of extensive panels of PCR assays in this study revealed that one of the positive specimens was quadruply infected; adenovirus, rhinovirus, and bocavirus and WU virus were all present. Further investigations that aim to systematically define the spectrum of viruses present in the respiratory tract are clearly warranted so that the possible roles that co-infections may play in disease pathogenesis can be explored.
Extremely high sequence divergence was observed in the capsid proteins VP1 and VP2 of WU virus and KI virus as compared to the other known polyomaviruses. This divergence may reflect a different “lifestyle” for the WU/KI branch as compared to known polyomaviruses. Our data demonstrating the presence of WU in respiratory secretions and its absence in urine samples suggest that the mode of transmission or the sites of persistence of WU may be distinct from the other human polyomaviruses. As such, the structure of the virion must be optimized to enable the virus to survive dramatically distinct physiological and environmental conditions. This may partially explain the observed sequence divergence in the capsid proteins.
Another question raised by this study relates to the potential antigenic cross reactivity of the WU capsid proteins. In terms of establishing the seroprevalence of WU itself and determining whether seroconversion accompanies acute infection with WU, it will be essential to conduct these studies with consideration for potential cross reactivity to KI, BK, JC, and SV40 antibodies. In addition, it is tantalizing to speculate whether serum antibodies to WU have the potential to cross react to SV40-derived antigens, and if so, whether they may at least partially account for some of the studies that report the presence of SV40 antibodies in the human population that is too young to have suffered exposure from contaminated polio vaccination [30–32].
In conclusion, we have identified and completely sequenced the genome of a novel polyomavirus. This virus appears to be geographically widespread in the human population as evidenced by the detection of 44 distinct cases in two continents. Based on preliminary analysis, WU and KI virus share some strikingly similar properties, including their complement of genes, phylogenetic relationship, and physical sites of detection in the human body. These data suggest that WU virus and KI virus define a novel branch within the Polyomaviridae family with unexplored biology and pathogenicity. Another implication of these results is that the diversity of viruses in this family may be far greater than currently realized. Further experimentation is now underway to determine the relative pathogenicity of WU virus in humans and to understand the molecular properties of the virus. Since the T antigen of WU is predicted to have transforming properties by analogy to other polyomavirus T antigens, one question currently under investigation is whether a subset of human tumors may be associated with WU.
Because BK and JC virus are frequently excreted in urine, we examined urine samples from patient cohorts in both St. Louis and Brisbane for the presence of WU virus by PCR. In the St. Louis cohort, urine samples from 200 adult patients participating in a study of polyomavirus infections in kidney transplant recipients were tested. For most patients, samples were tested at three time points: prior to transplant, 1 mo post transplant, and 4 mo post transplant, although for some patients the pre-transplant specimen was not available. Zero out of 501 samples tested were positive for the WU polyomavirus. As a control, using previously validated BK primers, we were able to amplify BK virus in a subset of these urine samples, confirming the integrity of the specimens themselves (unpublished data). Similarly, from the Brisbane cohort, none of the 226 urine samples tested were positive for WU virus.
Four distinct papillomavirus sequences were identified in two bat species, E. helvum (Eidolon helvum papillomavirus 8; EhPv-8) and T. perforatus (T. perforatus papillomavirus 1, 2 and 3; TpPv-1, -2, -3). Complete coding sequences were recovered for EhPv-8 (acc. no. KX434763, 6985 bp), TpPv-1 (acc. no. KX434764, 7180 bp) and TpPv-3 (acc. no. KX434767, 7083bp). EpPv-8 shared 63% aa similarity to E. helvum papillomavirus type 1 across the DNA helicase protein (E1). TpPv-1, TpPv-2, and TpPv-3 (acc. no. KX434766, partial genome) shared 44%, 47% and 44% aa similarity across the E1 protein with human papillomavirus 63, Miniopterus schrebersii papillomavirus 1, and Castor canadensis papillomavirus 1, respectively (Fig 7).
In cases where a cluster of patients with similar symptoms presents itself, there can be an investigation to look for epidemiological clues of the link between the cases. Additional information is garnered from the use of viral genome sequencing, making it possible to track origins of outbreaks, and to estimate how much of the observed human disease is attributable to foodborne infection by computerized linking of epidemiologic data to aligned viral genomic sequences (Verhoef et al., 2011). However, often the original source or evidence of it being food- or waterborne cannot be found, which means that outbreaks often are merely registered. Of the 941 viral disease outbreaks reported as foodborne in the joint ECDC-EFSA surveillance report of 2015, only 9.1% had robust evidence of food- or waterborne transmission (Eurosurveillance editorial team, 2015). Routine application of genotyping of HAV in newly diagnosed cases quadrupled the number of cases in which food was the most likely source of infection a 3 year enhanced surveillance study in The Netherlands, but this is not commonly done (Petrignani et al., 2014). In an investigation of 1794 food- and waterborne outbreaks in Korea, roughly 75% of the outbreaks reported in schools and public restaurants were attributed to an unknown origin (Moon et al., 2014). Availability and costs of molecular testing combined with sequencing, additional to the limited success of virus detection in food products, are likely further limiting their use in food and water surveillance. This is demonstrated by the fact that formal confirmation of a viral outbreak associated with food- and waterborne transmission still requires extensive epidemiological analysis or confirmation of a virus in the infected individual, or both (ESFA, 2016). However, due to the increase of genomic information of viruses, sequence data is increasingly used to support and strengthen outbreak investigations. Nevertheless, the surveillance programs for these viruses in the human food chain is limited, in contrast with the American CDC1 and the European ECDC2 surveillance programs for bacteria and parasitic pathogens causing food- and waterborne diseases (Deng et al., 2016) and does not have widespread coverage. As an example, to comply with European food safety regulations, shellfish, a well-known source of foodborne pathogens, need to be tested for enteric bacteria. However, it has been well documented that shellfish that pass quality control based on bacterial counts may still contain human pathogenic viruses (Rodriguez-Manzano et al., 2014). To be able to recognize food- waterborne viral disease outbreaks and stop underestimation of its disease burden there should be innovations in the current foodborne surveillance system.
Although the list of viruses causing acute gastroenteritis is long, norovirus ranks among the top causes of diarrheal disease (Ahmed et al., 2014). Reporting of outbreaks suggests that the food- and waterborne disease transmission route is relatively rare, but provides an underestimate, bearing in mind that it may be hard to recognize a food- and waterborne transmission route in community-acquired diarrheal disease. To quantify the burden of all diarrheal disease attributable to foodborne transmission, the World Health Organization commissioned a study that combined data from surveillance and exhaustive literature reviews with a systematic approach to calculation of the fraction of disease attributable to food contamination (Havelaar et al., 2015). This ranked the burden of norovirus illness among the top causes of foodborne disease, along with Campylobacter, and listed HAV associated disease among other significant causes of foodborne disease, along with Salmonella and Taenia solium.
For bacterial foodborne pathogens, the analysis of systematically collected surveillance data has been used as the basis of attribution analysis (Pires et al., 2009). A popular approach has been to quantify the proportion of foodborne disease of humans to their likely origin, by comparing diversity of strains found in human disease outbreaks with that found in animal and environmental reservoirs (Hald et al., 2007). While this model does not allow estimating the foodborne disease where food is a vehicle for person-to-person transmission, which is common for noroviruses, it has been used with some success to quantify the contribution of foodborne viral disease stemming from environmentally contaminated food (e.g., associated with shellfish; (Verhoef et al., 2015)). This builds from the observation that there is a large discrepancy between the norovirus variants in clinical settings and environmental samples (Tao et al., 2015; Kazama et al., 2016). Norovirus GII.4, found in clinical setting, is generally related to person-to-person transmission, however, several other norovirus genotypes and genogroups were found in environmental samples in the same area. However, food associated acute gastroenteritis is not limited to norovirus infections. In a large retrospective study of oyster-related acute gastroenteritis outbreaks in Osaka City in Japan 30.7% of the cases were attributed to other pathogens such aichivirus, astrovirus, sapovirus rotavirus A, and enteroviruses (Iritani et al., 2014). Furthermore, outbreaks can be caused by a mixture of these viruses and viral variants (Wang et al., 2015).
In 2008–2017, morbidity of Class B infectious diseases showed a significant downward trend, from 185.34/100,000 in 2008 to 54.36/100,000 in 2017 (χ2trend = 11,093.22, p < 0.05), with an annual morbidity of 90.39/100,000; morbidity of Class C infectious diseases showed a fluctuating upward trend, from 1352.97/100,000 in 2008 to 2549.03/100,000 in 2017 (χ2trend = 97,595.69, p < 0.05), with an average annual morbidity rate of 2412.47/100,000 (Table 1).
The top 5 reported Class B infectious diseases were dysentery, scarlet fever, measles, Influenza A (H1N1) and syphilis. The morbidity of measles, dysentery and syphilis showed a decline (measles: χ2trend = 10,156.59, p < 0.05; dysentery: χ2trend = 6301.75, p < 0.05; syphilis: χ2trend = 3376.99, p < 0.05); and that of scarlet fever was on the rise in recent years (χ2trend = 4185.20, p < 0.05). Influenza A (H1N1) was classified as a Class B infectious disease in 2009; 5805 cases of influenza A (H1N1) were reported in 2009, ranking first among Class B infectious diseases reported in the same year. This disease showed a decline in 2010 (χ2 = 5126.04, p < 0.05), and the number of cases reported was between 3 and 259 in 2010–2013. Since 1 January 2014, it was removed from Class B to Class C under the management of existing influenza (Figure 1).
The top 5 reported Class C infectious diseases were hand-foot-and-mouth disease (HFMD), other infectious diarrheal diseases, mumps, influenza and acute hemorrhagic conjunctivitis, among which the morbidity of HFMD, other infectious diarrheal diseases, and influenza were on the rise, while the morbidity of acute hemorrhagic conjunctivitis and mumps were decreasing year by year. In 2010, 11,789 cases of acute hemorrhagic conjunctivitis were reported, and thereafter the number of cases reported decreased rapidly (Figure 2).
In addition to noroviruses, kobuviruses, and parvoviruses, other viruses such as astroviruses, rotaviruses, novel circular replication-associated protein-encoding single-stranded (CRESS) DNA viruses, and Lyon-IARC polyomaviruses (LIPyVs), are suspected of having the ability to cause enteric disease in cats. Astroviruses (AstVs), family Astroviridae, are small, non-enveloped, spherical viruses of approximately 28–30 nm in diameter, with a positive single-stranded RNA genome of 6.8–7.3 kb in length organized in three ORFs (ORF1a, ORF1b, and ORF2) and a poly A tail at the 3′ end. AstVs have been identified in human beings and in a variety of terrestrial and marine mammals, as well as in several avian species, and they are classified into two genera, Mamastrovirus (AstVs of mammals) and Avastrovirus (AstVs of avians). AstVs are currently considered as one of the most common viruses associated with either mild or severe gastroenteritis in humans, mainly in young children and immunodeficient patients. Feline astrovirus (FAstV) was first identified by EM from the stool of a domestic kitten with diarrhea in the USA. Subsequent EM-based investigations revealed the presence of FAstV either in the stools of diarrheic or healthy cats. In the last few years, molecular tools have allowed characterizing in more detail the FAstVs infecting cats demonstrating the circulation of strains genetically closest to each other that have been classified within the species Mamastrovirus 2, formerly known as feline astrovirus. Sequence analysis of the complete ORF2 gene revealed that the feline strains clustered within two distinct groups that have been recently proposed as different genotypes (group 1 and 2). Furthermore, mamastroviruses more genetically closest in the RdRp region to AstVs previously found in foxes have occasionally been identified in fecal samples collected from healthy cats.
To date, FAstVs have been detected in stool samples of cats in UK, Australia, New Zealand, Italy, Hong Kong, South Korea, Portugal, Northeast China, and the USA with prevalence rates ranging from 4.8% to 28.6%. The role of AstV as enteric pathogen in cats still remains unclear, although experimental infection using the FAstV strain Bristol in specific pathogen–free (SPF) kittens induced enteritis and viral shedding. Furthermore, natural infection has been described in domestic cats with diarrhea either alone or in mixed infections mainly with FPV.
Rotaviruses (RVs) have been recognized as a major cause of human acute gastroenteritis since 1973. They primarily affect young children, accounting for almost 40% of hospital admissions for diarrhea and 200000 deaths worldwide. RVs (genus Rotavirus, family Reoviridae) are characterized by a 70–75 nm non-enveloped multi-layered virion with 11-segmented double-stranded RNA encoding six structural proteins (VP1–VP4, VP6, and VP7) and other five or six NSPs (e.g., NSP1–NSP5/NSP6). To date, nine rotavirus species (RVA to RVI) have been recognized, and a tentative tenth species (RVJ) has been described. Among these, RVA to RVC, RVE, RVH, and RVI are known to infect mammals, with RVA being the most prevalent. The genetic variability of the VP4 and VP7-encoding genes determines the binary RVA genotype classification system. Currently, 27 G (VP7) and 37 P genotypes (VP4) of RVA have been described in mammals and avian species. First evidence on the susceptibility of cats to feline rotavirus (FRV) infection was obtained serologically in 1978 by McNulty et al.. Subsequent experimental infections gave contrasting results, with some showing an association between rotavirus and reduced fecal quality as increased water content and not optimal conformation of feces, while others failed to give infection with evident clinical signs. Early epidemiological studies on rotavirus infection in cats has been conducted by serological assay and EM analysis. The serological studies reported prevalence ranging from 3.5% to 100%, while EM revealed rates from 5% to 6% either in diarrheic or healthy animals. In a large molecular survey performed in the UK, FRVs RNA has been detected with an overall prevalence of 3.0% (57/1727); statistical associations with diarrhea or age have not been found. Sequence analysis of the strains identified in cats showed the highest genetic correlation with RVs belonging to the A group. More recently, an I group rotavirus was detected in the feces of a diarrheic seventh-month old indoor cats. Phylogenetic analyses revealed that rotavirus I strain Felis catus shared a monophyletic root, being most closely related to the two currently known rotavirus species I strain detected in the feces of two sheltered dogs in Hungary. Overall, FRVs are currently considered to play a minor role in clinical disease and are not routinely screened in diarrheic cases in small-animal veterinary practices. However, whether and to what extent FRVs may impact feline health, deserves further study.
Mayor CRESS DNA viruses detected in animals are represented by circoviruses and cycloviruses belonging to the Circoviridae family. The susceptibility of carnivores to circoviruses infections have been previously demonstrated in domestic and wild canids. In dogs, these viruses have been found in association with clinical disease characterized by hemorrhagic gastroenteritis, severe necrotizing vasculitis, and granulomatous lymphadenitis. By converse, information on the epidemiology of circoviruses in the feline host is still limited. The identification of CRESS-DNA genomes in stool samples has been reported only on two occasions. By metagenomic sequencing, the complete genome of a cyclovirus strain (CyCVs-FD) was acquired assessing pooled fecal samples collected from clinically normal cats in a shelter in Davis, California. More recently, by molecular screening of twenty stool samples collected from diarrheic and healthy cats from a private cattery in Japan, viral DNA was detected in 71.4% (10/14) of animals with enteritis signs and in 50% (3/6) of asymptomatic cats. Full-genome sequence analysis of four strains revealed that these novel CRESS-DNA genomes, called feline stool associated circular viruses (FeSCVs), clustered within the family Circoviridae, but into a distinct clade to that of circovirus and cyclovirus. Further investigations aimed to acquire information on the genetic features, epidemiology and pathogenesis of these viruses are needed. Polyomaviruses (PyVs), belonging to the Polyomaviridae family, are non-enveloped double-stranded DNA viruses with a circular genome of approximately 5.0 kb in length. PyVs DNA have been occasionally identified in fish, birds, rodents, and primates. Interestingly, novel PyVs have been recently found in cat fecal samples during a diarrhea outbreak in Canada by using a metagenomic approach. Upon sequence analysis, the feline PyVs revealed the highest identity (97.0%) to members of the genus Alphapolyomavirus detected in saliva and skin samples of human origin and provisionally named Lyon IARC PyVs (LIPyVs). The etiologic role of LIPyV in feline diarrhea should be investigated as well as its zoonotic potential.
Although lower respiratory infections, including pneumonia, are one of the main causes of death worldwide, real-time surveillance systems and situational awareness are generally lacking.
In the year after the SARS outbreak in 2003, NHFPC developed a surveillance system for unexplained pneumonia to facilitate timely detection of airborne pathogens that form a severe threat to public health. Therefore, all Chinese health care facilities are required to report any patient who has a clinical diagnosis of pneumonia with an unknown causative pathogen and whose illness meets the following five criteria (2007 modified definition): (1) fever ≥38 °C; (2) radiologic characteristics consistent with pneumonia; (3) normal or reduced leukocyte count or low lymphocyte count in early clinical stage; (4) no improvement or worsening of the patient’s condition after first-line antibiotic treatment for 3–5 days; and (5) the pneumonia etiology cannot be attributed to an alternative laboratory or clinical diagnosis (clinicians are granted flexibility to determine how to interpret this criterion and specific tests are not specified) [22, 23]. Once the case is registered in NIDRIS, the data are further analysed in CIDARS as a type 1 disease, for which a fixed-threshold method (of 1 case) is applied. A real-time SMS is followed by a field investigation, whereby case samples are tested to rule out avian influenza, SARS and Middle East respiratory syndrome coronavirus (MERS-CoV). Although physicians are required to report unexplained pneumonia cases, considerable under-reporting occurs. The aim of this surveillance system is not to detect each unexplained pneumonia case but to focus on clusters that could indicate an (unknown) emerging infectious disease outbreak.
Unexplained pneumonia is not a notifiable condition in the Netherlands as it is in China. However, according to the Public Health Act (2008), each physician should notify a case or an unusual number of cases with an (unknown) infectious disease that forms a severe threat to public health. An example is the Q fever outbreak (2007); the unusual number of atypical pneumonia cases early in the outbreak were not detected by routine surveillance systems but by astute general practitioners (GPs). Both Dutch legislation and the Chinese pneumonia surveillance system aim for early notification of (unknown) emerging infectious disease outbreaks. However, in both countries, criteria for notification are not well defined and a considerable degree of under-ascertainment and under-reporting is likely. In the Netherlands, structural syndromic pneumonia surveillance is carried out using data extracted from electronic patient files maintained by sentinel GP practices, representing 7% of the Dutch population. Moreover, sentinel registration of pneumonia cases in nursing homes takes place. A separate virologic laboratory surveillance system provides information on circulating respiratory viruses. Since 2015, a pilot study has been carried out for hospitalized severe acute respiratory infections (SARI) patients. As it includes only two of 133 hospitals in the country at present, the obtained data is not yet reliable to provide early warning of infectious pneumonia outbreaks. Currently, no set threshold exists for unusual occurrence of pneumonia. Expert opinion determines which signals are discussed by the NEWC.
In China, if a notifiable infectious disease is clinically diagnosed and/or laboratory confirmed according to the unified national diagnostic criteria issued by the NHFPC, cases must be reported to the national China CDC, which collects and analyses the acquired data. The health care provider enters the case information using a standard form into the Notifiable Infectious Diseases Reporting Information System (NIDRIS), a web-based system that enables all healthcare institutions to report cases of notifiable infectious diseases. Approximately 5 million infectious disease cases are reported annually (≈ 385 cases per 100,000 citizens per year). Each China CDC level can analyse its own data in NIDRIS and data from subordinate levels within its own administrative boundaries.
In the Netherlands, if a notifiable infectious disease is suspected and/or laboratory tests confirms it, the case must be reported both by the attending physician and the laboratory to the regional PHS. The case information is collected and entered by the PHS into Osiris, a web-based database that transmits the data to RIVM for further analyses. In 2014, 13,863 notifiable disease cases were reported via Osiris to RIVM (≈ 815 cases per 100,000 citizen per year).
During the period of 2008–2017, a total of 32 types and 1,994,740 cases of notifiable diseases in children aged 0–14 years, including 266 deaths, were reported in Zhejiang Province, with an annual average morbidity rate of 2502.87/100,000 and an annual average mortality rate of 0.33/100,000. There were no cases and deaths involving plague, cholera, infectious atypical pneumonia, human infection with avian influenza, polio, anthrax, diphtheria and filariasis. No Class A infectious diseases were reported. Twenty-two types and 72,041 cases of Class B infectious diseases were reported, including 138 deaths; 10 types and 1,922,699 cases of Class C infectious diseases were reported, including 128 deaths.